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FaceBase Publications | FaceBase

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FaceBase Publications

The following are published journal articles based on data available in the FaceBase Consortium.

  1. Dynamic enhancer landscapes in human craniofacial development

    Rajderkar, Sudha Sunil; Paraiso, Kitt; Amaral, Maria Luisa; Kosicki, Michael; Cook, Laura E.; Darbellay, Fabrice; Spurrell, Cailyn H.; Osterwalder, Marco; Zhu, Yiwen; Wu, Han; Afzal, Sarah Yasmeen; Blow, Matthew J.; Kelman, Guy; Barozzi, Iros; Fukuda-Yuzawa, Yoko; Akiyama, Jennifer A.; Afzal, Veena; Tran, Stella; Plajzer-Frick, Ingrid; Novak, Catherine S.; Kato, Momoe; Hunter, Riana D.; Von Maydell, Kianna; Wang, Allen; Lin, Lin; Preissl, Sebastian; Lisgo, Steven; Ren, Bing; Dickel, Diane E.; Pennacchio, Len A.; Visel, Axel. Nature Communications. vol. 15(1), 2030. March 2024.

    The genetic basis of human facial variation and craniofacial birth defects remains poorly understood. Distant-acting transcriptional enhancers control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development. However, a lack of accurate maps of the genomic locations and cell type-resolved activities of craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combine histone modification, chromatin accessibility, and gene expression profiling of human craniofacial development with single-cell analyses of the developing mouse face to define the regulatory landscape of facial development at tissue- and single cell-resolution. We provide temporal activity profiles for 14,000 human developmental craniofacial enhancers. We find that 56% of human craniofacial enhancers share chromatin accessibility in the mouse and we provide cell population- and embryonic stage-resolved predictions of their in vivo activity. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development.

  2. Comparing 2D and 3D representations for face-based genetic syndrome diagnosis

    Bannister, Jordan J.; Wilms, Matthias; Aponte, J. David; Katz, David C.; Klein, Ophir D.; Bernier, Francois P.; Spritz, Richard A.; Hallgrímsson, Benedikt; Forkert, Nils D.. European Journal of Human Genetics. vol. 31(9), 1010–1016. September 2023.

  3. Privacy, bias and the clinical use of facial recognition technology: A survey of genetics professionals

    Aboujaoude, Elias; Light, Janice; Brown, Julia E. H.; Boscardin, W. John; Hallgrímsson, Benedikt; Klein, Ophir D.. American Journal of Medical Genetics Part C: Seminars in Medical Genetics. vol. 193(3), e32035. September 2023.

    Abstract Facial recognition technology (FRT) has been adopted as a precision medicine tool. The medical genetics field highlights both the clinical potential and privacy risks of this technology, putting the discipline at the forefront of a new digital privacy debate. Investigating how geneticists perceive the privacy concerns surrounding FRT can help shape the evolution and regulation of the field, and provide lessons for medicine and research more broadly. Five hundred and sixty‐two genetics clinicians and researchers were approached to fill out a survey, 105 responded, and 80% of these completed. The survey consisted of 48 questions covering demographics, relationship to new technologies, views on privacy, views on FRT, and views on regulation. Genetics professionals generally placed a high value on privacy, although specific views differed, were context‐specific, and covaried with demographic factors. Most respondents (88%) agreed that privacy is a basic human right, but only 37% placed greater weight on it than other values such as freedom of speech. Most respondents (80%) supported FRT use in genetics, but not necessarily for broader clinical use. A sizeable percentage (39%) were unaware of FRT’s lower accuracy rates in marginalized communities and of the mental health effects of privacy violations (62%), but most (76% and 75%, respectively) expressed concern when informed. Overall, women and those who self‐identified as politically progressive were more concerned about the lower accuracy rates in marginalized groups (88% vs. 64% and 83% vs. 63%, respectively). Younger geneticists were more wary than older geneticists about using FRT in genetics (28% compared to 56% “strongly” supported such use). There was an overall preference for more regulation, but respondents had low confidence in governments’ or technology companies’ ability to accomplish this. Privacy views are nuanced and context‐dependent. Support for privacy was high but not absolute, and clear deficits existed in awareness of crucial FRT‐related discrimination potential and mental health impacts. Education and professional guidelines may help to evolve views and practices within the field.

  4. Spatiotemporal single-cell regulatory atlas reveals neural crest lineage diversification and cellular function during tooth morphogenesis

    Jing, Junjun; Feng, Jifan; Yuan, Yuan; Guo, Tingwei; Lei, Jie; Pei, Fei; Ho, Thach-Vu; Chai, Yang. Nature Communications. vol. 13(1), 4803. August 2022.

    Cranial neural crest cells are an evolutionary innovation of vertebrates for craniofacial development and function, yet the mechanisms that govern the cell fate decisions of postmigratory cranial neural crest cells remain largely unknown. Using the mouse molar as a model, we perform single-cell transcriptome profiling to interrogate the cell fate diversification of postmigratory cranial neural crest cells. We reveal the landscape of transcriptional heterogeneity and define the specific cellular domains during the progression of cranial neural crest cell-derived dental lineage diversification, and find that each domain makes a specific contribution to distinct molar mesenchymal tissues. Furthermore, IGF signaling-mediated cell-cell interaction between the cellular domains highlights the pivotal role of autonomous regulation of the dental mesenchyme. Importantly, we reveal cell-type-specific gene regulatory networks in the dental mesenchyme and show that Foxp4 is indispensable for the differentiation of periodontal ligament. Our single-cell atlas provides comprehensive mechanistic insight into the cell fate diversification process of the cranial neural crest cell-derived odontogenic populations.

  5. Whole-genome sequencing reveals de-novo mutations associated with nonsyndromic cleft lip/palate

    Awotoye, Waheed; Mossey, Peter A.; Hetmanski, Jacqueline B.; Gowans, Lord J. J.; Eshete, Mekonen A.; Adeyemo, Wasiu L.; Alade, Azeez; Zeng, Erliang; Adamson, Olawale; Naicker, Thirona; Anand, Deepti; Adeleke, Chinyere; Busch, Tamara; Li, Mary; Petrin, Aline; Aregbesola, Babatunde S.; Braimah, Ramat O.; Oginni, Fadekemi O.; Oladele, Ayodeji O.; Oladayo, Abimbola; Kayali, Sami; Olotu, Joy; Hassan, Mohaned; Pape, John; Donkor, Peter; Arthur, Fareed K. N.; Obiri-Yeboah, Solomon; Sabbah, Daniel K.; Agbenorku, Pius; Plange-Rhule, Gyikua; Oti, Alexander Acheampong; Gogal, Rose A.; Beaty, Terri H.; Taub, Margaret; Marazita, Mary L.; Schnieders, Michael J.; Lachke, Salil A.; Adeyemo, Adebowale A.; Murray, Jeffrey C.; Butali, Azeez. Scientific Reports. vol. 12(1), 11743. July 2022.

    The majority (85%) of nonsyndromic cleft lip with or without cleft palate (nsCL/P) cases occur sporadically, suggesting a role for de novo mutations (DNMs) in the etiology of nsCL/P. To identify high impact protein-altering DNMs that contribute to the risk of nsCL/P, we conducted whole-genome sequencing (WGS) analyses in 130 African case-parent trios (affected probands and unaffected parents). We identified 162 high confidence protein-altering DNMs some of which are based on available evidence, contribute to the risk of nsCL/P. These include novel protein-truncating DNMs in the ACTL6A, ARHGAP10, MINK1, TMEM5 and TTN genes; as well as missense variants in ACAN, DHRS3, DLX6, EPHB2, FKBP10, KMT2D, RECQL4, SEMA3C, SEMA4D, SHH, TP63, and TULP4. Many of these protein-altering DNMs were predicted to be pathogenic. Analysis using mouse transcriptomics data showed that some of these genes are expressed during the development of primary and secondary palate. Gene-set enrichment analysis of the protein-altering DNMs identified palatal development and neural crest migration among the few processes that were significantly enriched. These processes are directly involved in the etiopathogenesis of clefting. The analysis of the coding sequence in the WGS data provides more evidence of the opportunity for novel findings in the African genome.

  6. FaceBase: A Community-Driven Hub for Data-Intensive Research

    Schuler, R.E.; Bugacov, A.; Hacia, J.G.; Ho, T.V.; Iwata, J.; Pearlman, L.; Samuels, B.D.; Williams, C.; Zhao, Z.; Kesselman, C.; Chai, Y.. Journal of Dental Research. , 002203452211079. July 2022.

    The FaceBase Consortium, funded by the National Institute of Dental and Craniofacial Research of the National Institutes of Health, was established in 2009 with the recognition that dental and craniofacial research are increasingly data-intensive disciplines. Data sharing is critical for the validation and reproducibility of results as well as to enable reuse of data. In service of these goals, data ought to be FAIR: Findable, Accessible, Interoperable, and Reusable. The FaceBase data repository and educational resources exemplify the FAIR principles and support a broad user community including researchers in craniofacial development, molecular genetics, and genomics. FaceBase demonstrates that a model in which researchers “self-curate” their data can be successful and scalable. We present the results of the first 2.5 y of FaceBase’s operations as an open community and summarize the data sets published during this period. We then describe a research highlight from work on the identification of regulatory networks and noncoding RNAs involved in cleft lip with/without cleft palate that both used and in turn contributed new findings to publicly available FaceBase resources. Collectively, FaceBase serves as a dynamic and continuously evolving resource to facilitate data-intensive research, enhance data reproducibility, and perform deep phenotyping across multiple species in dental and craniofacial research.

  7. Marsupials and Multi-Omics: Establishing New Comparative Models of Neural Crest Patterning and Craniofacial Development

    Newton, Axel H.. Frontiers in Cell and Developmental Biology. vol. 10, 941168. June 2022.

    Studies across vertebrates have revealed significant insights into the processes that drive craniofacial morphogenesis, yet we still know little about how distinct facial morphologies are patterned during development. Studies largely point to evolution in GRNs of cranial progenitor cell types such as neural crest cells, as the major driver underlying adaptive cranial shapes. However, this hypothesis requires further validation, particularly within suitable models amenable to manipulation. By utilizing comparative models between related species, we can begin to disentangle complex developmental systems and identify the origin of species-specific patterning. Mammals present excellent evolutionary examples to scrutinize how these differences arise, as sister clades of eutherians and marsupials possess suitable divergence times, conserved cranial anatomies, modular evolutionary patterns, and distinct developmental heterochrony in their NCC behaviours and craniofacial patterning. In this review, I lend perspectives into the current state of mammalian craniofacial biology and discuss the importance of establishing a new marsupial model, the fat-tailed dunnart, for comparative research. Through detailed comparisons with the mouse, we can begin to decipher mammalian conserved, and species-specific processes and their contribution to craniofacial patterning and shape disparity. Recent advances in single-cell multi-omics allow high-resolution investigations into the cellular and molecular basis of key developmental processes. As such, I discuss how comparative evolutionary application of these tools can provide detailed insights into complex cellular behaviours and expression dynamics underlying adaptive craniofacial evolution. Though in its infancy, the field of “comparative evo-devo-omics” presents unparalleled opportunities to precisely uncover how phenotypic differences arise during development.

  8. A unique form of collective epithelial migration is crucial for tissue fusion in the secondary palate and can overcome loss of epithelial apoptosis

    Teng, Teng; Teng, Camilla S.; Kaartinen, Vesa; Bush, Jeffrey O.. Development. vol. 149(10), dev200181. May 2022.

    ABSTRACT Tissue fusion frequently requires the removal of an epithelium that intervenes distinct primordia to form one continuous structure. In the mammalian secondary palate, a midline epithelial seam (MES) forms between two palatal shelves and must be removed to allow mesenchymal confluence. Abundant apoptosis and cell extrusion support their importance in MES removal. However, genetically disrupting the intrinsic apoptotic regulators BAX and BAK within the MES results in complete loss of cell death and cell extrusion, but successful removal of the MES. Novel static- and live-imaging approaches reveal that the MES is removed through streaming migration of epithelial trails and islands to reach the oral and nasal epithelial surfaces. Epithelial trail cells that express the basal epithelial marker ΔNp63 begin to express periderm markers, suggesting that migration is concomitant with differentiation. Live imaging reveals anisotropic actomyosin contractility within epithelial trails, and genetic ablation of actomyosin contractility results in dispersion of epithelial collectives and failure of normal MES migration. These findings demonstrate redundancy between cellular mechanisms of morphogenesis, and reveal a crucial and unique form of collective epithelial migration during tissue fusion.

  9. MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses

    Devine, Jay; Vidal-García, Marta; Liu, Wei; Neves, Amanda; Lo Vercio, Lucas D.; Green, Rebecca M.; Richbourg, Heather A.; Marchini, Marta; Unger, Colton M.; Nickle, Audrey C.; Radford, Bethany; Young, Nathan M.; Gonzalez, Paula N.; Schuler, Robert E.; Bugacov, Alejandro; Rolian, Campbell; Percival, Christopher J.; Williams, Trevor; Niswander, Lee; Calof, Anne L.; Lander, Arthur D.; Visel, Axel; Jirik, Frank R.; Cheverud, James M.; Klein, Ophir D.; Birnbaum, Ramon Y.; Merrill, Amy E.; Ackermann, Rebecca R.; Graf, Daniel; Hemberger, Myriam; Dean, Wendy; Forkert, Nils D.; Murray, Stephen A.; Westerberg, Henrik; Marcucio, Ralph S.; Hallgrímsson, Benedikt. Scientific Data. vol. 9(1), 230. May 2022.

    Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N = 10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase (, 10.25550/3-HXMC) and GitHub (

  10. Dissecting the Role of the Six1‐Associated Genes in Craniofacial Development and Disease

    Jourdeuil, Karyn; Baxi, Aparna B.; Tavares, Andre L.. The FASEB Journal. vol. 36(S1), fasebj.2022.36.S1.R4883. May 2022.

    Genetic variants of SIX1, a transcription factor implicated in many cellular processes during craniofacial development, and of its co-factor EYA1 underlie 50% of Branchio-oto-renal syndrome (BOR) cases. BOR is an autosomal dominant disorder characterized by various craniofacial defects, including malformed middle ear ossicles leading to conductive hearing loss. Interestingly, some BOR variants in SIX1 also underlie craniosynostosis (CRS) which indicates that SIX1 has a crucial role in craniofacial bone development. One important aspect of SIX1 function is that it interacts with co-factors that can induce or repress its transcriptional activity. In this work, we expand our knowledge of the Six1 gene regulatory network with respect to craniofacial development. We first used a Six1 knockout mouse line to assess gene expression profiles of dissected E10.5 mandibular arches which give rise to the middle ear ossicles and lower jaw, via RNA-sequencing. Our transcriptomic analysis led to the identification of 122 genes, including components of the FGF and WNT signaling pathways, known contributors to craniofacial bone development. In parallel, we used in vitro studies to characterize three novel co-factors of Six1: Sobp, Mcrs1 and Pa2g4 that were recently identified in Xenopus. Our findings indicate that Sobp and Mcrs1 are bona fide Six1 co-factors in mouse. We are now studying the genes identified in the RNA-seq and the novel Six1 co-factors using in vitro culture of mouse pre-osteoblastic cells (MC3T3-E1) to identify their role in bone formation. Altogether, our results identified novel Six1-associated genes that are potential candidate genes for the unresolved cases of BOR and CRS.

  11. Technological scouting of bi-material face masks: simulation of adherence using 3D Facial Norms

    Ficarella, Elisa; Natalicchio, Angelo; Spina, Roberto; Galantucci, Luigi Maria. Procedia CIRP. vol. 110, 259–264. 2022.

    During the COVID-19 pandemic started in March 2020, the need for personal protective equipment rapidly grew as it became mandatory. The availability of a set of faces can be of great utility in designing a face mask with proper adherence and comfortability in wearing and breathing. A 3D geometry of a face with user-defined anthropometric measures was generated with Blender, a powerful development tool for creating 3D images. Using 3D Facial Norms, a free online database, it was possible to compute the mean anthropometric measures for the age groups of 17-20, 20-30, and 30-40 years old and then generate the respective faces for both genders. The adherence of an innovative face mask was then simulated with the reverse engineering software considering both the face mask and the faces rigid.

  12. KDM6B interacts with TFDP1 to activate P53 signaling in regulating mouse palatogenesis

    Guo, Tingwei; Han, Xia; He, Jinzhi; Feng, Jifan; Jing, Junjun; Janečková, Eva; Lei, Jie; Ho, Thach-Vu; Xu, Jian; Chai, Yang. eLife. vol. 11, e74595. February 2022.

    Epigenetic regulation plays extensive roles in diseases and development. Disruption of epigenetic regulation not only increases the risk of cancer, but can also cause various developmental defects. However, the question of how epigenetic changes lead to tissue-specific responses during neural crest fate determination and differentiation remains understudied. Using palatogenesis as a model, we reveal the functional significance of Kdm6b , an H3K27me3 demethylase, in regulating mouse embryonic development. Our study shows that Kdm6b plays an essential role in cranial neural crest development, and loss of Kdm6b disturbs P53 pathway-mediated activity, leading to complete cleft palate along with cell proliferation and differentiation defects in mice. Furthermore, activity of H3K27me3 on the promoter of Trp53 is antagonistically controlled by Kdm6b , and Ezh2 in cranial neural crest cells. More importantly, without Kdm6b , the transcription factor TFDP1, which normally binds to the promoter of Trp53 , cannot activate Trp53 expression in palatal mesenchymal cells. Furthermore, the function of Kdm6b in activating Trp53 in these cells cannot be compensated for by the closely related histone demethylase Kdm6a . Collectively, our results highlight the important role of the epigenetic regulator KDM6B and how it specifically interacts with TFDP1 to achieve its functional specificity in regulating Trp53 expression, and further provide mechanistic insights into the epigenetic regulatory network during organogenesis.

  13. Lifelong single-cell profiling of cranial neural crest diversification in zebrafish

    Fabian, Peter; Tseng, Kuo-Chang; Thiruppathy, Mathi; Arata, Claire; Chen, Hung-Jhen; Smeeton, Joanna; Nelson, Nellie; Crump, J. Gage. Nature Communications. vol. 13(1), 13. January 2022.

    The cranial neural crest generates a huge diversity of derivatives, including the bulk of connective and skeletal tissues of the vertebrate head. How neural crest cells acquire such extraordinary lineage potential remains unresolved. By integrating single-cell transcriptome and chromatin accessibility profiles of cranial neural crest-derived cells across the zebrafish lifetime, we observe progressive and region-specific establishment of enhancer accessibility for distinct fates. Neural crest-derived cells rapidly diversify into specialized progenitors, including multipotent skeletal progenitors, stromal cells with a regenerative signature, fibroblasts with a unique metabolic signature linked to skeletal integrity, and gill-specific progenitors generating cell types for respiration. By retrogradely mapping the emergence of lineage-specific chromatin accessibility, we identify a wealth of candidate lineage-priming factors, including a Gata3 regulatory circuit for respiratory cell fates. Rather than multilineage potential being established during cranial neural crest specification, our findings support progressive and region-specific chromatin remodeling underlying acquisition of diverse potential.

  14. The Chromatin Regulator Ankrd11 Controls Palate and Cranial Bone Development

    Roth, Daniela Marta; Baddam, Pranidhi; Lin, Haiming; Vidal-García, Marta; Aponte, Jose David; De Souza, Sarah-Thea; Godziuk, Devyn; Watson, Adrianne Eve Scovil; Footz, Tim; Schachter, Nathan F.; Egan, Sean E.; Hallgrímsson, Benedikt; Graf, Daniel; Voronova, Anastassia. Frontiers in Cell and Developmental Biology. vol. 9, 645386. 2021.

    Epigenetic and chromatin regulation of craniofacial development remains poorly understood. Ankyrin Repeat Domain 11 (ANKRD11) is a chromatin regulator that has previously been shown to control neural stem cell fates via modulation of histone acetylation. ANKRD11 gene variants, or microdeletions of the 16q24.3 chromosomal region encompassing the ANKRD11 gene, cause KBG syndrome, a rare autosomal dominant congenital disorder with variable neurodevelopmental and craniofacial involvement. Craniofacial abnormalities include a distinct facial gestalt, delayed bone age, tooth abnormalities, delayed fontanelle closure, and frequently cleft or submucosal palate. Despite this, the dramatic phenotype and precise role of ANKRD11 in embryonic craniofacial development remain unexplored. Quantitative analysis of 3D images of KBG syndromic subjects shows an overall reduction in the size of the middle and lower face. Here, we report that mice with heterozygous deletion of Ankrd11 in neural crest cells (Ankrd11nchet) display a mild midfacial hypoplasia including reduced midfacial width and a persistent open fontanelle, both of which mirror KBG syndrome patient facial phenotypes. Mice with a homozygous Ankrd11 deletion in neural crest cells (Ankrd11ncko) die at birth. They show increased severity of several clinical manifestations described for KBG syndrome, such as cleft palate, retrognathia, midfacial hypoplasia, and reduced calvarial growth. At E14.5, Ankrd11 expression in the craniofacial complex is closely associated with developing bony structures, while expression at birth is markedly decreased. Conditional deletion of Ankrd11 leads to a reduction in ossification of midfacial bones, with several ossification centers failing to expand and/or fuse. Intramembranous bones show features of delayed maturation, with bone remodeling severely curtailed at birth. Palatal shelves remain hypoplastic at all developmental stages, with a local reduction in proliferation at E13.5. Our study identifies Ankrd11 as a critical regulator of intramembranous ossification and palate development and suggests that Ankrd11nchet and Ankrd11ncko mice may serve as pre-clinical models for KBG syndrome in humans.

  15. Mouse Dspp frameshift model of human dentinogenesis imperfecta

    Liang, Tian; Hu, Yuanyuan; Zhang, Hong; Xu, Qian; Smith, Charles E.; Zhang, Chuhua; Kim, Jung-Wook; Wang, Shih-Kai; Saunders, Thomas L.; Lu, Yongbo; Hu, Jan C.-C.; Simmer, James P.. Scientific Reports. vol. 11(1), 20653. December 2021.

    Abstract Non-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein ( DSPP ): 5′ mutations affecting an N-terminal targeting sequence and 3′ mutations that shift translation into the − 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp −1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a Dspp P19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. Dspp P19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. Dspp P19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp −1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp +/+ and Dspp P19L dentin. The Dspp −1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5′ and 3′ Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders.

  16. Large-scale open-source three-dimensional growth curves for clinical facial assessment and objective description of facial dysmorphism

    Matthews, Harold S.; Palmer, Richard L.; Baynam, Gareth S.; Quarrell, Oliver W.; Klein, Ophir D.; Spritz, Richard A.; Hennekam, Raoul C.; Walsh, Susan; Shriver, Mark; Weinberg, Seth M.; Hallgrimsson, Benedikt; Hammond, Peter; Penington, Anthony J.; Peeters, Hilde; Claes, Peter D.. Scientific Reports. vol. 11(1), 12175. December 2021.

    Abstract Craniofacial dysmorphism is associated with thousands of genetic and environmental disorders. Delineation of salient facial characteristics can guide clinicians towards a correct clinical diagnosis and understanding the pathogenesis of the disorder. Abnormal facial shape might require craniofacial surgical intervention, with the restoration of normal shape an important surgical outcome. Facial anthropometric growth curves or standards of single inter-landmark measurements have traditionally supported assessments of normal and abnormal facial shape, for both clinical and research applications. However, these fail to capture the full complexity of facial shape. With the increasing availability of 3D photographs, methods of assessment that take advantage of the rich information contained in such images are needed. In this article we derive and present open-source three-dimensional (3D) growth curves of the human face. These are sequences of age and sex-specific expected 3D facial shapes and statistical models of the variation around the expected shape, derived from 5443 3D images. We demonstrate the use of these growth curves for assessing patients and show that they identify normal and abnormal facial morphology independent from age-specific facial features. 3D growth curves can facilitate use of state-of-the-art 3D facial shape assessment by the broader clinical and biomedical research community. This advance in phenotype description will support clinical diagnosis and the understanding of disease pathogenesis including genotype–phenotype relations.

  17. The developing mouse coronal suture at single-cell resolution

    Farmer, D’Juan T.; Mlcochova, Hana; Zhou, Yan; Koelling, Nils; Wang, Guanlin; Ashley, Neil; Bugacov, Helena; Chen, Hung-Jhen; Parvez, Riana; Tseng, Kuo-Chang; Merrill, Amy E.; Maxson, Robert E.; Wilkie, Andrew O. M.; Crump, J. Gage; Twigg, Stephen R. F.. Nature Communications. vol. 12(1), 4797. December 2021.

    Abstract Sutures separate the flat bones of the skull and enable coordinated growth of the brain and overlying cranium. The coronal suture is most commonly fused in monogenic craniosynostosis, yet the unique aspects of its development remain incompletely understood. To uncover the cellular diversity within the murine embryonic coronal suture, we generated single-cell transcriptomes and performed extensive expression validation. We find distinct pre-osteoblast signatures between the bone fronts and periosteum, a ligament-like population above the suture that persists into adulthood, and a chondrogenic-like population in the dura mater underlying the suture. Lineage tracing reveals an embryonic Six2 + osteoprogenitor population that contributes to the postnatal suture mesenchyme, with these progenitors being preferentially affected in a Twist1 +/−; Tcf12 +/− mouse model of Saethre-Chotzen Syndrome. This single-cell atlas provides a resource for understanding the development of the coronal suture and the mechanisms for its loss in craniosynostosis.

  18. Single-cell analysis identifies a key role for Hhip in murine coronal suture development

    Holmes, Greg; Gonzalez-Reiche, Ana S.; Saturne, Madrikha; Motch Perrine, Susan M.; Zhou, Xianxiao; Borges, Ana C.; Shewale, Bhavana; Richtsmeier, Joan T.; Zhang, Bin; van Bakel, Harm; Jabs, Ethylin Wang. Nature Communications. vol. 12(1), 7132. December 2021.

    Craniofacial development depends on formation and maintenance of sutures between bones of the skull. In sutures, growth occurs at osteogenic fronts along the edge of each bone, and suture mesenchyme separates adjacent bones. Here, we perform single-cell RNA-seq analysis of the embryonic, wild type murine coronal suture to define its population structure. Seven populations at E16.5 and nine at E18.5 comprise the suture mesenchyme, osteogenic cells, and associated populations. Expression of Hhip, an inhibitor of hedgehog signaling, marks a mesenchymal population distinct from those of other neurocranial sutures. Tracing of the neonatal Hhip-expressing population shows that descendant cells persist in the coronal suture and contribute to calvarial bone growth. In Hhip-/- coronal sutures at E18.5, the osteogenic fronts are closely apposed and the suture mesenchyme is depleted with increased hedgehog signaling compared to those of the wild type. Collectively, these data demonstrate that Hhip is required for normal coronal suture development.

  19. Integrating GWAS and eQTL to predict genes and pathways for non-syndromic cleft lip with or without palate

    Yang, Jing; Yu, Xin; Zhu, Guirong; Wang, Ruimin; Lou, Shu; Zhu, Weihao; Fu, Chengyi; Liu, Jinsuo; Fan, Liwen; Li, Dandan; Shao, Qinghua; Ma, Lan; Wang, Lin; Wang, Zhendong; Pan, Yongchu. Oral Diseases. vol. 27(7), 1747–1754. October 2021.

    OBJECTIVE: To explore susceptibility genes and pathways for non-syndromic cleft lip with or without cleft palate (NSCL/P). MATERIALS AND METHODS: Two genome-wide association studies (GWAS) datasets, including 858 NSCL/P cases and 1,248 controls, were integrated with expression quantitative trait loci (eQTL) dataset identified by Genotype-Tissue Expression (GTEx) project in whole-blood samples. The expression of the candidate genes in mouse orofacial development was inquired from FaceBase. Protein-protein interaction (PPI) network was visualized to identify protein functions. Go and KEGG pathway analyses were performed to explore the underlying risk pathways. RESULTS: A total of 233 eQTL single-nucleotide polymorphisms (SNPs) in 432 candidate genes were identified to be associated with the risk of NSCL/P. One hundred and eighty-three susceptible genes were expressed in mouse orofacial development according to FaceBase. PPI network analysis highlighted that these genes involved in ubiquitin-mediated proteolysis (KCTD7, ASB1, UBOX5, ANAPC4) and DNA synthesis (XRCC3, RFC3, KAT5, RHNO1) were associated with the risk of NSCL/P. GO and KEGG pathway analyses revealed that the fatty acid metabolism pathway (ACADL, HSD17B12, ACSL5, PPT1, MCAT) played an important role in the development of NSCL/P. CONCLUSIONS: Our results identified novel susceptibility genes and pathways associated with the development of NSCL/P.

  20. Genome-wide copy number variations in a large cohort of bantu African children

    Yilmaz, Feyza; Null, Megan; Astling, David; Yu, Hung-Chun; Cole, Joanne; Santorico, Stephanie A.; Hallgrimsson, Benedikt; Manyama, Mange; Spritz, Richard A.; Hendricks, Audrey E.; Shaikh, Tamim H.. BMC medical genomics. vol. 14(1), 129. May 2021.

    BACKGROUND: Copy number variations (CNVs) account for a substantial proportion of inter-individual genomic variation. However, a majority of genomic variation studies have focused on single-nucleotide variations (SNVs), with limited genome-wide analysis of CNVs in large cohorts, especially in populations that are under-represented in genetic studies including people of African descent. METHODS: We carried out a genome-wide copy number analysis in \textgreater 3400 healthy Bantu Africans from Tanzania. Signal intensity data from high density (\textgreater 2.5 million probes) genotyping arrays were used for CNV calling with three algorithms including PennCNV, DNAcopy and VanillaICE. Stringent quality metrics and filtering criteria were applied to obtain high confidence CNVs. RESULTS: We identified over 400,000 CNVs larger than 1 kilobase (kb), for an average of 120 CNVs (SE = 2.57) per individual. We detected 866 large CNVs (≥ 300 kb), some of which overlapped genomic regions previously associated with multiple congenital anomaly syndromes, including Prader-Willi/Angelman syndrome (Type1) and 22q11.2 deletion syndrome. Furthermore, several of the common CNVs seen in our cohort (≥ 5%) overlap genes previously associated with developmental disorders. CONCLUSIONS: These findings may help refine the phenotypic outcomes and penetrance of variations affecting genes and genomic regions previously implicated in diseases. Our study provides one of the largest datasets of CNVs from individuals of African ancestry, enabling improved clinical evaluation and disease association of CNVs observed in research and clinical studies in African populations.

  21. Arid1a-Plagl1-Hh signaling is indispensable for differentiation-associated cell cycle arrest of tooth root progenitors

    Du, Jiahui; Jing, Junjun; Yuan, Yuan; Feng, Jifan; Han, Xia; Chen, Shuo; Li, Xiang; Peng, Weiqun; Xu, Jian; Ho, Thach-Vu; Jiang, Xinquan; Chai, Yang. Cell Reports. vol. 35(1), 108964. April 2021.

    Chromatin remodelers often show broad expression patterns in multiple cell types yet can elicit cell-specific effects in development and diseases. Arid1a binds DNA and regulates gene expression during tissue development and homeostasis. However, it is unclear how Arid1a achieves its functional specificity in regulating progenitor cells. Using the tooth root as a model, we show that loss of Arid1a impairs the differentiation-associated cell cycle arrest of tooth root progenitors through Hedgehog (Hh) signaling regulation, leading to shortened roots. Our data suggest that Plagl1, as a co-factor, endows Arid1a with its cell-type/spatial functional specificity. Furthermore, we show that loss of Arid1a leads to increased expression of Arid1b, which is also indispensable for odontoblast differentiation but is not involved in regulation of Hh signaling. This study expands our knowledge of the intricate interactions among chromatin remodelers, transcription factors, and signaling molecules during progenitor cell fate determination and lineage commitment.

  22. Anomalous incisor morphology indicates tissue-specific roles for Tfap2a and Tfap2b in tooth development

    Woodruff, Emily D.; Gutierrez, Galaxy C.; Van Otterloo, Eric; Williams, Trevor; Cohn, Martin J.. Developmental Biology. vol. 472, 67–74. April 2021.

    Mice possess two types of teeth that differ in their cusp patterns; incisors have one cusp and molars have multiple cusps. The patterning of these two types of teeth relies on fine-tuning of the reciprocal molecular signaling between dental epithelial and mesenchymal tissues during embryonic development. The AP-2 transcription factors, particularly Tfap2a and Tfap2b, are essential components of such epithelial-mesenchymal signaling interactions that coordinate craniofacial development in mice and other vertebrates, but little is known about their roles in the regulation of tooth development and shape. Here we demonstrate that incisors and molars differ in their temporal and spatial expression of Tfap2a and Tfap2b. At the bud stage, Tfap2a is expressed in both the epithelium and mesenchyme of the incisors and molars, but Tfap2b expression is restricted to the molar mesenchyme, only later appearing in the incisor epithelium. Tissue-specific deletions show that loss of the epithelial domain of Tfap2a and Tfap2b affects the number and spatial arrangement of the incisors, notably resulting in duplicated lower incisors. In contrast, deletion of these two genes in the mesenchymal domain has little effect on tooth development. Collectively these results implicate epithelial expression of Tfap2a and Tfap2b in regulating the extent of the dental lamina associated with patterning the incisors and suggest that these genes contribute to morphological differences between anterior (incisor) and posterior (molar) teeth within the mammalian dentition.

  23. Influence of Unilateral Cleft Lip Configuration on Long-Term Facial Averageness: A New Three-Dimensional Analysis Approach

    Neuhaus, Michael-Tobias; Zimmerer, Rüdiger; Zeller, Alexander-Nicolai; Jehn, Philipp; Gellrich, Nils-Claudius; Tavassol, Frank. Facial Plastic Surgery & Aesthetic Medicine. vol. 23(2), 132–139. April 2021.

    Background: Concepts regarding cleft lip and palate care vary widely globally, as there is no international consensus. Consequently, institutional surveillance of cleft concepts is essential. In addition to subjective expert rankings, three-dimensional (3D) photogrammetry of the face has become the gold standard for evaluation and objective assessment of facial structures. We evaluated cleft configuration preoperatively to determine the influence thereof on the long-term facial appearance/averageness of unilateral cleft lip patients. Methods: Plaster models of the patient’s labio-oral region and nose were made preoperatively, digitized, and cleft configuration assessed (cleft width, cleft-columella angle, nasal projection, heminasal width ratio, transverse lip length, labial height). Between 4 and 12 years after surgery, stereophotogrammetry (3D face scans) were captured to determine the patients’ individual facial averageness index (FAI) as a deviation from the population mean, using proportion indices (PIs) of facial landmarks and reference data provided by Farkas and FaceBase. Results: Cleft width (r = 0.77) and columella length correlated strongly with long-term facial averageness (r = 0.52). Decreasing FAI was seen with increasing patient age (r = -0.42), indicating increasing facial averageness. Other cleft properties showed weaker correlations. Cleft alveolus did not have any impact on FAI. Conclusions: The presented method allows objective measurement of facial appearance/averageness as well as preoperative cleft properties. It might be superior to subjective assessments of facial attractiveness/appearance. Objective cleft measurements are strong predictors for future facial averageness. Increasing averageness with growth and age suggests late corrective surgery.

  24. Was facial width-to-height ratio subject to sexual selection pressures? A life course approach

    Hodges-Simeon, Carolyn R.; Albert, Graham; Richardson, George B.; McHale, Timothy S.; Weinberg, Seth M.; Gurven, Michael; Gaulin, Steven J. C.. PLOS ONE. vol. 16(3), e0240284. March 2021.

    Sexual selection researchers have traditionally focused on adult sex differences; however, the schedule and pattern of sex-specific ontogeny can provide insights unobtainable from an exclusive focus on adults. Recently, it has been debated whether facial width-to-height ratio (fWHR; bi-zygomatic breadth divided by midface height) is a human secondary sexual characteristic (SSC). Here, we review current evidence, then address this debate using ontogenetic evidence, which has been under-explored in fWHR research. Facial measurements were collected from 3D surface images of males and females aged 3 to 40 (Study 1; US European-descent, n = 2449), and from 2D photographs of males and females aged 7 to 21 (Study 2; Bolivian Tsimane, n = 179), which were used to calculate three fWHR variants (which we call fWHR nasion , fWHR stomion , and fWHR brow ) and two other common facial masculinity ratios (facial width-to-lower-face-height ratio, fWHR lower , and cheekbone prominence). We test whether the observed pattern of facial development exhibits patterns indicative of SSCs, i.e., differential adolescent growth in either male or female facial morphology leading to an adult sex difference. Results showed that only fWHR lower exhibited both adult sex differences as well as the classic pattern of ontogeny for SSCs—greater lower-face growth in male adolescents relative to females. fWHR brow was significantly wider among both pre- and post-pubertal males in the Bolivian Tsimane sample; post-hoc analyses revealed that the effect was driven by large sex differences in brow height, with females having higher placed brows than males across ages. In both samples, all fWHR measures were inversely associated with age; that is, human facial growth is characterized by greater relative elongation in the mid-face and lower face relative to facial width. This trend continues even into middle adulthood. BMI was also a positive predictor of most of the ratios across ages, with greater BMI associated with wider faces. Researchers collecting data on fWHR should target fWHR lower and fWHR brow and should control for both age and BMI. Researchers should also compare ratio approaches with multivariate techniques, such as geometric morphometrics, to examine whether the latter have greater utility for understanding the evolution of facial sexual dimorphism.

  25. A landmark-free morphometrics pipeline for high-resolution phenotyping: application to a mouse model of Down syndrome

    Toussaint, Nicolas; Redhead, Yushi; Vidal-García, Marta; Lo Vercio, Lucas; Liu, Wei; Fisher, Elizabeth M. C.; Hallgrímsson, Benedikt; Tybulewicz, Victor L. J.; Schnabel, Julia A.; Green, Jeremy B. A.. Development (Cambridge, England). vol. 148(18), dev188631. March 2021.

    Characterising phenotypes often requires quantification of anatomical shape. Quantitative shape comparison (morphometrics) traditionally uses manually located landmarks and is limited by landmark number and operator accuracy. Here, we apply a landmark-free method to characterise the craniofacial skeletal phenotype of the Dp1Tyb mouse model of Down syndrome and a population of the Diversity Outbred (DO) mouse model, comparing it with a landmark-based approach. We identified cranial dysmorphologies in Dp1Tyb mice, especially smaller size and brachycephaly (front-back shortening), homologous to the human phenotype. Shape variation in the DO mice was partly attributable to allometry (size-dependent shape variation) and sexual dimorphism. The landmark-free method performed as well as, or better than, the landmark-based method but was less labour-intensive, required less user training and, uniquely, enabled fine mapping of local differences as planar expansion or shrinkage. Its higher resolution pinpointed reductions in interior mid-snout structures and occipital bones in both the models that were not otherwise apparent. We propose that this landmark-free pipeline could make morphometrics widely accessible beyond its traditional niches in zoology and palaeontology, especially in characterising developmental mutant phenotypes.

  26. Timing of Mouse Molar Formation Is Independent of Jaw Length Including Retromolar Space

    Ko, Daisy Jihyung; Kelly, Tess; Thompson, Lacey; Uppal, Jasmene K.; Rostampour, Nasim; Webb, Mark Adam; Zhu, Ning; Belev, George; Mondal, Prosanta; Cooper, David M. L.; Boughner, Julia C.. Journal of Developmental Biology. vol. 9(1), 8. March 2021.

    For humans and other mammals to eat effectively, teeth must develop properly inside the jaw. Deciphering craniodental integration is central to explaining the timely formation of permanent molars, including third molars which are often impacted in humans, and to clarifying how teeth and jaws fit, function and evolve together. A factor long-posited to influence molar onset time is the jaw space available for each molar organ to form within. Here, we tested whether each successive molar initiates only after a minimum threshold of space is created via jaw growth. We used synchrotron-based micro-CT scanning to assess developing molars in situ within jaws of C57BL/6J mice aged E10 to P32, encompassing molar onset to emergence. We compared total jaw, retromolar and molar lengths, and molar onset times, between upper and lower jaws. Initiation time and developmental duration were comparable between molar upper and lower counterparts despite shorter, slower-growing retromolar space in the upper jaw, and despite size differences between upper and lower molars. Timing of molar formation appears unmoved by jaw length including space. Conditions within the dental lamina likely influence molar onset much more than surrounding jaw tissues. We theorize that molar initiation is contingent on sufficient surface area for the physical reorganization of dental epithelium and its invagination of underlying mesenchyme.

  27. Effects of Male Facial Masculinity on Perceived Attractiveness

    Ekrami, Omid; Claes, Peter; Shriver, Mark D.; Weinberg, Seth M.; Marazita, Mary L.; Walsh, Susan; Van Dongen, Stefan. Adaptive Human Behavior and Physiology. vol. 7(1), 73–88. March 2021.

  28. Fluctuating Asymmetry and Sexual Dimorphism in Human Facial Morphology: A Multi-Variate Study

    Ekrami, Omid; Claes, Peter; Van Assche, Ellen; Shriver, Mark D.; Weinberg, Seth M.; Marazita, Mary L.; Walsh, Susan; Van Dongen, Stefan. Symmetry. vol. 13(2), 304. February 2021.

    (1) Background: Fluctuating asymmetry is often used as an indicator of developmental instability, and is proposed as a signal of genetic quality. The display of prominent masculine phenotypic features, which are a direct result of high androgen levels, is also believed to be a sign of genetic quality, as these hormones may act as immunosuppressants. Fluctuating asymmetry and masculinity are therefore expected to covary. However, there is lack of strong evidence in the literature regarding this hypothesis. (2) Materials and methods: In this study, we examined a large dataset of high-density 3D facial scans of 1260 adults (630 males and 630 females). We mapped a high-density 3D facial mask onto the facial scans in order to obtain a high number of quasi-landmarks on the faces. Multi-dimensional measures of fluctuating asymmetry were extracted from the landmarks using Principal Component Analysis, and masculinity/femininity scores were obtained for each face using Partial Least Squares. The possible correlation between these two qualities was then examined using Pearson’s coefficient and Canonical Correlation Analysis. (3) Results: We found no correlation between fluctuating asymmetry and masculinity in men. However, a weak but significant correlation was found between average fluctuating asymmetry and masculinity in women, in which feminine faces had higher levels of fluctuating asymmetry on average. This correlation could possibly point to genetic quality as an underlying mechanism for both asymmetry and masculinity; however, it might also be driven by other fitness or life history traits, such as fertility. (4) Conclusions: Our results question the idea that fluctuating asymmetry and masculinity should be (more strongly) correlated in men, which is in line with the recent literature. Future studies should possibly focus more on the evolutionary relevance of the observed correlation in women.

  29. The Intersection of the Genetic Architectures of Orofacial Clefts and Normal Facial Variation

    Indencleef, Karlijne; Hoskens, Hanne; Lee, Myoung Keun; White, Julie D.; Liu, Chenxing; Eller, Ryan J.; Naqvi, Sahin; Wehby, George L.; Moreno Uribe, Lina M.; Hecht, Jacqueline T.; Long, Ross E.; Christensen, Kaare; Deleyiannis, Frederic W.; Walsh, Susan; Shriver, Mark D.; Richmond, Stephen; Wysocka, Joanna; Peeters, Hilde; Shaffer, John R.; Marazita, Mary L.; Hens, Greet; Weinberg, Seth M.; Claes, Peter. Frontiers in Genetics. vol. 12, 626403. February 2021.

    Unaffected relatives of individuals with non-syndromic cleft lip with or without cleft palate (NSCL/P) show distinctive facial features. The presence of this facial endophenotype is potentially an expression of underlying genetic susceptibility to NSCL/P in the larger unselected population. To explore this hypothesis, we first partitioned the face into 63 partially overlapping regions representing global-to-local facial morphology and then defined endophenotypic traits by contrasting the 3D facial images from 264 unaffected parents of individuals with NSCL/P versus 3,171 controls. We observed distinct facial features between parents and controls across 59 global-to-local facial segments at nominal significance ( p ≤ 0.05) and 52 segments at Bonferroni corrected significance ( p < 1.2 × 10 –3 ), respectively. Next, we quantified these distinct facial features as univariate traits in another dataset of 8,246 unaffected European individuals and performed a genome-wide association study. We identified 29 independent genetic loci that were associated ( p < 5 × 10 –8 ) with at least one of the tested endophenotypic traits, and nine genetic loci also passed the study-wide threshold ( p < 8.47 × 10 –10 ). Of the 29 loci, 22 were in proximity of loci previously associated with normal facial variation, 18 were near genes that show strong evidence in orofacial clefting (OFC), and another 10 showed some evidence in OFC. Additionally, polygenic risk scores for NSCL/P showed associations with the endophenotypic traits. This study thus supports the hypothesis of a shared genetic architecture of normal facial development and OFC.

  30. Impact of low-frequency coding variants on human facial shape

    Liu, Dongjing; Alhazmi, Nora; Matthews, Harold; Lee, Myoung Keun; Li, Jiarui; Hecht, Jacqueline T.; Wehby, George L.; Moreno, Lina M.; Heike, Carrie L.; Roosenboom, Jasmien; Feingold, Eleanor; Marazita, Mary L.; Claes, Peter; Liao, Eric C.; Weinberg, Seth M.; Shaffer, John R.. Scientific Reports. vol. 11(1), 748. January 2021.

    The contribution of low-frequency variants to the genetic architecture of normal-range facial traits is unknown. We studied the influence of low-frequency coding variants (MAF \textless 1%) in 8091 genes on multi-dimensional facial shape phenotypes in a European cohort of 2329 healthy individuals. Using three-dimensional images, we partitioned the full face into 31 hierarchically arranged segments to model facial morphology at multiple levels, and generated multi-dimensional phenotypes representing the shape variation within each segment. We used MultiSKAT, a multivariate kernel regression approach to scan the exome for face-associated low-frequency variants in a gene-based manner. After accounting for multiple tests, seven genes (AR, CARS2, FTSJ1, HFE, LTB4R, TELO2, NECTIN1) were significantly associated with shape variation of the cheek, chin, nose and mouth areas. These genes displayed a wide range of phenotypic effects, with some impacting the full face and others affecting localized regions. The missense variant rs142863092 in NECTIN1 had a significant effect on chin morphology and was predicted bioinformatically to have a deleterious effect on protein function. Notably, NECTIN1 is an established craniofacial gene that underlies a human syndrome that includes a mandibular phenotype. We further showed that nectin1a mutations can affect zebrafish craniofacial development, with the size and shape of the mandibular cartilage altered in mutant animals. Findings from this study expanded our understanding of the genetic basis of normal-range facial shape by highlighting the role of low-frequency coding variants in several novel genes.

  31. Ror2-mediated non-canonical Wnt signaling regulates Cdc42 and cell proliferation during tooth root development

    Ma, Yuanyuan; Jing, Junjun; Feng, Jifan; Yuan, Yuan; Wen, Quan; Han, Xia; He, Jinzhi; Chen, Shuo; Ho, Thach-Vu; Chai, Yang. Development (Cambridge, England). vol. 148(2), dev196360. January 2021.

    The control of size and shape is an important part of regulatory process during organogenesis. Tooth formation is a highly complex process that fine-tunes the size and shape of the tooth, which are crucial for its physiological functions. Each tooth consists of a crown and one or more roots. Despite comprehensive knowledge of the mechanism that regulates early tooth crown development, we have limited understanding of the mechanism regulating root patterning and size during development. Here, we show that Ror2-mediated non-canonical Wnt signaling in the dental mesenchyme plays a crucial role in cell proliferation, and thereby regulates root development size in mouse molars. Furthermore, Cdc42 acts as a potential downstream mediator of Ror2 signaling in root formation. Importantly, activation of Cdc42 can restore cell proliferation and partially rescue the root development size defects in Ror2 mutant mice. Collectively, our findings provide novel insights into the function of Ror2-mediated non-canonical Wnt signaling in regulating tooth morphogenesis, and suggest potential avenues for dental tissue engineering.

  32. Insights into the genetic architecture of the human face

    White, Julie D.; Indencleef, Karlijne; Naqvi, Sahin; Eller, Ryan J.; Hoskens, Hanne; Roosenboom, Jasmien; Lee, Myoung Keun; Li, Jiarui; Mohammed, Jaaved; Richmond, Stephen; Quillen, Ellen E.; Norton, Heather L.; Feingold, Eleanor; Swigut, Tomek; Marazita, Mary L.; Peeters, Hilde; Hens, Greet; Shaffer, John R.; Wysocka, Joanna; Walsh, Susan; Weinberg, Seth M.; Shriver, Mark D.; Claes, Peter. Nature Genetics. vol. 53(1), 45–53. January 2021.

  33. A developmental stage-specific network approach for studying dynamic co-regulation of transcription factors and microRNAs during craniofacial development

    Yan, Fangfang; Jia, Peilin; Yoshioka, Hiroki; Suzuki, Akiko; Iwata, Junichi; Zhao, Zhongming. Development (Cambridge, England). vol. 147(24), dev192948. December 2020.

    Craniofacial development is regulated through dynamic and complex mechanisms that involve various signaling cascades and gene regulations. Disruption of such regulations can result in craniofacial birth defects. Here, we propose the first developmental stage-specific network approach by integrating two crucial regulators, transcription factors (TFs) and microRNAs (miRNAs), to study their co-regulation during craniofacial development. Specifically, we used TFs, miRNAs and non-TF genes to form feed-forward loops (FFLs) using genomic data covering mouse embryonic days E10.5 to E14.5. We identified key novel regulators (TFs Foxm1, Hif1a, Zbtb16, Myog, Myod1 and Tcf7, and miRNAs miR-340-5p and miR-129-5p) and target genes (Col1a1, Sgms2 and Slc8a3) expression of which changed in a developmental stage-dependent manner. We found that the Wnt-FoxO-Hippo pathway (from E10.5 to E11.5), tissue remodeling (from E12.5 to E13.5) and miR-129-5p-mediated Col1a1 regulation (from E10.5 to E14.5) might play crucial roles in craniofacial development. Enrichment analyses further suggested their functions. Our experiments validated the regulatory roles of miR-340-5p and Foxm1 in the Wnt-FoxO-Hippo subnetwork, as well as the role of miR-129-5p in the miR-129-5p-Col1a1 subnetwork. Thus, our study helps understand the comprehensive regulatory mechanisms for craniofacial development.

  34. Spatiotemporal cellular movement and fate decisions during first pharyngeal arch morphogenesis

    Yuan, Yuan; Loh, Yong-Hwee Eddie; Han, Xia; Feng, Jifan; Ho, Thach-Vu; He, Jinzhi; Jing, Junjun; Groff, Kimberly; Wu, Alan; Chai, Yang. Science Advances. vol. 6(51), eabb0119. December 2020.

    Cranial neural crest (CNC) cells contribute to different cell types during embryonic development. It is unknown whether postmigratory CNC cells undergo dynamic cellular movement and how the process of cell fate decision occurs within the first pharyngeal arch (FPA). Our investigations demonstrate notable heterogeneity within the CNC cells, refine the patterning domains, and identify progenitor cells within the FPA. These progenitor cells undergo fate bifurcation that separates them into common progenitors and mesenchymal cells, which are characterized by Cdk1 and Spry2/Notch2 expression, respectively. The common progenitors undergo further bifurcations to restrict them into osteogenic/odontogenic and chondrogenic/fibroblast lineages. Disruption of a patterning domain leads to specific mandible and tooth defects, validating the binary cell fate restriction process. Different from the compartment model of mandibular morphogenesis, our data redefine heterogeneous cellular domains within the FPA, reveal dynamic cellular movement in time, and describe a sequential series of binary cell fate decision-making process.

  35. Automated syndrome diagnosis by three-dimensional facial imaging

    Hallgrímsson, Benedikt; Aponte, J. David; Katz, David C.; Bannister, Jordan J.; Riccardi, Sheri L.; Mahasuwan, Nick; McInnes, Brenda L.; Ferrara, Tracey M.; Lipman, Danika M.; Neves, Amanda B.; Spitzmacher, Jared A. J.; Larson, Jacinda R.; Bellus, Gary A.; Pham, Anh M.; Aboujaoude, Elias; Benke, Timothy A.; Chatfield, Kathryn C.; Davis, Shanlee M.; Elias, Ellen R.; Enzenauer, Robert W.; French, Brooke M.; Pickler, Laura L.; Shieh, Joseph T. C.; Slavotinek, Anne; Harrop, A. Robertson; Innes, A. Micheil; McCandless, Shawn E.; McCourt, Emily A.; Meeks, Naomi J. L.; Tartaglia, Nicole R.; Tsai, Anne C.-H.; Wyse, J. Patrick H.; Bernstein, Jonathan A.; Sanchez-Lara, Pedro A.; Forkert, Nils D.; Bernier, Francois P.; Spritz, Richard A.; Klein, Ophir D.. Genetics in Medicine. vol. 22(10), 1682–1693. October 2020.

    Deep phenotyping is an emerging trend in precision medicine for genetic disease. The shape of the face is affected in 30–40% of known genetic syndromes. Here, we determine whether syndromes can be diagnosed from 3D images of human faces.

  36. FaceBase 3: analytical tools and FAIR resources for craniofacial and dental research

    Samuels, Bridget D.; Aho, Robert; Brinkley, James F.; Bugacov, Alejandro; Feingold, Eleanor; Fisher, Shannon; Gonzalez-Reiche, Ana S.; Hacia, Joseph G.; Hallgrimsson, Benedikt; Hansen, Karissa; Harris, Matthew P.; Ho, Thach-Vu; Holmes, Greg; Hooper, Joan E.; Jabs, Ethylin Wang; Jones, Kenneth L.; Kesselman, Carl; Klein, Ophir D.; Leslie, Elizabeth J.; Li, Hong; Liao, Eric C.; Long, Hannah; Lu, Na; Maas, Richard L.; Marazita, Mary L.; Mohammed, Jaaved; Prescott, Sara; Schuler, Robert; Selleri, Licia; Spritz, Richard A.; Swigut, Tomek; van Bakel, Harm; Visel, Axel; Welsh, Ian; Williams, Cristina; Williams, Trevor J.; Wysocka, Joanna; Yuan, Yuan; Chai, Yang. Development (Cambridge, England). vol. 147(18), dev191213. September 2020.

    The FaceBase Consortium was established by the National Institute of Dental and Craniofacial Research in 2009 as a ’big data’ resource for the craniofacial research community. Over the past decade, researchers have deposited hundreds of annotated and curated datasets on both normal and disordered craniofacial development in FaceBase, all freely available to the research community on the FaceBase Hub website. The Hub has developed numerous visualization and analysis tools designed to promote integration of multidisciplinary data while remaining dedicated to the FAIR principles of data management (findability, accessibility, interoperability and reusability) and providing a faceted search infrastructure for locating desired data efficiently. Summaries of the datasets generated by the FaceBase projects from 2014 to 2019 are provided here. FaceBase 3 now welcomes contributions of data on craniofacial and dental development in humans, model organisms and cell lines. Collectively, the FaceBase Consortium, along with other NIH-supported data resources, provide a continuously growing, dynamic and current resource for the scientific community while improving data reproducibility and fulfilling data sharing requirements.

  37. Runx2+ Niche Cells Maintain Incisor Mesenchymal Tissue Homeostasis through IGF Signaling

    Chen, Shuo; Jing, Junjun; Yuan, Yuan; Feng, Jifan; Han, Xia; Wen, Quan; Ho, Thach-Vu; Lee, Chelsea; Chai, Yang. Cell Reports. vol. 32(6), 108007. August 2020.

    Stem cell niches provide a microenvironment to support the self-renewal and multi-lineage differentiation of stem cells. Cell-cell interactions within the niche are essential for maintaining tissue homeostasis. However, the niche cells supporting mesenchymal stem cells (MSCs) are largely unknown. Using single-cell RNA sequencing, we show heterogeneity among Gli1+ MSCs and identify a subpopulation of Runx2+/Gli1+ cells in the adult mouse incisor. These Runx2+/Gli1+ cells are strategically located between MSCs and transit-amplifying cells (TACs). They are not stem cells but help to maintain the MSC niche via IGF signaling to regulate TAC proliferation, differentiation, and incisor growth rate. ATAC-seq and chromatin immunoprecipitation reveal that Runx2 directly binds to Igfbp3 in niche cells. This Runx2-mediated IGF signaling is crucial for regulating the MSC niche and maintaining tissue homeostasis to support continuous growth of the adult mouse incisor, providing a model for analysis of the molecular regulation of the MSC niche.

  38. 3D Analysis of Facial Morphology of a Colombian Population Compared to Adult Caucasians

    Bravo-Hammett, Sonia; Nucci, Ludovica; Christou, Teti; Aristizabal, Juan Fernando; Kau, Chung How. European Journal of Dentistry. vol. 14(3), 342–351. July 2020.

    OBJECTIVE: The purpose of this study involves describing the facial morphology of a Colombian population with three-dimensional (3D) imaging, and comparing their facial morphology with the Caucasian to create a database for this ethnicity. MATERIALS AND METHODS: The study, which included 135 subjects selected from the Valle University in Cali, Colombia, and 535 Caucasian subjects selected from the FaceBase-Data (1U01DE024449-01), was funded by the National Institute of Dental and Craniofacial Research. All images were taken in the natural head position (NHP) and captured using a stereo-photogrammetric camera system (3dMDface) to obtain a 3D image of each patient. The subjects were between 19 to 31 years of age, with a normal body mass index (BMI), and no craniofacial deformities. All images were plotted and analyzed using the 3dMDVultus software to calculate linear and angular measurements. Standard deviation (SD) and means were calculated for each measurement and analyzed using t-test for different samples. RESULTS: The Hispanic population had wider eyes, more protruded upper and lower lips, wider face, and greater mandibular width. Caucasian females had a more acute full profile, larger middle third, and less protrusive lips. Understanding the facial morphology of different populations would help to establish a better diagnosis and treatment planning for each ethnicity. CONCLUSIONS: From this study, the following conclusions may be drawn:1. The mean values of Colombian males showed greater measurements than females in the majority of measurements.2. Caucasian females had a more acute full profile, larger middle and lower third, and less protrusive lips.3. This study showed significant ethnic differences in the linear and angular measurements, showing us the importance of considering these findings in the diagnosis and treatment planning when a Hispanic population is involved. Soft tissue values should reflect the norms according to each ethnic population in order to achieve treatment goals.

  39. Integrated Transcriptome and Network Analysis Reveals Spatiotemporal Dynamics of Calvarial Suturogenesis

    Holmes, Greg; Gonzalez-Reiche, Ana S.; Lu, Na; Zhou, Xianxiao; Rivera, Joshua; Kriti, Divya; Sebra, Robert; Williams, Anthony A.; Donovan, Michael J.; Potter, S. Steven; Pinto, Dalila; Zhang, Bin; van Bakel, Harm; Jabs, Ethylin Wang. Cell Reports. vol. 32(1), 107871. July 2020.

    Craniofacial abnormalities often involve sutures, the growth centers of the skull. To characterize the organization and processes governing their development, we profile the murine frontal suture, a model for sutural growth and fusion, at the tissue- and single-cell level on embryonic days (E)16.5 and E18.5. For the wild-type suture, bulk RNA sequencing (RNA-seq) analysis identifies mesenchyme-, osteogenic front-, and stage-enriched genes and biological processes, as well as alternative splicing events modifying the extracellular matrix. Single-cell RNA-seq analysis distinguishes multiple subpopulations, of which five define a mesenchyme-osteoblast differentiation trajectory and show variation along the anteroposterior axis. Similar analyses of in vivo mouse models of impaired frontal suturogenesis in Saethre-Chotzen and Apert syndromes, Twist1+/- and Fgfr2+/S252W, demonstrate distinct transcriptional changes involving angiogenesis and ribogenesis, respectively. Co-expression network analysis reveals gene expression modules from which we validate key driver genes regulating osteoblast differentiation. Our study provides a global approach to gain insights into suturogenesis.

  40. Phosphotungstic acid‐enhanced microCT: Optimized protocols for embryonic and early postnatal mice

    Lesciotto, Kate M.; Motch Perrine, Susan M.; Kawasaki, Mizuho; Stecko, Timothy; Ryan, Timothy M.; Kawasaki, Kazuhiko; Richtsmeier, Joan T.. Developmental Dynamics. vol. 249(4), 573–585. April 2020.

    Background: Given the need for descriptive and increasingly mechanistic morphological analyses, contrast-enhanced microcomputed tomography (microCT) represents perhaps the best method for visualizing 3D biological soft tissues in situ. Although staining protocols using phosphotungstic acid (PTA) have been published with beautiful visualizations of soft tissue structures, these protocols are often aimed at highly specific research questions and are applicable to a limited set of model organisms, specimen ages, or tissue types. We provide detailed protocols for micro-level visualization of soft tissue structures in mice at several embryonic and early postnatal ages using PTA-enhanced microCT. Results: Our protocols produce microCT scans that enable visualization and quantitative analyses of whole organisms, individual tissues, and organ systems while preserving 3D morphology and relationships with surrounding structures, with minimal soft tissue shrinkage. Of particular note, both internal and external features of the murine heart, lungs, and liver, as well as embryonic cartilage, are captured at high resolution. Conclusion: These protocols have broad applicability to mouse models for a variety of diseases and conditions. Minor experimentation in the staining duration can expand this protocol to additional age groups, permitting ontogenetic studies of internal organs and soft tissue structures within their 3D in situ position.

  41. A Multivariate Approach to Determine the Dimensionality of Human Facial Asymmetry

    Ekrami, Omid; Claes, Peter; White, Julie D.; Weinberg, Seth M.; Marazita, Mary L.; Walsh, Susan; Shriver, Mark D.; Van Dongen, Stefan. Symmetry. vol. 12(3), 348. March 2020.

    Many studies have suggested that developmental instability (DI) could lead to asymmetric development, otherwise known as fluctuating asymmetry (FA). Several attempts to unravel the biological meaning of FA have been made, yet the main step in estimating FA is to remove the effects of directional asymmetry (DA), which is defined as the average bilateral asymmetry at the population level. Here, we demonstrate in a multivariate context that the conventional method of DA correction does not adequately compensate for the effects of DA in other dimensions of asymmetry. This appears to be due to the presence of between-individual variation along the DA dimension. Consequently, we propose to decompose asymmetry into its different orthogonal dimensions, where we introduce a new measure of asymmetry, namely fluctuating directional asymmetry (F-DA). This measure describes individual variation in the dimension of DA, and can be used to adequately correct the asymmetry measurements for the presence of DA. We provide evidence that this measure can be useful in disentangling the different dimensions of asymmetry, and further studies on this measure can provide valuable insight into the underlying biological processes leading to these different asymmetry dimensions.

  42. Unilateral Cleft Lip Nasal Deformity: Three-Dimensional Analysis of the Primary Deformity and Longitudinal Changes following Primary Correction of the Nasal Foundation

    Tse, Raymond W.; Knight, Robert; Oestreich, Makinna; Rosser, Mica; Mercan, Ezgi. Plastic and Reconstructive Surgery. vol. 145(1), 185–199. January 2020.

  43. Dynamic activation of Wnt, Fgf, and Hh signaling during soft palate development

    Janečková, Eva; Feng, Jifan; Li, Jingyuan; Rodriguez, Gabriela; Chai, Yang. PloS One. vol. 14(10), e0223879. 2019.

    The soft palate is a key component of the oropharyngeal complex that is critical for swallowing, breathing, hearing and speech. However, complete functional restoration in patients with cleft soft palate remains a challenging task. New insights into the molecular signaling network governing the development of soft palate will help to overcome these clinical challenges. In this study, we investigated whether key signaling pathways required for hard palate development are also involved in soft palate development in mice. We described the dynamic expression patterns of signaling molecules from well-known pathways, such as Wnt, Hh, and Fgf, during the development of the soft palate. We found that Wnt signaling is active throughout the development of soft palate myogenic sites, predominantly in cells of cranial neural crest (CNC) origin neighboring the myogenic cells, suggesting that Wnt signaling may play a significant role in CNC-myogenic cell-cell communication during myogenic differentiation in the soft palate. Hh signaling is abundantly active in early palatal epithelium, some myogenic cells, and the CNC-derived cells adjacent to the myogenic cells. Hh signaling gradually diminishes during the later stages of soft palate development, indicating its involvement mainly in early embryonic soft palate development. Fgf signaling is expressed most prominently in CNC-derived cells in the myogenic sites and persists until later stages of embryonic soft palate development. Collectively, our results highlight a network of Wnt, Hh, and Fgf signaling that may be involved in the development of the soft palate, particularly soft palate myogenesis. These findings provide a foundation for future studies on the functional significance of these signaling pathways individually and collectively in regulating soft palate development.

  44. Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

    Wu, Meng; Kriti, Divya; van Bakel, Harm; Jabs, Ethylin Wang; Holmes, Greg. Journal of Visualized Experiments: JoVE. (154) December 2019.

    Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel’s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

  45. A role for G protein-coupled receptor 137b in bone remodeling in mouse and zebrafish

    Urso, K.; Caetano-Lopes, J.; Lee, P. Y.; Yan, J.; Henke, K.; Sury, M.; Liu, H.; Zgoda, M.; Jacome-Galarza, C.; Nigrovic, P. A.; Duryea, J.; Harris, M. P.; Charles, J. F.. Bone. vol. 127, 104–113. October 2019.

    G protein-coupled receptor 137b (GPR137b) is an orphan seven-pass transmembrane receptor of unknown function. In mouse, Gpr137b is highly expressed in osteoclasts in vivo and is upregulated during in vitro differentiation. To elucidate the role that GPR137b plays in osteoclasts, we tested the effect of GPR137b deficiency on osteoclast maturation and resorbing activity. We used CRISPR/Cas9 gene editing in mouse-derived ER-Hoxb8 immortalized myeloid progenitors to generate GPR137b-deficient osteoclast precursors. Decreasing Gpr137b in these precursors led to increased osteoclast differentiation and bone resorption activity. To explore the role of GPR137b during skeletal development, we generated zebrafish deficient for the ortholog gpr137ba. Gpr137ba-deficient zebrafish are viable and fertile and do not display overt morphological defects as adults. However, analysis of osteoclast function in gpr137ba-/- mutants demonstrated increased bone resorption. Micro-computed tomography evaluation of vertebral bone mass and morphology demonstrated that gpr137ba-deficiency altered the angle of the neural arch, a skeletal site with high osteoclast activity. Vital staining of gpr137ba-/- fish with calcein and alizarin red indicated that bone formation in the mutants is also increased, suggesting high bone turnover. These results identify GPR137b as a conserved negative regulator of osteoclast activity essential for normal resorption and patterning of the skeleton. Further, these data suggest that coordination of osteoclast and osteoblast activity is a conserved process among vertebrates and may have similar regulation.

  46. Regulatory Mechanisms of Soft Palate Development and Malformations

    Li, J.; Rodriguez, G.; Han, X.; Janečková, E.; Kahng, S.; Song, B.; Chai, Y.. Journal of Dental Research. vol. 98(9), 959–967. August 2019.

    Orofacial clefting is the most common congenital craniofacial malformation, appearing in approximately 1 in 700 live births. Orofacial clefting includes several distinct anatomic malformations affecting the upper lip and hard and soft palate. The etiology of orofacial clefting is multifactorial, including genetic or environmental factors or their combination. A large body of work has focused on the molecular etiology of cleft lip and clefts of the hard palate, but study of the underlying etiology of soft palate clefts is an emerging field. Recent advances in the understanding of soft palate development suggest that it may be regulated by distinct pathways from those implicated in hard palate development. Soft palate clefting leads to muscle misorientation and oropharyngeal deficiency and adversely affects speech, swallowing, breathing, and hearing. Hence, there is an important need to investigate the regulatory mechanisms of soft palate development. Significantly, the anatomy, function, and development of soft palatal muscles are similar in humans and mice, rendering the mouse an excellent model for investigating molecular and cellular mechanisms of soft palate clefts. Cranial neural crest-derived cells provide important regulatory cues to guide myogenic progenitors to differentiate into muscles in the soft palate. Signals from the palatal epithelium also play key roles via tissue-tissue interactions mediated by Tgf-β, Wnt, Fgf, and Hh signaling molecules. Additionally, mutations in transcription factors, such as Dlx5, Tbx1, and Tbx22, have been associated with soft palate clefting in humans and mice, suggesting that they play important regulatory roles during soft palate development. Finally, we highlight the importance of distinguishing specific types of soft palate defects in patients and developing relevant animal models for each of these types to improve our understanding of the regulatory mechanism of soft palate development. This knowledge will provide a foundation for improving treatment for patients in the future.

  47. Facial recognition from DNA using face-to-DNA classifiers

    Sero, Dzemila; Zaidi, Arslan; Li, Jiarui; White, Julie D.; Zarzar, Tomás B. González; Marazita, Mary L.; Weinberg, Seth M.; Suetens, Paul; Vandermeulen, Dirk; Wagner, Jennifer K.; Shriver, Mark D.; Claes, Peter. Nature Communications. vol. 10(1), 2557. June 2019.

    Facial recognition from DNA refers to the identification or verification of unidentified biological material against facial images with known identity. One approach to establish the identity of unidentified biological material is to predict the face from DNA, and subsequently to match against facial images. However, DNA phenotyping of the human face remains challenging. Here, another proof of concept to biometric authentication is established by using multiple face-to-DNA classifiers, each classifying given faces by a DNA-encoded aspect (sex, genomic background, individual genetic loci), or by a DNA-inferred aspect (BMI, age). Face-to-DNA classifiers on distinct DNA aspects are fused into one matching score for any given face against DNA. In a globally diverse, and subsequently in a homogeneous cohort, we demonstrate preliminary, but substantial true (83%, 80%) over false (17%, 20%) matching in verification mode. Consequences of future efforts include forensic applications, necessitating careful consideration of ethical and legal implications for privacy in genomic databases.

  48. Genome-wide analysis of dental caries and periodontitis combining clinical and self-reported data

    Shungin, Dmitry; Haworth, Simon; Divaris, Kimon; Agler, Cary S.; Kamatani, Yoichiro; Keun Lee, Myoung; Grinde, Kelsey; Hindy, George; Alaraudanjoki, Viivi; Pesonen, Paula; Teumer, Alexander; Holtfreter, Birte; Sakaue, Saori; Hirata, Jun; Yu, Yau-Hua; Ridker, Paul M.; Giulianini, Franco; Chasman, Daniel I.; Magnusson, Patrik K. E.; Sudo, Takeaki; Okada, Yukinori; Völker, Uwe; Kocher, Thomas; Anttonen, Vuokko; Laitala, Marja-Liisa; Orho-Melander, Marju; Sofer, Tamar; Shaffer, John R.; Vieira, Alexandre; Marazita, Mary L.; Kubo, Michiaki; Furuichi, Yasushi; North, Kari E.; Offenbacher, Steve; Ingelsson, Erik; Franks, Paul W.; Timpson, Nicholas J.; Johansson, Ingegerd. Nature Communications. vol. 10(1), 2773. June 2019.

    Dental caries and periodontitis account for a vast burden of morbidity and healthcare spending, yet their genetic basis remains largely uncharacterized. Here, we identify self-reported dental disease proxies which have similar underlying genetic contributions to clinical disease measures and then combine these in a genome-wide association study meta-analysis, identifying 47 novel and conditionally-independent risk loci for dental caries. We show that the heritability of dental caries is enriched for conserved genomic regions and partially overlapping with a range of complex traits including smoking, education, personality traits and metabolic measures. Using cardio-metabolic traits as an example in Mendelian randomization analysis, we estimate causal relationships and provide evidence suggesting that the processes contributing to dental caries may have undesirable downstream effects on health.

  49. The molecular anatomy of mammalian upper lip and primary palate fusion at single cell resolution

    Li, Hong; Jones, Kenneth L.; Hooper, Joan E.; Williams, Trevor. Development (Cambridge, England). vol. 146(12), dev174888. June 2019.

    The mammalian lip and primary palate form when coordinated growth and morphogenesis bring the nasal and maxillary processes into contact, and the epithelia co-mingle, remodel and clear from the fusion site to allow mesenchyme continuity. Although several genes required for fusion have been identified, an integrated molecular and cellular description of the overall process is lacking. Here, we employ single cell RNA sequencing of the developing mouse face to identify ectodermal, mesenchymal and endothelial populations associated with patterning and fusion of the facial prominences. This analysis indicates that key cell populations at the fusion site exist within the periderm, basal epithelial cells and adjacent mesenchyme. We describe the expression profiles that make each population unique, and the signals that potentially integrate their behaviour. Overall, these data provide a comprehensive high-resolution description of the various cell populations participating in fusion of the lip and primary palate, as well as formation of the nasolacrimal groove, and they furnish a powerful resource for those investigating the molecular genetics of facial development and facial clefting that can be mined for crucial mechanistic information concerning this prevalent human birth defect.

  50. 3D stereophotogrammetry versus traditional craniofacial anthropometry: Comparing measurements from the 3D facial norms database to Farkas’s North American norms

    Weinberg, Seth M.. American Journal of Orthodontics and Dentofacial Orthopedics. vol. 155(5), 693–701. May 2019.

  51. MeshMonk: Open-source large-scale intensive 3D phenotyping

    White, Julie D.; Ortega-Castrillón, Alejandra; Matthews, Harold; Zaidi, Arslan A.; Ekrami, Omid; Snyders, Jonatan; Fan, Yi; Penington, Tony; Van Dongen, Stefan; Shriver, Mark D.; Claes, Peter. Scientific Reports. vol. 9(1), 6085. April 2019.

    Dense surface registration, commonly used in computer science, could aid the biological sciences in accurate and comprehensive quantification of biological phenotypes. However, few toolboxes exist that are openly available, non-expert friendly, and validated in a way relevant to biologists. Here, we report a customizable toolbox for reproducible high-throughput dense phenotyping of 3D images, specifically geared towards biological use. Given a target image, a template is first oriented, repositioned, and scaled to the target during a scaled rigid registration step, then transformed further to fit the specific shape of the target using a non-rigid transformation. As validation, we use n = 41 3D facial images to demonstrate that the MeshMonk registration is accurate, with 1.26 mm average error, across 19 landmarks, between placements from manual observers and using the MeshMonk toolbox. We also report no variation in landmark position or centroid size significantly attributable to landmarking method used. Though validated using 19 landmarks, the MeshMonk toolbox produces a dense mesh of vertices across the entire surface, thus facilitating more comprehensive investigations of 3D shape variation. This expansion opens up exciting avenues of study in assessing biological shapes to better understand their phenotypic variation, genetic and developmental underpinnings, and evolutionary history.

  52. novoCaller: a Bayesian network approach for de novo variant calling from pedigree and population sequence data

    Mohanty, Anwoy Kumar; Vuzman, Dana; Francioli, Laurent; Cassa, Christopher; Brigham Genomic Medicine; Undiagnosed Diseases Network; Brigham; Women’s Hospital FaceBase Project; Toth-Petroczy, Agnes; Sunyaev, Shamil. Bioinformatics (Oxford, England). vol. 35(7), 1174–1180. April 2019.

    MOTIVATION: De novo mutations (i.e. newly occurring mutations) are a pre-dominant cause of sporadic dominant monogenic diseases and play a significant role in the genetics of complex disorders. De novo mutation studies also inform population genetics models and shed light on the biology of DNA replication and repair. Despite the broad interest, there is room for improvement with regard to the accuracy of de novo mutation calling. RESULTS: We designed novoCaller, a Bayesian variant calling algorithm that uses information from read-level data both in the pedigree and in unrelated samples. The method was extensively tested using large trio-sequencing studies, and it consistently achieved over 97% sensitivity. We applied the algorithm to 48 trio cases of suspected rare Mendelian disorders as part of the Brigham Genomic Medicine gene discovery initiative. Its application resulted in a significant reduction in the resources required for manual inspection and experimental validation of the calls. Three de novo variants were found in known genes associated with rare disorders, leading to rapid genetic diagnosis of the probands. Another 14 variants were found in genes that are likely to explain the phenotype, and could lead to novel disease-gene discovery. AVAILABILITY AND IMPLEMENTATION: Source code implemented in C++ and Python can be downloaded from SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  53. The developmental-genetics of canalization

    Hallgrimsson, Benedikt; Green, Rebecca M.; Katz, David C.; Fish, Jennifer L.; Bernier, Francois P.; Roseman, Charles C.; Young, Nathan M.; Cheverud, James M.; Marcucio, Ralph S.. Seminars in Cell & Developmental Biology. vol. 88, 67–79. April 2019.

  54. Integrating gene regulatory pathways into differential network analysis of gene expression data

    Grimes, Tyler; Potter, S. Steven; Datta, Somnath. Scientific Reports. vol. 9(1), 5479. April 2019.

    The advent of next-generation sequencing has introduced new opportunities in analyzing gene expression data. Research in systems biology has taken advantage of these opportunities by gleaning insights into gene regulatory networks through the analysis of gene association networks. Contrasting networks from different populations can reveal the many different roles genes fill, which can lead to new discoveries in gene function. Pathologies can also arise from aberrations in these gene-gene interactions. Exposing these network irregularities provides a new avenue for understanding and treating diseases. A general framework for integrating known gene regulatory pathways into a differential network analysis between two populations is proposed. The framework importantly allows for any gene-gene association measure to be used, and inference is carried out through permutation testing. A simulation study investigates the performance in identifying differentially connected genes when incorporating known pathways, even if the pathway knowledge is partially inaccurate. Another simulation study compares the general framework with four state-of-the-art methods. Two RNA-seq datasets are analyzed to illustrate the use of this framework in practice. In both examples, the analysis reveals genes and pathways that are known to be biologically significant along with potentially novel findings that may be used to motivate future research.

  55. Association of low-frequency genetic variants in regulatory regions with nonsyndromic orofacial clefts

    Shaffer, John R.; LeClair, Jessica; Carlson, Jenna C.; Feingold, Eleanor; Buxó, Carmen J.; Christensen, Kaare; Deleyiannis, Frederic W. B.; Field, L. Leigh; Hecht, Jacqueline T.; Moreno, Lina; Orioli, Ieda M.; Padilla, Carmencita; Vieira, Alexandre R.; Wehby, George L.; Murray, Jeffrey C.; Weinberg, Seth M.; Marazita, Mary L.; Leslie, Elizabeth J.. American Journal of Medical Genetics. Part A. vol. 179(3), 467–474. March 2019.

    Genome-wide scans have shown that common risk alleles for orofacial clefts (OFC) tend to be located in noncoding regulatory elements and cumulatively explain only part of the heritability of OFCs. Low-frequency variants may account for some of the "missing" heritability. Therefore, we scanned low-frequency variants located within putative craniofacial enhancers to identify novel OFC risk variants and implicate new regulatory elements in OFC pathogenesis. Analyses were performed in a multiethnic sample of 1,995 cases of cleft lip with or without cleft palate (CL/P), 221 cases with cleft palate (CP) only, and 1,576 unaffected controls. One hundred and nineteen putative craniofacial enhancers identified from ChIP-Seq studies in craniofacial tissues or cell lines contained multiple low-frequency (0.01-1%) variants, which we genotyped in participants using a custom Illumina panel. Two complementary statistical approaches, sequence kernel association test and combined multivariate and collapsing, were used to test association of the aggregated low-frequency variants across each enhancer region with CL/P and CP. We discovered a significant association between CP and a branchial arch enhancer near FOXP1 (mm60; p-value = .0002). Additionally, we observed a suggestive association between CL/P and a forebrain enhancer near FOXE1 (hs1717; p-value = .001). These findings suggest that low-frequency variants in craniofacial enhancer regions contribute to the complex etiology of nonsyndromic OFCs.

  56. miRNA analysis with Prost! reveals evolutionary conservation of organ-enriched expression and post-transcriptional modifications in three-spined stickleback and zebrafish

    Desvignes, Thomas; Batzel, Peter; Sydes, Jason; Eames, B. Frank; Postlethwait, John H.. Scientific Reports. vol. 9(1), 3913. March 2019.

    MicroRNAs (miRNAs) can have organ-specific expression and functions; they can originate from dedicated miRNA genes, from non-canonical miRNA genes, or from mirror-miRNA genes and can also experience post-transcriptional variation. It remains unclear, however, which mechanisms of miRNA production or modification are organ-specific and the extent of their evolutionary conservation. To address these issues, we developed the software Prost! (PRocessing Of Short Transcripts), which, among other features, helps quantify mature miRNAs, accounts for post-transcriptional processing, such as nucleotide editing, and identifies mirror-miRNAs. Here, we applied Prost! to annotate and analyze miRNAs in three-spined stickleback (Gasterosteus aculeatus), a model fish for evolutionary biology reported to have a miRNome larger than most teleost fish. Zebrafish (Danio rerio), a distantly related teleost with a well-known miRNome, served as comparator. Our results provided evidence for the existence of 286 miRNA genes and 382 unique mature miRNAs (excluding mir430 gene duplicates and the vaultRNA-derived mir733), which doesn’t represent a miRNAome larger than other teleost miRNomes. In addition, small RNA sequencing data from brain, heart, testis, and ovary in both stickleback and zebrafish identified suites of mature miRNAs that display organ-specific enrichment, many of which are evolutionarily-conserved in the brain and heart in both species. These data also supported the hypothesis that evolutionarily-conserved, organ-specific mechanisms may regulate post-transcriptional variations in miRNA sequence. In both stickleback and zebrafish, miR2188-5p was edited frequently with similar nucleotide changes in the seed sequence with organ specific editing rates, highest in the brain. In summary, Prost! is a new tool to identify and understand small RNAs, to help clarify a species’ miRNA biology as shown here for an important model for the evolution of developmental mechanisms, and to provide insight into organ-enriched expression and the evolutionary conservation of miRNA post-transcriptional modifications.

  57. Detection of de novo copy number deletions from targeted sequencing of trios

    Fu, Jack M.; Leslie, Elizabeth J.; Scott, Alan F.; Murray, Jeffrey C.; Marazita, Mary L.; Beaty, Terri H.; Scharpf, Robert B.; Ruczinski, Ingo. Bioinformatics (Oxford, England). vol. 35(4), 571–578. February 2019.

    MOTIVATION: De novo copy number deletions have been implicated in many diseases, but there is no formal method to date that identifies de novo deletions in parent-offspring trios from capture-based sequencing platforms. RESULTS: We developed Minimum Distance for Targeted Sequencing (MDTS) to fill this void. MDTS has similar sensitivity (recall), but a much lower false positive rate compared to less specific CNV callers, resulting in a much higher positive predictive value (precision). MDTS also exhibited much better scalability. AVAILABILITY AND IMPLEMENTATION: MDTS is freely available as open source software from the Bioconductor repository. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  58. Transcriptomics reveals complex kinetics of dorsal-ventral patterning gene expression in the mandibular arch

    Sharma, Praveer P.; MacLean, Adam L.; Meinecke, Lina; Clouthier, David E.; Nie, Qing; Schilling, Thomas F.. Genesis (New York, N.Y.: 2000). vol. 57(1), e23275. January 2019.

    The mandibular or first pharyngeal arch forms the upper and lower jaws in all gnathostomes. A gene regulatory network that defines ventral, intermediate, and dorsal domains along the dorsal-ventral (D-V) axis of the arch has emerged from studies in zebrafish and mice, but the temporal dynamics of this process remain unclear. To define cell fate trajectories in the arches we have performed quantitative gene expression analyses of D-V patterning genes in pharyngeal arch primordia in zebrafish and mice. Using NanoString technology to measure transcript numbers per cell directly we show that, in many cases, genes expressed in similar D-V domains and induced by similar signals vary dramatically in their temporal profiles. This suggests that cellular responses to D-V patterning signals are likely shaped by the baseline kinetics of target gene expression. Furthermore, similarities in the temporal dynamics of genes that occupy distinct pathways suggest novel shared modes of regulation. Incorporating these gene expression kinetics into our computational models for the mandibular arch improves the accuracy of patterning, and facilitates temporal comparisons between species. These data suggest that the magnitude and timing of target gene expression help diversify responses to patterning signals during craniofacial development.

  59. An integrated clinical program and crowdsourcing strategy for genomic sequencing and Mendelian disease gene discovery

    Haghighi, Alireza; Krier, Joel B.; Toth-Petroczy, Agnes; Cassa, Christopher A.; Frank, Natasha Y.; Carmichael, Nikkola; Fieg, Elizabeth; Bjonnes, Andrew; Mohanty, Anwoy; Briere, Lauren C.; Lincoln, Sharyn; Lucia, Stephanie; Gupta, Vandana A.; Söylemez, Onuralp; Sutti, Sheila; Kooshesh, Kameron; Qiu, Haiyan; Fay, Christopher J.; Perroni, Victoria; Valerius, Jamie; Hanna, Meredith; Frank, Alexander; Ouahed, Jodie; Snapper, Scott B.; Pantazi, Angeliki; Chopra, Sameer S.; Leshchiner, Ignaty; Stitziel, Nathan O.; Feldweg, Anna; Mannstadt, Michael; Loscalzo, Joseph; Sweetser, David A.; Liao, Eric; Stoler, Joan M.; Nowak, Catherine B.; Sanchez-Lara, Pedro A.; Klein, Ophir D.; Perry, Hazel; Patsopoulos, Nikolaos A.; Raychaudhuri, Soumya; Goessling, Wolfram; Green, Robert C.; Seidman, Christine E.; MacRae, Calum A.; Sunyaev, Shamil R.; Maas, Richard L.; Vuzman, Dana; Undiagnosed Diseases Network, Brigham; Women’s Hospital FaceBase Project, Brigham Genomic Medicine (BGM). NPJ genomic medicine. vol. 3, 21. 2018.

    Despite major progress in defining the genetic basis of Mendelian disorders, the molecular etiology of many cases remains unknown. Patients with these undiagnosed disorders often have complex presentations and require treatment by multiple health care specialists. Here, we describe an integrated clinical diagnostic and research program using whole-exome and whole-genome sequencing (WES/WGS) for Mendelian disease gene discovery. This program employs specific case ascertainment parameters, a WES/WGS computational analysis pipeline that is optimized for Mendelian disease gene discovery with variant callers tuned to specific inheritance modes, an interdisciplinary crowdsourcing strategy for genomic sequence analysis, matchmaking for additional cases, and integration of the findings regarding gene causality with the clinical management plan. The interdisciplinary gene discovery team includes clinical, computational, and experimental biomedical specialists who interact to identify the genetic etiology of the disease, and when so warranted, to devise improved or novel treatments for affected patients. This program effectively integrates the clinical and research missions of an academic medical center and affords both diagnostic and therapeutic options for patients suffering from genetic disease. It may therefore be germane to other academic medical institutions engaged in implementing genomic medicine programs.

  60. Associations Between Genetic Data and Quantitative Assessment of Normal Facial Asymmetry

    Rolfe, Sara; Lee, Su-In; Shapiro, Linda. Frontiers in Genetics. vol. 9, 659. 2018.

    Human facial asymmetry is due to a complex interaction of genetic and environmental factors. To identify genetic influences on facial asymmetry, we developed a method for automated scoring that summarizes local morphology features and their spatial distribution. A genome-wide association study using asymmetry scores from two local symmetry features was conducted and significant genetic associations were identified for one asymmetry feature, including genes thought to play a role in craniofacial disorders and development: NFATC1, SOX5, NBAS, and TCF7L1. These results provide evidence that normal variation in facial asymmetry may be impacted by common genetic variants and further motivate the development of automated summaries of complex phenotypes.

  61. Modeling craniofacial development reveals spatiotemporal constraints on robust patterning of the mandibular arch

    Meinecke, Lina; Sharma, Praveer P.; Du, Huijing; Zhang, Lei; Nie, Qing; Schilling, Thomas F.. PLoS computational biology. vol. 14(11), e1006569. November 2018.

    How does pattern formation occur accurately when confronted with tissue growth and stochastic fluctuations (noise) in gene expression? Dorso-ventral (D-V) patterning of the mandibular arch specifies upper versus lower jaw skeletal elements through a combination of Bone morphogenetic protein (Bmp), Endothelin-1 (Edn1), and Notch signaling, and this system is highly robust. We combine NanoString experiments of early D-V gene expression with live imaging of arch development in zebrafish to construct a computational model of the D-V mandibular patterning network. The model recapitulates published genetic perturbations in arch development. Patterning is most sensitive to changes in Bmp signaling, and the temporal order of gene expression modulates the response of the patterning network to noise. Thus, our integrated systems biology approach reveals non-intuitive features of the complex signaling system crucial for craniofacial development, including novel insights into roles of gene expression timing and stochasticity in signaling and gene regulation.

  62. Genome-wide interaction studies identify sex-specific risk alleles for nonsyndromic orofacial clefts

    Carlson, Jenna C.; Nidey, Nichole L.; Butali, Azeez; Buxo, Carmen J.; Christensen, Kaare; Deleyiannis, Frederic W.-D.; Hecht, Jacqueline T.; Field, L. Leigh; Moreno-Uribe, Lina M.; Orioli, Ieda M.; Poletta, Fernando A.; Padilla, Carmencita; Vieira, Alexandre R.; Weinberg, Seth M.; Wehby, George L.; Feingold, Eleanor; Murray, Jeffrey C.; Marazita, Mary L.; Leslie, Elizabeth J.. Genetic Epidemiology. vol. 42(7), 664–672. October 2018.

    Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial birth defect in humans and is notable for its apparent sexual dimorphism where approximately twice as many males are affected as females. The sources of this disparity are largely unknown, but interactions between genetic and sex effects are likely contributors. We examined gene-by-sex (G × S) interactions in a worldwide sample of 2,142 NSCL/P cases and 1,700 controls recruited from 13 countries. First, we performed genome-wide joint tests of the genetic (G) and G × S effects genome-wide using logistic regression assuming an additive genetic model and adjusting for 18 principal components of ancestry. We further interrogated loci with suggestive results from the joint test ( p \textless 1.00 × 10 -5 ) by examining the G × S effects from the same model. Out of the 133 loci with suggestive results ( p \textless 1.00 × 10 -5 ) for the joint test, we observed one genome-wide significant G × S effect in the 10q21 locus (rs72804706; p = 6.69 × 10 -9 ; OR = 2.62 CI [1.89, 3.62]) and 16 suggestive G × S effects. At the intergenic 10q21 locus, the risk of NSCL/P is estimated to increase with additional copies of the minor allele for females, but the opposite effect for males. Our observation that the impact of genetic variants on NSCL/P risk differs for males and females may further our understanding of the genetic architecture of NSCL/P and the sex differences underlying clefts and other birth defects.

  63. Midface and upper airway dysgenesis in FGFR2-related craniosynostosis involves multiple tissue-specific and cell cycle effects

    Holmes, Greg; O’Rourke, Courtney; Motch Perrine, Susan M.; Lu, Na; van Bakel, Harm; Richtsmeier, Joan T.; Jabs, Ethylin Wang. Development (Cambridge, England). vol. 145(19), dev166488. October 2018.

    Midface dysgenesis is a feature of more than 200 genetic conditions in which upper airway anomalies frequently cause respiratory distress, but its etiology is poorly understood. Mouse models of Apert and Crouzon craniosynostosis syndromes exhibit midface dysgenesis similar to the human conditions. They carry activating mutations of Fgfr2, which is expressed in multiple craniofacial tissues during development. Magnetic resonance microscopy of three mouse models of Apert and Crouzon syndromes revealed decreased nasal passage volume in all models at birth. Histological analysis suggested overgrowth of the nasal cartilage in the two Apert syndrome mouse models. We used tissue-specific gene expression and transcriptome analysis to further dissect the structural, cellular and molecular alterations underlying midface and upper airway dysgenesis in Apert Fgfr2+/S252W mutants. Cartilage thickened progressively during embryogenesis because of increased chondrocyte proliferation in the presence of Fgf2 Oral epithelium expression of mutant Fgfr2, which resulted in a distinctive nasal septal fusion defect, and premature facial suture fusion contributed to the overall dysmorphology. Midface dysgenesis in Fgfr2-related craniosynostosis is a complex phenotype arising from the combined effects of aberrant signaling in multiple craniofacial tissues.

  64. Zebrafish type I collagen mutants faithfully recapitulate human type I collagenopathies

    Gistelinck, Charlotte; Kwon, Ronald Y.; Malfait, Fransiska; Symoens, Sofie; Harris, Matthew P.; Henke, Katrin; Hawkins, Michael B.; Fisher, Shannon; Sips, Patrick; Guillemyn, Brecht; Bek, Jan Willem; Vermassen, Petra; De Saffel, Hanna; Witten, Paul Eckhard; Weis, MaryAnn; De Paepe, Anne; Eyre, David R.; Willaert, Andy; Coucke, Paul J.. Proceedings of the National Academy of Sciences of the United States of America. vol. 115(34), E8037–E8046. August 2018.

    The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of osteogenesis imperfecta (OI) and the Ehlers-Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and a zebrafish model resembling human EDS, which harbors a number of soft connective tissues defects, typical of EDS. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen-related disease models. Our studies reveal a high degree of intergenotype variability in phenotypic expressivity that closely correlates with associated OI severity. Furthermore, we demonstrate the potential for select mutations to give rise to phenotypic variability, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the future potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention.

  65. Prmt1 regulates craniofacial bone formation upstream of Msx1

    Gou, Yongchao; Li, Jingyuan; Wu, Jian; Gupta, Rahul; Cho, Ihnbae; Ho, Thach-Vu; Chai, Yang; Merrill, Amy; Wang, Jun; Xu, Jian. Mechanisms of Development. vol. 152, 13–20. August 2018.

    Protein arginine methylation has been recently identified as an important form of post-translational modification (PTM). It is carried out by the protein arginine methyltransferase (PRMT) family of enzymes, which in mammals consists of nine members. Among them, PRMT1 is the major arginine methyltransferase and participates in transcription, signal transduction, development and cancer. The function of PRMT1 in craniofacial development remains unclear. We generated Wnt1-Cre;Prmt1fl/fl mice with cranial neural crest (CNC)-specific deletion of Prmt1 and compared CNC-derived craniofacial bones from newborn control and Wnt1-Cre;Prmt1fl/fl mice. The size, surface area and volume of the premaxilla, maxilla, palatine bone, frontal bone, and mandible were analyzed using three-dimensional (3D) micro-computed tomography (microCT). We found that Prmt1 deficiency led to alterations in craniofacial bones including the premaxilla, maxilla, palatine bone, frontal bone, and mandible, as well as defects in the incisor and alveolar bone, recapitulating changes seen in Msx1-deficient mice. We further determined that Prmt1 depletion resulted in significant downregulation of Msx1 in calvaria-derived preosteoblast and primordium of frontal bone and mandible. Our study reveals critical roles of PRMT1 in the formation of CNC-derived craniofacial bones and suggests that Prmt1 is an upstream regulator of Msx1 in craniofacial bone development.

  66. A Combined PLS and Negative Binomial Regression Model for Inferring Association Networks from Next-Generation Sequencing Count Data

    Pesonen, Maiju; Nevalainen, Jaakko; Potter, Steven; Datta, Somnath; Datta, Susmita. IEEE/ACM transactions on computational biology and bioinformatics. vol. 15(3), 760–773. June 2018.

    A major challenge of genomics data is to detect interactions displaying functional associations from large-scale observations. In this study, a new cPLS-algorithm combining partial least squares approach with negative binomial regression is suggested to reconstruct a genomic association network for high-dimensional next-generation sequencing count data. The suggested approach is applicable to the raw counts data, without requiring any further pre-processing steps. In the settings investigated, the cPLS-algorithm outperformed the two widely used comparative methods, graphical lasso, and weighted correlation network analysis. In addition, cPLS is able to estimate the full network for thousands of genes without major computational load. Finally, we demonstrate that cPLS is capable of finding biologically meaningful associations by analyzing an example data set from a previously published study to examine the molecular anatomy of the craniofacial development.

  67. High-Resolution Epigenomic Atlas of Human Embryonic Craniofacial Development

    Wilderman, Andrea; VanOudenhove, Jennifer; Kron, Jeffrey; Noonan, James P.; Cotney, Justin. Cell Reports. vol. 23(5), 1581–1597. May 2018.

  68. Mapping genetic variants for cranial vault shape in humans

    Roosenboom, Jasmien; Lee, Myoung Keun; Hecht, Jacqueline T.; Heike, Carrie L.; Wehby, George L.; Christensen, Kaare; Feingold, Eleanor; Marazita, Mary L.; Maga, A. Murat; Shaffer, John R.; Weinberg, Seth M.. PLOS ONE. vol. 13(4), e0196148. April 2018.

  69. Genome-wide mapping of global-to-local genetic effects on human facial shape

    Claes, Peter; Roosenboom, Jasmien; White, Julie D.; Swigut, Tomek; Sero, Dzemila; Li, Jiarui; Lee, Myoung Keun; Zaidi, Arslan; Mattern, Brooke C.; Liebowitz, Corey; Pearson, Laurel; González, Tomás; Leslie, Elizabeth J.; Carlson, Jenna C.; Orlova, Ekaterina; Suetens, Paul; Vandermeulen, Dirk; Feingold, Eleanor; Marazita, Mary L.; Shaffer, John R.; Wysocka, Joanna; Shriver, Mark D.; Weinberg, Seth M.. Nature Genetics. vol. 50(3), 414–423. March 2018.

    Genome-wide association scans of complex multipartite traits like the human face typically use preselected phenotypic measures. Here we report a data-driven approach to phenotyping facial shape at multiple levels of organization, allowing for an open-ended description of facial variation while preserving statistical power. In a sample of 2,329 persons of European ancestry, we identified 38 loci, 15 of which replicated in an independent European sample (n = 1,719). Four loci were completely new. For the others, additional support (n = 9) or pleiotropic effects (n = 2) were found in the literature, but the results reported here were further refined. All 15 replicated loci highlighted distinctive patterns of global-to-local genetic effects on facial shape and showed enrichment for active chromatin elements in human cranial neural crest cells, suggesting an early developmental origin of the facial variation captured. These results have implications for studies of facial genetics and other complex morphological traits.

  70. Facial shape manifestations of growth faltering in Tanzanian children

    Cole, Joanne B.; Manyama, Mange F.; Nikitovic, Dejana; Gonzalez, Paula N.; Liberton, Denise K.; Wilson, Warren M.; Rolian, Campbell; Larson, Jacinda R.; Kimwaga, Emmanuel; Mathayo, Joshua; Roseman, Charles C.; Santorico, Stephanie A.; Lukowiak, Ken; Spritz, Richard A.; Hallgrimsson, Benedikt. Journal of Anatomy. vol. 232(2), 250–262. February 2018.

    Variation in the shape of the human face and in stature is determined by complex interactions between genetic and environmental influences. One such environmental influence is malnourishment, which can result in growth faltering, usually diagnosed by means of comparing an individual’s stature with a set of age-appropriate standards. These standards for stature, however, are typically ascertained in groups where people are at low risk for growth faltering. Moreover, genetic differences among populations with respect to stature are well established, further complicating the generalizability of stature-based diagnostic tools. In a large sample of children aged 5-19 years, we obtained high-resolution genomic data, anthropometric measures and 3D facial images from individuals within and around the city of Mwanza, Tanzania. With genome-wide complex trait analysis, we partitioned genetic and environmental variance for growth outcomes and facial shape. We found that children with growth faltering have faces that look like those of older and taller children, in a direction opposite to the expected allometric trajectory, and in ways predicted by the environmental portion of covariance at the community and individual levels. The environmental variance for facial shape varied subtly but significantly among communities, whereas genetic differences were minimal. These results reveal that facial shape preserves information about exposure to undernourishment, with important implications for refining assessments of nutritional status in children and the developmental-genetics of craniofacial variation alike.

  71. Body size and allometric variation in facial shape in children

    Larson, Jacinda R.; Manyama, Mange F.; Cole, Joanne B.; Gonzalez, Paula N.; Percival, Christopher J.; Liberton, Denise K.; Ferrara, Tracey M.; Riccardi, Sheri L.; Kimwaga, Emmanuel A.; Mathayo, Joshua; Spitzmacher, Jared A.; Rolian, Campbell; Jamniczky, Heather A.; Weinberg, Seth M.; Roseman, Charles C.; Klein, Ophir; Lukowiak, Ken; Spritz, Richard A.; Hallgrimsson, Benedikt. American Journal of Physical Anthropology. vol. 165(2), 327–342. February 2018.

    OBJECTIVES: Morphological integration, or the tendency for covariation, is commonly seen in complex traits such as the human face. The effects of growth on shape, or allometry, represent a ubiquitous but poorly understood axis of integration. We address the question of to what extent age and measures of size converge on a single pattern of allometry for human facial shape. METHODS: Our study is based on two large cross-sectional cohorts of children, one from Tanzania and the other from the United States (N = 7,173). We employ 3D facial imaging and geometric morphometrics to relate facial shape to age and anthropometric measures. RESULTS: The two populations differ significantly in facial shape, but the magnitude of this difference is small relative to the variation within each group. Allometric variation for facial shape is similar in both populations, representing a small but significant proportion of total variation in facial shape. Different measures of size are associated with overlapping but statistically distinct aspects of shape variation. Only half of the size-related variation in facial shape can be explained by the first principal component of four size measures and age while the remainder associates distinctly with individual measures. CONCLUSIONS: Allometric variation in the human face is complex and should not be regarded as a singular effect. This finding has important implications for how size is treated in studies of human facial shape and for the developmental basis for allometric variation more generally.

  72. Small teleost fish provide new insights into human skeletal diseases

    Witten, P. E.; Harris, M. P.; Huysseune, A.; Winkler, C.. Methods in Cell Biology. vol. 138, 321–346. 2017.

    Small teleost fish such as zebrafish and medaka are increasingly studied as models for human skeletal diseases. Efficient new genome editing tools combined with advances in the analysis of skeletal phenotypes provide new insights into fundamental processes of skeletal development. The skeleton among vertebrates is a highly conserved organ system, but teleost fish and mammals have evolved unique traits or have lost particular skeletal elements in each lineage. Several unique features of the skeleton relate to the extremely small size of early fish embryos and the small size of adult fish used as models. A detailed analysis of the plethora of interesting skeletal phenotypes in zebrafish and medaka pushes available skeletal imaging techniques to their respective limits and promotes the development of new imaging techniques. Impressive numbers of zebrafish and medaka mutants with interesting skeletal phenotypes have been characterized, complemented by transgenic zebrafish and medaka lines. The advent of efficient genome editing tools, such as TALEN and CRISPR/Cas9, allows to introduce targeted deficiencies in genes of model teleosts to generate skeletal phenotypes that resemble human skeletal diseases. This review will also discuss other attractive aspects of the teleost skeleton. This includes the capacity for lifelong tooth replacement and for the regeneration of dermal skeletal elements, such as scales and fin rays, which further increases the value of zebrafish and medaka models for skeletal research.

  73. Developmental nonlinearity drives phenotypic robustness

    Green, Rebecca M.; Fish, Jennifer L.; Young, Nathan M.; Smith, Francis J.; Roberts, Benjamin; Dolan, Katie; Choi, Irene; Leach, Courtney L.; Gordon, Paul; Cheverud, James M.; Roseman, Charles C.; Williams, Trevor J.; Marcucio, Ralph S.; Hallgrímsson, Benedikt. Nature Communications. vol. 8(1), 1970. December 2017.

    Robustness to perturbation is a fundamental feature of complex organisms. Mutations are the raw material for evolution, yet robustness to their effects is required for species survival. The mechanisms that produce robustness are poorly understood. Nonlinearities are a ubiquitous feature of development that may link variation in development to phenotypic robustness. Here, we manipulate the gene dosage of a signaling molecule, Fgf8, a critical regulator of vertebrate development. We demonstrate that variation in Fgf8 expression has a nonlinear relationship to phenotypic variation, predicting levels of robustness among genotypes. Differences in robustness are not due to gene expression variance or dysregulation, but emerge from the nonlinearity of the genotype–phenotype curve. In this instance, embedded features of development explain robustness differences. How such features vary in natural populations and relate to genetic variation are key questions for unraveling the origin and evolvability of this feature of organismal development.

  74. SERCA directs cell migration and branching across species and germ layers

    Bower, Danielle V.; Lansdale, Nick; Navarro, Sonia; Truong, Thai V.; Bower, Dan J.; Featherstone, Neil C.; Connell, Marilyn G.; Al Alam, Denise; Frey, Mark R.; Trinh, Le A.; Fernandez, G. Esteban; Warburton, David; Fraser, Scott E.; Bennett, Daimark; Jesudason, Edwin C.. Biology Open. vol. 6(10), 1458–1471. October 2017.

    Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca2+ reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca2+ levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca2+ differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca2+ in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca2+ signaling, established by SERCA, in iterative budding.

  75. Genetic Screen for Postembryonic Development in the Zebrafish (Danio rerio): Dominant Mutations Affecting Adult Form

    Henke, Katrin; Daane, Jacob M.; Hawkins, M. Brent; Dooley, Christopher M.; Busch-Nentwich, Elisabeth M.; Stemple, Derek L.; Harris, Matthew P.. Genetics. vol. 207(2), 609–623. October 2017.

    Large-scale forward genetic screens have been instrumental for identifying genes that regulate development, homeostasis, and regeneration, as well as the mechanisms of disease. The zebrafish, Danio rerio, is an established genetic and developmental model used in genetic screens to uncover genes necessary for early development. However, the regulation of postembryonic development has received less attention as these screens are more labor intensive and require extensive resources. The lack of systematic interrogation of late development leaves large aspects of the genetic regulation of adult form and physiology unresolved. To understand the genetic control of postembryonic development, we performed a dominant screen for phenotypes affecting the adult zebrafish. In our screen, we identified 72 adult viable mutants showing changes in the shape of the skeleton as well as defects in pigmentation. For efficient mapping of these mutants and mutation identification, we devised a new mapping strategy based on identification of mutant-specific haplotypes. Using this method in combination with a candidate gene approach, we were able to identify linked mutations for 22 out of 25 mutants analyzed. Broadly, our mutational analysis suggests that there are key genes and pathways associated with late development. Many of these pathways are shared with humans and are affected in various disease conditions, suggesting constraint in the genetic pathways that can lead to change in adult form. Taken together, these results show that dominant screens are a feasible and productive means to identify mutations that can further our understanding of gene function during postembryonic development and in disease.

  76. ARHGAP29 Mutation Is Associated with Abnormal Oral Epithelial Adhesions

    Paul, B. J.; Palmer, K.; Sharp, J. C.; Pratt, C. H.; Murray, S. A.; Dunnwald, M.. Journal of Dental Research. vol. 96(11), 1298–1305. October 2017.

    Nonsyndromic cleft lip and/or palate (NSCL/P) is a prevalent birth defect of complex etiology. Previous studies identified mutations in ARHGAP29 associated with an increased risk for NSCL/P. To investigate the effects of ARHGAP29 in vivo, we generated a novel murine allele by inserting a point mutation identified in a patient with NSCL/P. This single-nucleotide variation of ARHGAP29 translates to an early nonsense mutation (K326X), presumably resulting in loss-of-function (LoF). Embryos from Arhgap29K326X/+ intercrosses were harvested at various time points. No homozygous Arhgap29K326X animals were found in the 45 analyzed litters, assessed as early as embryonic day 8.5 (e8.5). Coronal sectioning of e13.5 and e14.5 heads revealed that 59% of Arhgap29K326X/+ mice ( n = 37) exhibited improper epithelial contact between developing oral structures, while none were observed in wild types ( n = 10). In addition, Arhgap29K326X/+ embryos exhibited a significantly higher percentage of maxillary epithelium in contact with mandibular epithelium. Immunofluorescent analyses of the periderm and oral adhesions revealed the presence of Arhgap29 in periderm cells. These cells were p63 negative, keratin 17 positive, and keratin 6 positive and present at sites of adhesion, although occasionally disorganized. Oral adhesions did not appear to impair palatogenesis, as all analyzed Arhgap29K326X/+ embryos showed confluent palatal mesenchyme and epithelium at e18.5 ( n = 16), and no mice were found with a cleft at birth. Collectively, our data demonstrate that ARHGAP29 is required for embryonic survival and that heterozygosity for LoF variants of Arhgap29 increases the incidence and length of oral adhesions at a critical time point during orofacial development. In conclusion, we validate the LoF nature of the human K326X mutation in vivo and reveal a previously unknown effect of Arhgap29 in murine craniofacial development.

  77. ERMrest: A Collaborative Data Catalog with Fine Grain Access Control

    Czajkowski, Karl; Kesselman, Carl; Schuler, Robert. Proceedings ... IEEE International Conference on eScience. IEEE International Conference on eScience. vol. 2017, 510–517. October 2017.

    Creating and maintaining an accurate description of data assets and the relationships between assets is a critical aspect of making data findable, accessible, interoperable, and reusable (FAIR). Typically, such metadata are created and maintained in a data catalog by a curator as part of data publication. However, allowing metadata to be created and maintained by data producers as the data is generated rather then waiting for publication can have significant advantages in terms of productivity and repeatability. The responsibilities for metadata management need not fall on any one individual, but rather may be delegated to appropriate members of a collaboration, enabling participants to edit or maintain specific attributes, to describe relationships between data elements, or to correct errors. To support such collaborative data editing, we have created ERMrest, a relational data service for the Web that enables the creation, evolution and navigation of complex models used to describe and structure diverse file or relational data objects. A key capability of ERMrest is its ability to control operations down to the level of individual data elements, i.e. fine-grained access control, so that many different modes of data-oriented collaboration can be supported. In this paper we introduce ERMrest and describe its fine-grained access control capabilities that support collaborative editing. ERMrest is in daily use in many data driven collaborations and we describe a sample policy that is based on a common biocuration pattern.

  78. Experiences with Deriva: An Asset Management Platform for Accelerating eScience

    Bugacov, Alejandro; Czajkowski, Karl; Kesselman, Carl; Kumar, Anoop; Schuler, Robert E.; Tangmunarunkit, Hongsuda. Proceedings ... IEEE International Conference on eScience. IEEE International Conference on eScience. vol. 2017, 79–88. October 2017.

    The pace of discovery in eScience is increasingly dependent on a scientist’s ability to acquire, curate, integrate, analyze, and share large and diverse collections of data. It is all too common for investigators to spend inordinate amounts of time developing ad hoc procedures to manage their data. In previous work, we presented Deriva, a Scientific Asset Management System, designed to accelerate data driven discovery. In this paper, we report on the use of Deriva in a number of substantial and diverse eScience applications. We describe the lessons we have learned, both from the perspective of the Deriva technology, as well as the ability and willingness of scientists to incorporate Scientific Asset Management into their daily workflows.

  79. Closing the Gap: Mouse Models to Study Adhesion in Secondary Palatogenesis

    Lough, K. J.; Byrd, K. M.; Spitzer, D. C.; Williams, S. E.. Journal of Dental Research. vol. 96(11), 1210–1220. October 2017.

    Secondary palatogenesis occurs when the bilateral palatal shelves (PS), arising from maxillary prominences, fuse at the midline, forming the hard and soft palate. This embryonic phenomenon involves a complex array of morphogenetic events that require coordinated proliferation, apoptosis, migration, and adhesion in the PS epithelia and underlying mesenchyme. When the delicate process of craniofacial morphogenesis is disrupted, the result is orofacial clefting, including cleft lip and cleft palate (CL/P). Through human genetic and animal studies, there are now hundreds of known genetic alternations associated with orofacial clefts; so, it is not surprising that CL/P is among the most common of all birth defects. In recent years, in vitro cell-based assays, ex vivo palate cultures, and genetically engineered animal models have advanced our understanding of the developmental and cell biological pathways that contribute to palate closure. This is particularly true for the areas of PS patterning and growth as well as medial epithelial seam dissolution during palatal fusion. Here, we focus on epithelial cell-cell adhesion, a critical but understudied process in secondary palatogenesis, and provide a review of the available tools and mouse models to better understand this phenomenon.

  80. Composition and dosage of a multipartite enhancer cluster control developmental expression of Ihh (Indian hedgehog)

    Will, Anja J.; Cova, Giulia; Osterwalder, Marco; Chan, Wing-Lee; Wittler, Lars; Brieske, Norbert; Heinrich, Verena; de Villartay, Jean-Pierre; Vingron, Martin; Klopocki, Eva; Visel, Axel; Lupiáñez, Darío G.; Mundlos, Stefan. Nature Genetics. vol. 49(10), 1539–1545. October 2017.

    Copy number variations (CNVs) often include noncoding sequences and putative enhancers, but how these rearrangements induce disease is poorly understood. Here we investigate CNVs involving the regulatory landscape of IHH (encoding Indian hedgehog), which cause multiple, highly localized phenotypes including craniosynostosis and synpolydactyly. We show through transgenic reporter and genome-editing studies in mice that Ihh is regulated by a constellation of at least nine enhancers with individual tissue specificities in the digit anlagen, growth plates, skull sutures and fingertips. Consecutive deletions, resulting in growth defects of the skull and long bones, showed that these enhancers function in an additive manner. Duplications, in contrast, caused not only dose-dependent upregulation but also misexpression of Ihh, leading to abnormal phalanges, fusion of sutures and syndactyly. Thus, precise spatiotemporal control of developmental gene expression is achieved by complex multipartite enhancer ensembles. Alterations in the composition of such clusters can result in gene misexpression and disease.

  81. Measuring Symmetry in Children With Cleft Lip. Part 3: Quantifying Nasal Symmetry and Nasal Normalcy Before and After Unilateral Cleft Lip Repair

    Liang, Shu; Shapiro, Linda; Tse, Raymond. The Cleft Palate-Craniofacial Journal: Official Publication of the American Cleft Palate-Craniofacial Association. vol. 54(5), 602–611. September 2017.

    OBJECTIVE: The purpose of this project was to develop objective computer-based methods to measure nasal asymmetry and abnormality in children undergoing treatment of unilateral cleft lip (UCL) and to determine the correlation of these measures to clinical expectations. PARTICIPANTS: Thirty infants with UCL undergoing cleft lip repair; 27 children with UCL aged 8 to 10 years who had previously undergone cleft lip repair; 3 control infants; 3 control children aged 8 to 10 years. INTERVENTIONS: To measure nasal symmetry, we used a process of depth mapping and calculated the Depth Area Difference. To measure abnormality, we used the reconstruction error from Principle Component Analysis (PCA) that was based upon characteristics of a dataset of over 2000 images of normal control subjects. MAIN OUTCOME MEASURES: Depth Area Difference and PCA Reconstruction Error for cleft type, changes with surgery, and individual subjects ranked according to cleft severity were assessed. RESULTS: Significant differences in Depth Area Difference and PCA Reconstruction Error were found between cleft types and found before and after surgery. Nasal symmetry and normalcy scores for infants with UCL approached those of controls after surgery, and there was a strong correlation with ranked cleft severity. For older children, measures of nasal symmetry and abnormality were better than infants prior to repair but worse than infants following UCL repair. CONCLUSIONS: Our computer-based 3D analysis of nasal symmetry and normalcy correlated with clinical expectations. Automated processing made measurement convenient. Use of these measures may help to objectively measure cleft severity and treatment outcome.

  82. Germline Chd8 haploinsufficiency alters brain development in mouse

    Gompers, Andrea L.; Su-Feher, Linda; Ellegood, Jacob; Copping, Nycole A.; Riyadh, M. Asrafuzzaman; Stradleigh, Tyler W.; Pride, Michael C.; Schaffler, Melanie D.; Wade, A. Ayanna; Catta-Preta, Rinaldo; Zdilar, Iva; Louis, Shreya; Kaushik, Gaurav; Mannion, Brandon J.; Plajzer-Frick, Ingrid; Afzal, Veena; Visel, Axel; Pennacchio, Len A.; Dickel, Diane E.; Lerch, Jason P.; Crawley, Jacqueline N.; Zarbalis, Konstantinos S.; Silverman, Jill L.; Nord, Alex S.. Nature Neuroscience. vol. 20(8), 1062–1073. August 2017.

    The chromatin remodeling gene CHD8 represents a central node in neurodevelopmental gene networks implicated in autism. We examined the impact of germline heterozygous frameshift Chd8 mutation on neurodevelopment in mice. Chd8+/del5 mice displayed normal social interactions with no repetitive behaviors but exhibited cognitive impairment correlated with increased regional brain volume, validating that phenotypes of Chd8+/del5 mice overlap pathology reported in humans with CHD8 mutations. We applied network analysis to characterize neurodevelopmental gene expression, revealing widespread transcriptional changes in Chd8+/del5 mice across pathways disrupted in neurodevelopmental disorders, including neurogenesis, synaptic processes and neuroimmune signaling. We identified a co-expression module with peak expression in early brain development featuring dysregulation of RNA processing, chromatin remodeling and cell-cycle genes enriched for promoter binding by Chd8, and we validated increased neuronal proliferation and developmental splicing perturbation in Chd8+/del5 mice. This integrative analysis offers an initial picture of the consequences of Chd8 haploinsufficiency for brain development.

  83. Utility of quantitative micro-computed tomographic analysis in zebrafish to define gene function during skeletogenesis

    Charles, Julia F.; Sury, Meera; Tsang, Kelly; Urso, Katia; Henke, Katrin; Huang, Yue; Russell, Ruby; Duryea, Jeffrey; Harris, Matthew P.. Bone. vol. 101, 162–171. August 2017.

    The zebrafish is a powerful experimental model to investigate the genetic and morphologic basis of vertebrate development. Analysis of skeletogenesis in this fish is challenging as a result of the small size of the developing and adult zebrafish. Many of the bones of small fishes such as the zebrafish and medaka are quite thin, precluding many standard assays of bone quality and morphometrics commonly used on bones of larger animals. Microcomputed tomography (microCT) is a common imaging technique used for detailed analysis of the skeleton of the zebrafish and determination of mutant phenotypes. However, the utility of this modality for analysis of the zebrafish skeleton, and the effect of inherent variation among individual zebrafish, including variables such as sex, age and strain, is not well understood. Given the increased use and accessibility of microCT, we set out to define the sensitivity of microCT methods in developing and adult zebrafish. We assessed skeletal shape and density measures in the developing vertebrae and parasphenoid of the skull base. We found most skeletal variables are tightly correlated to standard length, but that at later growth stages (\textgreater3months) there are age dependent effects on some skeletal measures. Further we find modest strain but not sex differences in skeletal measures. These data suggest that the appropriate control for assessing mutant phenotypes should be age and strain matched, ideally a wild-type sibling. By analyzing two mutants exhibiting skeletal dysplasia, we show that microCT imaging can be a sensitive method to quantify distinct skeletal parameters of adults. Finally, as developing zebrafish skeletons remain difficult to resolve by radiographic means, we define a contrast agent specific for bone that enhances resolution at early stages, permitting detailed morphometric analysis of the forming skeleton. This increased capability for detection extends the use of this imaging modality to leverage the zebrafish model to understand the development causes of skeletal dysplasias.

  84. Gene expression profile data for mouse facial development

    Leach, Sonia M.; Feng, Weiguo; Williams, Trevor. Data in Brief. vol. 13, 242–247. August 2017.

    This article contains data related to the research articles "Spatial and Temporal Analysis of Gene Expression during Growth and Fusion of the Mouse Facial Prominences" (Feng et al., 2009) [1] and "Systems Biology of facial development: contributions of ectoderm and mesenchyme" (Hooper et al., 2017 In press) [2]. Embryonic mammalian craniofacial development is a complex process involving the growth, morphogenesis, and fusion of distinct facial prominences into a functional whole. Aberrant gene regulation during this process can lead to severe craniofacial birth defects, including orofacial clefting. As a means to understand the genes involved in facial development, we had previously dissected the embryonic mouse face into distinct prominences: the mandibular, maxillary or nasal between E10.5 and E12.5. The prominences were then processed intact, or separated into ectoderm and mesenchyme layers, prior analysis of RNA expression using microarrays (Feng et al., 2009, Hooper et al., 2017 in press) [1], [2]. Here, individual gene expression profiles have been built from these datasets that illustrate the timing of gene expression in whole prominences or in the separated tissue layers. The data profiles are presented as an indexed and clickable list of the genes each linked to a graphical image of that gene׳s expression profile in the ectoderm, mesenchyme, or intact prominence. These data files will enable investigators to obtain a rapid assessment of the relative expression level of any gene on the array with respect to time, tissue, prominence, and expression trajectory.

  85. Association studies of low-frequency coding variants in nonsyndromic cleft lip with or without cleft palate

    Leslie, Elizabeth J.; Carlson, Jenna C.; Shaffer, John R.; Buxó, Carmen J.; Castilla, Eduardo E.; Christensen, Kaare; Deleyiannis, Frederic W. B.; Field, Leigh L.; Hecht, Jacqueline T.; Moreno, Lina; Orioli, Ieda M.; Padilla, Carmencita; Vieira, Alexandre R.; Wehby, George L.; Feingold, Eleanor; Weinberg, Seth M.; Murray, Jeffrey C.; Marazita, Mary L.. American Journal of Medical Genetics. Part A. vol. 173(6), 1531–1538. June 2017.

    Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a group of common human birth defects with complex etiology. Although genome-wide association studies have successfully identified a number of risk loci, these loci only account for about 20% of the heritability of orofacial clefts. The "missing" heritability may be found in rare variants, copy number variants, or interactions. In this study, we investigated the role of low-frequency variants genotyped in 1995 cases and 1626 controls on the Illumina HumanCore + Exome chip. We performed two statistical tests, Sequence Kernel Association Test (SKAT) and Combined Multivariate and Collapsing (CMC) method using two minor allele frequency cutoffs (1% and 5%). We found that a burden of low-frequency coding variants in N4BP2, CDSN, PRTG, and AHRR were associated with increased risk of NSCL/P. Low-frequency variants in other genes were associated with decreased risk of NSCL/P. These results demonstrate that low-frequency variants contribute to the genetic etiology of NSCL/P.

  86. Systems biology of facial development: contributions of ectoderm and mesenchyme

    Hooper, Joan E.; Feng, Weiguo; Li, Hong; Leach, Sonia M.; Phang, Tzulip; Siska, Charlotte; Jones, Kenneth L.; Spritz, Richard A.; Hunter, Lawrence E.; Williams, Trevor. Developmental Biology. vol. 426(1), 97–114. June 2017.

    The rapid increase in gene-centric biological knowledge coupled with analytic approaches for genomewide data integration provides an opportunity to develop systems-level understanding of facial development. Experimental analyses have demonstrated the importance of signaling between the surface ectoderm and the underlying mesenchyme are coordinating facial patterning. However, current transcriptome data from the developing vertebrate face is dominated by the mesenchymal component, and the contributions of the ectoderm are not easily identified. We have generated transcriptome datasets from critical periods of mouse face formation that enable gene expression to be analyzed with respect to time, prominence, and tissue layer. Notably, by separating the ectoderm and mesenchyme we considerably improved the sensitivity compared to data obtained from whole prominences, with more genes detected over a wider dynamic range. From these data we generated a detailed description of ectoderm-specific developmental programs, including pan-ectodermal programs, prominence- specific programs and their temporal dynamics. The genes and pathways represented in these programs provide mechanistic insights into several aspects of ectodermal development. We also used these data to identify co-expression modules specific to facial development. We then used 14 co-expression modules enriched for genes involved in orofacial clefts to make specific mechanistic predictions about genes involved in tongue specification, in nasal process patterning and in jaw development. Our multidimensional gene expression dataset is a unique resource for systems analysis of the developing face; our co-expression modules are a resource for predicting functions of poorly annotated genes, or for predicting roles for genes that have yet to be studied in the context of facial development; and our analytic approaches provide a paradigm for analysis of other complex developmental programs.

  87. Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites

    Anderson, Courtney M.; Hu, Jianxin; Thomas, Reuben; Gainous, T. Blair; Celona, Barbara; Sinha, Tanvi; Dickel, Diane E.; Heidt, Analeah B.; Xu, Shan-Mei; Bruneau, Benoit G.; Pollard, Katherine S.; Pennacchio, Len A.; Black, Brian L.. Development (Cambridge, England). vol. 144(7), 1235–1241. April 2017.

    Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here, we identify a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by the SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription in vivo Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo.

  88. Rapid automated landmarking for morphometric analysis of three-dimensional facial scans

    Li, Mao; Cole, Joanne B.; Manyama, Mange; Larson, Jacinda R.; Liberton, Denise K.; Riccardi, Sheri L.; Ferrara, Tracey M.; Santorico, Stephanie A.; Bannister, Jordan J.; Forkert, Nils D.; Spritz, Richard A.; Mio, Washington; Hallgrimsson, Benedikt. Journal of Anatomy. vol. 230(4), 607–618. April 2017.

    Automated phenotyping is essential for the creation of large, highly standardized datasets from anatomical imaging data. Such datasets can support large-scale studies of complex traits or clinical studies related to precision medicine or clinical trials. We have developed a method that generates three-dimensional landmark data that meet the requirements of standard geometric morphometric analyses. The method is robust and can be implemented without high-performance computing resources. We validated the method using both direct comparison to manual landmarking on the same individuals and also analyses of the variation patterns and outlier patterns in a large dataset of automated and manual landmark data. Direct comparison of manual and automated landmarks reveals that automated landmark data are less variable, but more highly integrated and reproducible. Automated data produce covariation structure that closely resembles that of manual landmarks. We further find that while our method does produce some landmarking errors, they tend to be readily detectable and can be fixed by adjusting parameters used in the registration and control-point steps. Data generated using the method described here have been successfully used to study the genomic architecture of facial shape in two different genome-wide association studies of facial shape.

  89. Genome-wide meta-analyses of nonsyndromic orofacial clefts identify novel associations between FOXE1 and all orofacial clefts, and TP63 and cleft lip with or without cleft palate

    Leslie, Elizabeth J.; Carlson, Jenna C.; Shaffer, John R.; Butali, Azeez; Buxó, Carmen J.; Castilla, Eduardo E.; Christensen, Kaare; Deleyiannis, Fred W. B.; Leigh Field, L.; Hecht, Jacqueline T.; Moreno, Lina; Orioli, Ieda M.; Padilla, Carmencita; Vieira, Alexandre R.; Wehby, George L.; Feingold, Eleanor; Weinberg, Seth M.; Murray, Jeffrey C.; Beaty, Terri H.; Marazita, Mary L.. Human Genetics. vol. 136(3), 275–286. March 2017.

    Nonsyndromic orofacial clefts (OFCs) are a heterogeneous group of common craniofacial birth defects with complex etiologies that include genetic and environmental risk factors. OFCs are commonly categorized as cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP), which have historically been analyzed as distinct entities. Genes for both CL/P and CP have been identified via multiple genome-wide linkage and association studies (GWAS); however, altogether, known variants account for a minority of the estimated heritability in risk to these craniofacial birth defects. We performed genome-wide meta-analyses of CL/P, CP, and all OFCs across two large, multiethnic studies. We then performed population-specific meta-analyses in sub-samples of Asian and European ancestry. In addition to observing associations with known variants, we identified a novel genome-wide significant association between SNPs located in an intronic TP63 enhancer and CL/P (p = 1.16 × 10-8). Several novel loci with compelling candidate genes approached genome-wide significance on 4q21.1 (SHROOM3), 12q13.13 (KRT18), and 8p21 (NRG1). In the analysis of all OFCs combined, SNPs near FOXE1 reached genome-wide significance (p = 1.33 × 10-9). Our results support the highly heterogeneous nature of OFCs and illustrate the utility of meta-analysis for discovering new genetic risk factors.

  90. Intraflagellar transport 88 (IFT88) is crucial for craniofacial development in mice and is a candidate gene for human cleft lip and palate

    Tian, Hua; Feng, Jifan; Li, Jingyuan; Ho, Thach-Vu; Yuan, Yuan; Liu, Yang; Brindopke, Frederick; Figueiredo, Jane C.; Magee, William; Sanchez-Lara, Pedro A.; Chai, Yang. Human Molecular Genetics. vol. 26(5), 860–872. March 2017.

    Ciliopathies are pleiotropic human diseases resulting from defects of the primary cilium, and these patients often have cleft lip and palate. IFT88 is required for the assembly and function of the primary cilia, which mediate the activity of key developmental signaling pathways. Through whole exome sequencing of a family of three affected siblings with isolated cleft lip and palate, we discovered that they share a novel missense mutation in IFT88 (c.915G \textgreater C, p.E305D), suggesting this gene should be considered a candidate for isolated orofacial clefting. In order to evaluate the function of IFT88 in regulating craniofacial development, we generated Wnt1-Cre;Ift88fl/fl mice to eliminate Ift88 specifically in cranial neural crest (CNC) cells. Wnt1-Cre;Ift88fl/flpups died at birth due to severe craniofacial defects including bilateral cleft lip and palate and tongue agenesis, following the loss of the primary cilia in the CNC-derived palatal mesenchyme. Loss of Ift88 also resulted in a decrease in neural crest cell proliferation during early stages of palatogenesis as well as a downregulation of the Shh signaling pathway in the palatal mesenchyme. Importantly, Osr2KI-Cre;Ift88fl/flmice, in which Ift88 is lost specifically in the palatal mesenchyme, exhibit isolated cleft palate. Taken together, our results demonstrate that IFT88 has a highly conserved function within the primary cilia of the CNC-derived mesenchyme in the lip and palate region in mice and is a strong candidate as an orofacial clefting gene in humans.

  91. Cellular and molecular mechanisms of tooth root development

    Li, Jingyuan; Parada, Carolina; Chai, Yang. Development (Cambridge, England). vol. 144(3), 374–384. February 2017.

    The tooth root is an integral, functionally important part of our dentition. The formation of a functional root depends on epithelial-mesenchymal interactions and integration of the root with the jaw bone, blood supply and nerve innervations. The root development process therefore offers an attractive model for investigating organogenesis. Understanding how roots develop and how they can be bioengineered is also of great interest in the field of regenerative medicine. Here, we discuss recent advances in understanding the cellular and molecular mechanisms underlying tooth root formation. We review the function of cellular structure and components such as Hertwig’s epithelial root sheath, cranial neural crest cells and stem cells residing in developing and adult teeth. We also highlight how complex signaling networks together with multiple transcription factors mediate tissue-tissue interactions that guide root development. Finally, we discuss the possible role of stem cells in establishing the crown-to-root transition, and provide an overview of root malformations and diseases in humans.

  92. Human Facial Shape and Size Heritability and Genetic Correlations

    Cole, Joanne B.; Manyama, Mange; Larson, Jacinda R.; Liberton, Denise K.; Ferrara, Tracey M.; Riccardi, Sheri L.; Li, Mao; Mio, Washington; Klein, Ophir D.; Santorico, Stephanie A.; Hallgrímsson, Benedikt; Spritz, Richard A.. Genetics. vol. 205(2), 967–978. February 2017.

    The human face is an array of variable physical features that together make each of us unique and distinguishable. Striking familial facial similarities underscore a genetic component, but little is known of the genes that underlie facial shape differences. Numerous studies have estimated facial shape heritability using various methods. Here, we used advanced three-dimensional imaging technology and quantitative human genetics analysis to estimate narrow-sense heritability, heritability explained by common genetic variation, and pairwise genetic correlations of 38 measures of facial shape and size in normal African Bantu children from Tanzania. Specifically, we fit a linear mixed model of genetic relatedness between close and distant relatives to jointly estimate variance components that correspond to heritability explained by genome-wide common genetic variation and variance explained by uncaptured genetic variation, the sum representing total narrow-sense heritability. Our significant estimates for narrow-sense heritability of specific facial traits range from 28 to 67%, with horizontal measures being slightly more heritable than vertical or depth measures. Furthermore, for over half of facial traits, \textgreater90% of narrow-sense heritability can be explained by common genetic variation. We also find high absolute genetic correlation between most traits, indicating large overlap in underlying genetic loci. Not surprisingly, traits measured in the same physical orientation (i.e., both horizontal or both vertical) have high positive genetic correlations, whereas traits in opposite orientations have high negative correlations. The complex genetic architecture of facial shape informs our understanding of the intricate relationships among different facial features as well as overall facial development.

  93. Exome sequencing provides additional evidence for the involvement of ARHGAP29 in Mendelian orofacial clefting and extends the phenotypic spectrum to isolated cleft palate

    Liu, Huan; Busch, Tamara; Eliason, Steven; Anand, Deepti; Bullard, Steven; Gowans, Lord J. J.; Nidey, Nichole; Petrin, Aline; Augustine-Akpan, Eno-Abasi; Saadi, Irfan; Dunnwald, Martine; Lachke, Salil A.; Zhu, Ying; Adeyemo, Adebowale; Amendt, Brad; Roscioli, Tony; Cornell, Robert; Murray, Jeffrey; Butali, Azeez. Birth Defects Research. vol. 109(1), 27–37. January 2017.

    BACKGROUND: Recent advances in genomics methodologies, in particular the availability of next-generation sequencing approaches have made it possible to identify risk loci throughout the genome, in particular the exome. In the current study, we present findings from an exome study conducted in five affected individuals of a multiplex family with cleft palate only. METHODS: The GEnome MINIng (GEMINI) pipeline was used to functionally annotate the single nucleotide polymorphisms, insertions and deletions. Filtering methods were applied to identify variants that are clinically relevant and present in affected individuals at minor allele frequencies (≤1%) in the 1000 Genomes Project single nucleotide polymorphism database, Exome Aggregation Consortium, and Exome Variant Server databases. The bioinformatics tool Systems Tool for Craniofacial Expression-Based Gene Discovery was used to prioritize cleft candidates in our list of variants, and Sanger sequencing was used to validate the presence of identified variants in affected and unaffected relatives. RESULTS: Our analyses approach narrowed the candidates down to the novel missense variant in ARHGAP29 (GenBank: NM_004815.3, NP_004806.3;c.1654T\textgreaterC [p.Ser552Pro]. A functional assay in zebrafish embryos showed that the encoded protein lacks the activity possessed by its wild-type counterpart, and migration assays revealed that keratinocytes transfected with wild-type ARHGAP29 migrated faster than counterparts transfected with the p.Ser552Pro ARHGAP29 variant or empty vector (control). CONCLUSION: These findings reveal ARHGAP29 to be a regulatory protein essential for proper development of the face, identifies an amino acid that is key for this, and provides a potential new diagnostic tool.Birth Defects Research 109:27-37, 2017. © 2016 Wiley Periodicals, Inc.

  94. Shared molecular networks in orofacial and neural tube development

    Kousa, Youssef A.; Mansour, Tamer A.; Seada, Haitham; Matoo, Samaneh; Schutte, Brian C.. Birth Defects Research. vol. 109(2), 169–179. January 2017.

    BACKGROUND: Single genetic variants can affect multiple tissues during development. Thus it is possible that disruption of shared gene regulatory networks might underlie syndromic presentations. In this study, we explore this idea through examination of two critical developmental programs that control orofacial and neural tube development and identify shared regulatory factors and networks. Identification of these networks has the potential to yield additional candidate genes for poorly understood developmental disorders and assist in modeling and perhaps managing risk factors to prevent morbidly and mortality. METHODS: We reviewed the literature to identify genes common between orofacial and neural tube defects and development. We then conducted a bioinformatic analysis to identify shared molecular targets and pathways in the development of these tissues. Finally, we examine publicly available RNA-Seq data to identify which of these genes are expressed in both tissues during development. RESULTS: We identify common regulatory factors in orofacial and neural tube development. Pathway enrichment analysis shows that folate, cancer and hedgehog signaling pathways are shared in neural tube and orofacial development. Developing neural tissues differentially express mouse exencephaly and cleft palate genes, whereas developing orofacial tissues were enriched for both clefting and neural tube defect genes. CONCLUSION: These data suggest that key developmental factors and pathways are shared between orofacial and neural tube defects. We conclude that it might be most beneficial to focus on common regulatory factors and pathways to better understand pathology and develop preventative measures for these birth defects. Birth Defects Research 109:169-179, 2017. © 2016 Wiley Periodicals, Inc.

  95. Using the 3D Facial Norms Database to investigate craniofacial sexual dimorphism in healthy children, adolescents, and adults

    Kesterke, Matthew J.; Raffensperger, Zachary D.; Heike, Carrie L.; Cunningham, Michael L.; Hecht, Jacqueline T.; Kau, Chung How; Nidey, Nichole L.; Moreno, Lina M.; Wehby, George L.; Marazita, Mary L.; Weinberg, Seth M.. Biology of Sex Differences. vol. 7, 23. 2016.

    BACKGROUND: Although craniofacial sex differences have been extensively studied in humans, relatively little is known about when various dimorphic features manifest during postnatal life. Using cross-sectional data derived from the 3D Facial Norms data repository, we tested for sexual dimorphism of craniofacial soft-tissue morphology at different ages. METHODS: One thousand five hundred fifty-five individuals, pre-screened for craniofacial conditions, between 3 and 25 years of age were placed in to one of six age-defined categories: early childhood, late childhood, puberty, adolescence, young adult, and adult. At each age group, sex differences were tested by ANCOVA for 29 traditional soft-tissue anthropometric measurements collected from 3D facial scans. Additionally, sex differences in shape were tested using a geometric morphometric analysis of 24 3D facial landmarks. RESULTS: Significant (p \textless 0.05) sex differences were observed in every age group for measurements covering multiple aspects of the craniofacial complex. The magnitude of the dimorphism generally increased with age, with large spikes in the nasal, cranial, and facial measurements observed after puberty. Significant facial shape differences (p \textless 0.05) were also seen at each age, with some dimorphic features already present in young children (eye fissure inclination) and others emerging only after puberty (mandibular position). CONCLUSIONS: Several craniofacial soft-tissue sex differences were already present in the youngest age group studied, indicating that these differences emerged prior to 3 years of age. The results paint a complex and heterogeneous picture, with different groups of traits exhibiting distinct patterns of dimorphism during ontogeny. The definitive adult male and female facial shape was present following puberty, but arose from numerous distinct changes taking place at earlier stages.

  96. Sutures Possess Strong Regenerative Capacity for Calvarial Bone Injury

    Park, Shery; Zhao, Hu; Urata, Mark; Chai, Yang. Stem Cells and Development. vol. 25(23), 1801–1807. December 2016.

    Repair of calvarial bony defects remains challenging for craniofacial surgeons. Injury experiments on animal calvarial bones are widely used to study healing mechanisms and test tissue engineering approaches. Previously, we identified Gli1+ cells within the calvarial sutures as stem cells supporting calvarial bone turnover and injury repair. In this study, we tested the regenerative capacity of the suture region compared with other areas of calvarial bone. Injuries were made to mouse sagittal sutures or other areas of the calvarial bone at varying distances from the suture. Samples were collected at different time points after injury for evaluation. MicroCT and histological analyses were conducted. EdU incorporation analysis was performed to assay cell proliferation. Gli1-CreERT2;Tdtomatoflox mice were used to trace the fate of Gli1+ stem cells after injury. Calvarial sutures possess much stronger regeneration capability than the nonsuture bony areas of the calvaria. The healing rate of the calvarial bone is inversely proportional to the distance between the suture and injury site: injuries closer to the suture heal faster. After complete removal of the sagittal suture, regeneration and restoration of normal organization occur within 6 weeks. Gli1+ cells within the suture mesenchyme are the cellular source for injury repair and bone regeneration. These results demonstrate that calvarial bone healing is not an evenly distributed event on the calvarial surface. Sutures contain stem cells and are the origin of calvarial bone tissue regeneration. Therefore, current practice in calvarial surgery needs to be reevaluated and modified. These findings also necessitate the design of new approaches for repairing calvarial bony defects.

  97. Development Shapes a Consistent Inbreeding Effect in Mouse Crania of Different Line Crosses

    Pavličev, Mihaela; Mitteroecker, Philipp; Gonzalez, Paula M.; Rolian, Campbell; Jamniczky, Heather; Villena, Fernando Pardo-Manuel; Marcucio, Ralph; Spritz, Richard; Hallgrimsson, Benedikt. Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution. vol. 326(8), 474–488. December 2016.

    Development translates genetic variation into a multivariate pattern of phenotypic variation, distributing it among traits in a nonuniform manner. As developmental processes are largely shared within species, this suggests that heritable phenotypic variation will be patterned similarly, in spite of the different segregating alleles. To investigate developmental effect on the variational pattern in the shape of the mouse skull across genetically differentiated lines, we employed the full set of reciprocal crosses (a.k.a. diallel) between eight inbred mouse strains of the Collaborative Cross Project. We used geometric morphometrics and multivariate analysis to capture cranial size and shape changes in 8 parentals and their 54 F1 crosses. The high heterozygosity generated in the F1 crosses allowed us to compare the multivariate deviations of the F1 phenotypes from the expected midparental phenotypes in different haplotype combinations. In contrast to body weight, we found a high degree of nonadditive deviation in craniofacial shape. Whereas the phenotypic and genetic divergence of parental strains manifested in high dimensionality of additive effects, the nonadditive deviations exhibited lesser dimensionality and in particular a strikingly coherent direction in shape space. We interpret this finding as evidence for a strong structuring effect of a relatively small set of developmental processes on the mapping of genetic to phenotypic variation.

  98. The Ties That Bind: Mapping the Dynamic Enhancer-Promoter Interactome

    Spurrell, Cailyn H.; Dickel, Diane E.; Visel, Axel. Cell. vol. 167(5), 1163–1166. November 2016.

    Coupling chromosome conformation capture to molecular enrichment for promoter-containing DNA fragments enables the systematic mapping of interactions between individual distal regulatory sequences and their target genes. In this Minireview, we describe recent progress in the application of this technique and related complementary approaches to gain insight into the lineage- and cell-type-specific dynamics of interactions between regulators and gene promoters.

  99. The 3D Facial Norms Database: Part 1. A Web-Based Craniofacial Anthropometric and Image Repository for the Clinical and Research Community

    Weinberg, Seth M.; Raffensperger, Zachary D.; Kesterke, Matthew J.; Heike, Carrie L.; Cunningham, Michael L.; Hecht, Jacqueline T.; Kau, Chung How; Murray, Jeffrey C.; Wehby, George L.; Moreno, Lina M.; Marazita, Mary L.. The Cleft Palate-Craniofacial Journal. vol. 53(6), 185–197. November 2016.

    With the current widespread use of three-dimensional (3D) facial surface imaging in clinical and research environments, there is a growing demand for high-quality craniofacial norms based on 3D imaging technology. The principal goal of the 3D Facial Norms (3DFN) project was to create an interactive, Web-based repository of 3D facial images and measurements. Unlike other repositories, users can gain access to both summary-level statistics and individual-level data, including 3D facial landmark coordinates, 3D-derived anthropometric measurements, 3D facial surface images, and genotypes from every individual in the dataset. The 3DFN database currently consists of 2454 male and female participants ranging in age from 3 to 40 years. The subjects were recruited at four US sites and screened for a history of craniofacial conditions. The goal of this article is to introduce readers to the 3DFN repository by providing a general overview of the project, explaining the rationale behind the creation of the database, and describing the methods used to collect the data. Sex- and age-specific summary statistics (means and standard deviations) and growth curves for every anthropometric measurement in the 3DFN dataset are provided as a supplement available online. These summary statistics and growth curves can aid clinicians in the assessment of craniofacial dysmorphology.

  100. Modeling craniofacial and skeletal congenital birth defects to advance therapies

    Neben, Cynthia L.; Roberts, Ryan R.; Dipple, Katrina M.; Merrill, Amy E.; Klein, Ophir D.. Human Molecular Genetics. vol. 25(R2), R86–R93. October 2016.

    Craniofacial development is an intricate process of patterning, morphogenesis, and growth that involves many tissues within the developing embryo. Genetic misregulation of these processes leads to craniofacial malformations, which comprise over one-third of all congenital birth defects. Significant advances have been made in the clinical management of craniofacial disorders, but currently very few treatments specifically target the underlying molecular causes. Here, we review recent studies in which modeling of craniofacial disorders in primary patient cells, patient-derived induced pluripotent stem cells (iPSCs), and mice have enhanced our understanding of the etiology and pathophysiology of these disorders while also advancing therapeutic avenues for their prevention.

  101. Zika Virus Infection Induces Cranial Neural Crest Cells to Produce Cytokines at Levels Detrimental for Neurogenesis

    Bayless, Nicholas L.; Greenberg, Rachel S.; Swigut, Tomek; Wysocka, Joanna; Blish, Catherine A.. Cell Host & Microbe. vol. 20(4), 423–428. October 2016.

    Zika virus (ZIKV) infection during pregnancy is linked to microcephaly, which is attributed to infection of developing brain structures. ZIKV infects neural progenitor cells in vitro, though its effects on other developmentally relevant stem cell populations, including cranial neural crest cells (CNCCs), have not been assessed. CNCCs give rise to most cranial bones and exert paracrine effects on the developing brain. Here, we report that CNCCs are productively infected by ZIKV, but not by the related dengue virus. ZIKV-infected CNCCs undergo limited apoptosis but secrete cytokines that promote death and drive aberrant differentiation of neural progenitor cultures. Addition of two such cytokines, LIF or VEGF, at levels comparable to those secreted by ZIKV-infected CNCCs is sufficient to recapitulate premature neuronal differentiation and apoptotic death of neural progenitors. Thus, our results suggest that CNCC infection by ZIKV may contribute to associated embryopathies through signaling crosstalk between developing face and brain structures.

  102. Genetic structure of phenotypic robustness in the collaborative cross mouse diallel panel

    Gonzalez, P. N.; Pavlicev, M.; Mitteroecker, P.; Pardo-Manuel de Villena, F.; Spritz, R. A.; Marcucio, R. S.; Hallgrímsson, B.. Journal of Evolutionary Biology. vol. 29(9), 1737–1751. September 2016.

    Developmental stability and canalization describe the ability of developmental systems to minimize phenotypic variation in the face of stochastic micro-environmental effects, genetic variation and environmental influences. Canalization is the ability to minimize the effects of genetic or environmental effects, whereas developmental stability is the ability to minimize the effects of micro-environmental effects within individuals. Despite much attention, the mechanisms that underlie these two components of phenotypic robustness remain unknown. We investigated the genetic structure of phenotypic robustness in the collaborative cross (CC) mouse reference population. We analysed the magnitude of fluctuating asymmetry (FA) and among-individual variation of cranial shape in reciprocal crosses among the eight parental strains, using geometric morphometrics and a diallel analysis based on a Bayesian approach. Significant differences among genotypes were found for both measures, although they were poorly correlated at the level of individuals. An overall positive effect of inbreeding was found for both components of variation. The strain CAST/EiJ exerted a positive additive effect on FA and, to a lesser extent, among-individual variance. Sex- and other strain-specific effects were not significant. Neither FA nor among-individual variation was associated with phenotypic extremeness. Our results support the existence of genetic variation for both developmental stability and canalization. This finding is important because robustness is a key feature of developmental systems. Our finding that robustness is not related to phenotypic extremeness is consistent with theoretical work that suggests that its relationship to stabilizing selection is not straightforward.

  103. HIPSTR and thousands of lncRNAs are heterogeneously expressed in human embryos, primordial germ cells and stable cell lines

    Yunusov, Dinar; Anderson, Leticia; DaSilva, Lucas Ferreira; Wysocka, Joanna; Ezashi, Toshihiko; Roberts, R. Michael; Verjovski-Almeida, Sergio. Scientific Reports. vol. 6, 32753. September 2016.

    Eukaryotic genomes are transcribed into numerous regulatory long non-coding RNAs (lncRNAs). Compared to mRNAs, lncRNAs display higher developmental stage-, tissue-, and cell-subtype-specificity of expression, and are generally less abundant in a population of cells. Despite the progress in single-cell-focused research, the origins of low population-level expression of lncRNAs in homogeneous populations of cells are poorly understood. Here, we identify HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel lncRNA gene in the developmentally regulated TFAP2A locus. HIPSTR has evolutionarily conserved expression patterns, its promoter is most active in undifferentiated cells, and depletion of HIPSTR in HEK293 and in pluripotent H1BP cells predominantly affects the genes involved in early organismal development and cell differentiation. Most importantly, we find that HIPSTR is specifically induced and heterogeneously expressed in the 8-cell-stage human embryos during the major wave of embryonic genome activation. We systematically explore the phenomenon of cell-to-cell variation of gene expression and link it to low population-level expression of lncRNAs, showing that, similar to HIPSTR, the expression of thousands of lncRNAs is more highly heterogeneous than the expression of mRNAs in the individual, otherwise indistinguishable cells of totipotent human embryos, primordial germ cells, and stable cell lines.

  104. Transcriptional Networks Controlled by NKX2-1 in the Development of Forebrain GABAergic Neurons

    Sandberg, Magnus; Flandin, Pierre; Silberberg, Shanni; Su-Feher, Linda; Price, James D.; Hu, Jia Sheng; Kim, Carol; Visel, Axel; Nord, Alex S.; Rubenstein, John L. R.. Neuron. vol. 91(6), 1260–1275. September 2016.

    The embryonic basal ganglia generates multiple projection neurons and interneuron subtypes from distinct progenitor domains. Combinatorial interactions of transcription factors and chromatin are thought to regulate gene expression. In the medial ganglionic eminence, the NKX2-1 transcription factor controls regional identity and, with LHX6, is necessary to specify pallidal projection neurons and forebrain interneurons. Here, we dissected the molecular functions of NKX2-1 by defining its chromosomal binding, regulation of gene expression, and epigenetic state. NKX2-1 binding at distal regulatory elements led to a repressed epigenetic state and transcriptional repression in the ventricular zone. Conversely, NKX2-1 is required to establish a permissive chromatin state and transcriptional activation in the sub-ventricular and mantle zones. Moreover, combinatorial binding of NKX2-1 and LHX6 promotes transcriptionally permissive chromatin and activates genes expressed in cortical migrating interneurons. Our integrated approach provides a foundation for elucidating transcriptional networks guiding the development of the MGE and its descendants.

  105. Genetic dissection of the α-globin super-enhancer in vivo

    Hay, Deborah; Hughes, Jim R; Babbs, Christian; Davies, James O J; Graham, Bryony J; Hanssen, Lars L P; Kassouf, Mira T; Oudelaar, A Marieke; Sharpe, Jacqueline A; Suciu, Maria C; Telenius, Jelena; Williams, Ruth; Rode, Christina; Li, Pik-Shan; Pennacchio, Len A; Sloane-Stanley, Jacqueline A; Ayyub, Helena; Butler, Sue; Sauka-Spengler, Tatjana; Gibbons, Richard J; Smith, Andrew J H; Wood, William G; Higgs, Douglas R. Nature Genetics. vol. 48(8), 895–903. August 2016.

    Douglas Higgs and colleagues functionally test the α-globin super-enhancer in mice by genetically deleting its constituent enhancers. They find that the individual regulatory elements seem to act independently and in an additive way with respect to hematological phenotype, gene expression, and chromatin structure and conformation.

  106. Pharyngeal morphogenesis requires fras1-itga8-dependent epithelial-mesenchymal interaction

    Talbot, Jared Coffin; Nichols, James T.; Yan, Yi-Lin; Leonard, Isaac F.; BreMiller, Ruth A.; Amacher, Sharon L.; Postlethwait, John H.; Kimmel, Charles B.. Developmental Biology. vol. 416(1), 136–148. August 2016.

    Both Fras1 and Itga8 connect mesenchymal cells to epithelia by way of an extracellular ’Fraser protein complex’ that functions in signaling and adhesion; these proteins are vital to the development of several vertebrate organs. We previously found that zebrafish fras1 mutants have craniofacial defects, specifically, shortened symplectic cartilages and cartilage fusions that spare joint elements. During a forward mutagenesis screen, we identified a new zebrafish mutation, b1161, that we show here disrupts itga8, as confirmed using CRISPR-generated itga8 alleles. fras1 and itga8 single mutants and double mutants have similar craniofacial phenotypes, a result expected if loss of either gene disrupts function of the Fraser protein complex. Unlike fras1 mutants or other Fraser-related mutants, itga8 mutants do not show blistered tail fins. Thus, the function of the Fraser complex differs in the craniofacial skeleton and the tail fin. Focusing on the face, we find that itga8 mutants consistently show defective outpocketing of a late-forming portion of the first pharyngeal pouch, and variably express skeletal defects, matching previously characterized fras1 mutant phenotypes. In itga8 and fras1 mutants, skeletal severity varies markedly between sides, indicating that both mutants have increased developmental instability. Whereas fras1 is expressed in epithelia, we show that itga8 is expressed complementarily in facial mesenchyme. Paired with the observed phenotypic similarity, this expression indicates that the genes function in epithelial-mesenchymal interactions. Similar interactions between Fras1 and Itga8 have previously been found in mouse kidney, where these genes both regulate Nephronectin (Npnt) protein abundance. We find that zebrafish facial tissues express both npnt and the Fraser gene fibrillin2b (fbn2b), but their transcript levels do not depend on fras1 or itga8 function. Using a revertible fras1 allele, we find that the critical window for fras1 function in the craniofacial skeleton is between 1.5 and 3 days post fertilization, which coincides with the onset of fras1-dependent and itga8-dependent morphogenesis. We propose a model wherein Fras1 and Itga8 interact during late pharyngeal pouch morphogenesis to sculpt pharyngeal arches through epithelial-mesenchymal interactions, thereby stabilizing the developing craniofacial skeleton.

  107. Multi-species Ontologies of the Craniofacial Musculoskeletal System

    Mejino, Jose L. V.; Detwiler, Landon T.; Cox, Timothy C.; Brinkley, James F.. CEUR workshop proceedings. vol. 1747, https://ceur––1747/IP03_ICBO2016.pdf. August 2016.

    We created the Ontology of Craniofacial Development and Malformation (OCDM) [1] to provide a unifying framework for organizing and integrating craniofacial data ranging from genes to clinical phenotypes from multi-species. Within this framework we focused on spatio-structural representation of anatomical entities related to craniofacial development and malformation, such as craniosynostosis and midface hypoplasia. Animal models are used to support human studies and so we built multi-species ontologies that would allow for cross-species correlation of anatomical information. For this purpose we first developed and enhanced the craniofacial component of the human musculoskeletal system in the Foundational Model of Anatomy Ontology (FMA)[2], and then imported this component, which we call the Craniofacial Human Ontology (CHO), into the OCDM. The CHO was then used as a template to create the anatomy for the mouse, the Craniofacial Mouse Ontology (CMO) as well as for the zebrafish, the Craniofacial Zebrafish Ontology (CZO).

  108. Genome-Wide Association Study Reveals Multiple Loci Influencing Normal Human Facial Morphology

    Shaffer, John R.; Orlova, Ekaterina; Lee, Myoung Keun; Leslie, Elizabeth J.; Raffensperger, Zachary D.; Heike, Carrie L.; Cunningham, Michael L.; Hecht, Jacqueline T.; Kau, Chung How; Nidey, Nichole L.; Moreno, Lina M.; Wehby, George L.; Murray, Jeffrey C.; Laurie, Cecelia A.; Laurie, Cathy C.; Cole, Joanne; Ferrara, Tracey; Santorico, Stephanie; Klein, Ophir; Mio, Washington; Feingold, Eleanor; Hallgrimsson, Benedikt; Spritz, Richard A.; Marazita, Mary L.; Weinberg, Seth M.. PLoS genetics. vol. 12(8), e1006149. August 2016.

    Numerous lines of evidence point to a genetic basis for facial morphology in humans, yet little is known about how specific genetic variants relate to the phenotypic expression of many common facial features. We conducted genome-wide association meta-analyses of 20 quantitative facial measurements derived from the 3D surface images of 3118 healthy individuals of European ancestry belonging to two US cohorts. Analyses were performed on just under one million genotyped SNPs (Illumina OmniExpress+Exome v1.2 array) imputed to the 1000 Genomes reference panel (Phase 3). We observed genome-wide significant associations (p \textless 5 x 10-8) for cranial base width at 14q21.1 and 20q12, intercanthal width at 1p13.3 and Xq13.2, nasal width at 20p11.22, nasal ala length at 14q11.2, and upper facial depth at 11q22.1. Several genes in the associated regions are known to play roles in craniofacial development or in syndromes affecting the face: MAFB, PAX9, MIPOL1, ALX3, HDAC8, and PAX1. We also tested genotype-phenotype associations reported in two previous genome-wide studies and found evidence of replication for nasal ala length and SNPs in CACNA2D3 and PRDM16. These results provide further evidence that common variants in regions harboring genes of known craniofacial function contribute to normal variation in human facial features. Improved understanding of the genes associated with facial morphology in healthy individuals can provide insights into the pathways and mechanisms controlling normal and abnormal facial morphogenesis.

  109. Genomewide Association Study of African Children Identifies Association of SCHIP1 and PDE8A with Facial Size and Shape

    Cole, Joanne B.; Manyama, Mange; Kimwaga, Emmanuel; Mathayo, Joshua; Larson, Jacinda R.; Liberton, Denise K.; Lukowiak, Ken; Ferrara, Tracey M.; Riccardi, Sheri L.; Li, Mao; Mio, Washington; Prochazkova, Michaela; Williams, Trevor; Li, Hong; Jones, Kenneth L.; Klein, Ophir D.; Santorico, Stephanie A.; Hallgrimsson, Benedikt; Spritz, Richard A.. PLoS genetics. vol. 12(8), e1006174. August 2016.

    The human face is a complex assemblage of highly variable yet clearly heritable anatomic structures that together make each of us unique, distinguishable, and recognizable. Relatively little is known about the genetic underpinnings of normal human facial variation. To address this, we carried out a large genomewide association study and two independent replication studies of Bantu African children and adolescents from Mwanza, Tanzania, a region that is both genetically and environmentally relatively homogeneous. We tested for genetic association of facial shape and size phenotypes derived from 3D imaging and automated landmarking of standard facial morphometric points. SNPs within genes SCHIP1 and PDE8A were associated with measures of facial size in both the GWAS and replication cohorts and passed a stringent genomewide significance threshold adjusted for multiple testing of 34 correlated traits. For both SCHIP1 and PDE8A, we demonstrated clear expression in the developing mouse face by both whole-mount in situ hybridization and RNA-seq, supporting their involvement in facial morphogenesis. Ten additional loci demonstrated suggestive association with various measures of facial shape. Our findings, which differ from those in previous studies of European-derived whites, augment understanding of the genetic basis of normal facial development, and provide insights relevant to both human disease and forensics.

  110. Discovery and characterization of spontaneous mouse models of craniofacial dysmorphology

    Palmer, Kristina; Fairfield, Heather; Borgeia, Suhaib; Curtain, Michelle; Hassan, Mohamed G.; Dionne, Louise; Yong Karst, Son; Coombs, Harold; Bronson, Roderick T.; Reinholdt, Laura G.; Bergstrom, David E.; Donahue, Leah Rae; Cox, Timothy C.; Murray, Stephen A.. Developmental Biology. vol. 415(2), 216–227. July 2016.

    Craniofacial abnormalities are among the most common features of human genetic syndromes and disorders. The etiology of these conditions is often complex, influenced by both genetic context and the environment. Frequently, craniofacial abnormalities present as part of a syndrome with clear comorbid phenotypes, providing additional insight into mechanisms of the causative gene or pathway. The mouse has been a key tool in our understanding of the genetic mechanisms of craniofacial development and disease, and can provide excellent models for human craniofacial abnormalities. While powerful genetic engineering tools in the mouse have contributed significantly our understanding of craniofacial development and dysmorphology, forward genetic approaches provide an unbiased means to identify new genes and pathways. Moreover, spontaneous mutations can occur on any number of genetic backgrounds, potentially revealing critical genes that require a specific genetic context. Here we report discovery and phenotyping of 43 craniofacial mouse models, derived primarily from a screen for spontaneous mutations in production colonies at the Jackson Laboratory. We identify the causative gene for 33 lines, including novel genes in pathways not previously connected to craniofacial development, and novel alleles of known genes that present with unique phenotypes. Together with our detailed characterization, this work provides a valuable gene discovery resource for the craniofacial community, and a rich source of mouse models for further investigation.

  111. The old and new face of craniofacial research: How animal models inform human craniofacial genetic and clinical data

    Van Otterloo, Eric; Williams, Trevor; Artinger, Kristin Bruk. Developmental Biology. vol. 415(2), 171–187. July 2016.

    The craniofacial skeletal structures that comprise the human head develop from multiple tissues that converge to form the bones and cartilage of the face. Because of their complex development and morphogenesis, many human birth defects arise due to disruptions in these cellular populations. Thus, determining how these structures normally develop is vital if we are to gain a deeper understanding of craniofacial birth defects and devise treatment and prevention options. In this review, we will focus on how animal model systems have been used historically and in an ongoing context to enhance our understanding of human craniofacial development. We do this by first highlighting "animal to man" approaches; that is, how animal models are being utilized to understand fundamental mechanisms of craniofacial development. We discuss emerging technologies, including high throughput sequencing and genome editing, and new animal repository resources, and how their application can revolutionize the future of animal models in craniofacial research. Secondly, we highlight "man to animal" approaches, including the current use of animal models to test the function of candidate human disease variants. Specifically, we outline a common workflow deployed after discovery of a potentially disease causing variant based on a select set of recent examples in which human mutations are investigated in vivo using animal models. Collectively, these topics will provide a pipeline for the use of animal models in understanding human craniofacial development and disease for clinical geneticist and basic researchers alike.

  112. The FaceBase Consortium: a comprehensive resource for craniofacial researchers

    Brinkley, James F.; Fisher, Shannon; Harris, Matthew P.; Holmes, Greg; Hooper, Joan E.; Jabs, Ethylin Wang; Jones, Kenneth L.; Kesselman, Carl; Klein, Ophir D.; Maas, Richard L.; Marazita, Mary L.; Selleri, Licia; Spritz, Richard A.; van Bakel, Harm; Visel, Axel; Williams, Trevor J.; Wysocka, Joanna; FaceBase Consortium; Chai, Yang. Development (Cambridge, England). vol. 143(14), 2677–2688. July 2016.

    The FaceBase Consortium, funded by the National Institute of Dental and Craniofacial Research, National Institutes of Health, is designed to accelerate understanding of craniofacial developmental biology by generating comprehensive data resources to empower the research community, exploring high-throughput technology, fostering new scientific collaborations among researchers and human/computer interactions, facilitating hypothesis-driven research and translating science into improved health care to benefit patients. The resources generated by the FaceBase projects include a number of dynamic imaging modalities, genome-wide association studies, software tools for analyzing human facial abnormalities, detailed phenotyping, anatomical and molecular atlases, global and specific gene expression patterns, and transcriptional profiling over the course of embryonic and postnatal development in animal models and humans. The integrated data visualization tools, faceted search infrastructure, and curation provided by the FaceBase Hub offer flexible and intuitive ways to interact with these multidisciplinary data. In parallel, the datasets also offer unique opportunities for new collaborations and training for researchers coming into the field of craniofacial studies. Here, we highlight the focus of each spoke project and the integration of datasets contributed by the spokes to facilitate craniofacial research.

  113. Fishing for Function in the Human Gene Pool

    Barozzi, Iros; Visel, Axel; Dickel, Diane E.. Trends in genetics: TIG. vol. 32(7), 392–394. July 2016.

    Identification and characterization of causal non-coding variants in human genomes is challenging and requires substantial experimental resources. A new study by Tehranchi et al. describes a cost-effective approach for accurate mapping of molecular quantitative trait loci (QTLs) from pooled samples, a powerful way to link disease-associated changes to molecular functions.

  114. A multi-ethnic genome-wide association study identifies novel loci for non-syndromic cleft lip with or without cleft palate on 2p24.2, 17q23 and 19q13

    Leslie, Elizabeth J.; Carlson, Jenna C.; Shaffer, John R.; Feingold, Eleanor; Wehby, George; Laurie, Cecelia A.; Jain, Deepti; Laurie, Cathy C.; Doheny, Kimberly F.; McHenry, Toby; Resick, Judith; Sanchez, Carla; Jacobs, Jennifer; Emanuele, Beth; Vieira, Alexandre R.; Neiswanger, Katherine; Lidral, Andrew C.; Valencia-Ramirez, Luz Consuelo; Lopez-Palacio, Ana Maria; Valencia, Dora Rivera; Arcos-Burgos, Mauricio; Czeizel, Andrew E.; Field, L. Leigh; Padilla, Carmencita D.; Cutiongco-de la Paz, Eva Maria C.; Deleyiannis, Frederic; Christensen, Kaare; Munger, Ronald G.; Lie, Rolv T.; Wilcox, Allen; Romitti, Paul A.; Castilla, Eduardo E.; Mereb, Juan C.; Poletta, Fernando A.; Orioli, Iêda M.; Carvalho, Flavia M.; Hecht, Jacqueline T.; Blanton, Susan H.; Buxó, Carmen J.; Butali, Azeez; Mossey, Peter A.; Adeyemo, Wasiu L.; James, Olutayo; Braimah, Ramat O.; Aregbesola, Babatunde S.; Eshete, Mekonen A.; Abate, Fikre; Koruyucu, Mine; Seymen, Figen; Ma, Lian; de Salamanca, Javier Enríquez; Weinberg, Seth M.; Moreno, Lina; Murray, Jeffrey C.; Marazita, Mary L.. Human Molecular Genetics. vol. 25(13), 2862–2872. July 2016.

    Orofacial clefts (OFCs), which include non-syndromic cleft lip with or without cleft palate (CL/P), are among the most common birth defects in humans, affecting approximately 1 in 700 newborns. CL/P is phenotypically heterogeneous and has a complex etiology caused by genetic and environmental factors. Previous genome-wide association studies (GWASs) have identified at least 15 risk loci for CL/P. As these loci do not account for all of the genetic variance of CL/P, we hypothesized the existence of additional risk loci. We conducted a multiethnic GWAS in 6480 participants (823 unrelated cases, 1700 unrelated controls and 1319 case-parent trios) with European, Asian, African and Central and South American ancestry. Our GWAS revealed novel associations on 2p24 near FAM49A, a gene of unknown function (P = 4.22 × 10-8), and 19q13 near RHPN2, a gene involved in organizing the actin cytoskeleton (P = 4.17 × 10-8). Other regions reaching genome-wide significance were 1p36 (PAX7), 1p22 (ARHGAP29), 1q32 (IRF6), 8q24 and 17p13 (NTN1), all reported in previous GWASs. Stratification by ancestry group revealed a novel association with a region on 17q23 (P = 2.92 × 10-8) among individuals with European ancestry. This region included several promising candidates including TANC2, an oncogene required for development, and DCAF7, a scaffolding protein required for craniofacial development. In the Central and South American ancestry group, significant associations with loci previously identified in Asian or European ancestry groups reflected their admixed ancestry. In summary, we have identified novel CL/P risk loci and suggest new genes involved in craniofacial development, confirming the highly heterogeneous etiology of OFCs.

  115. A unique stylopod patterning mechanism by Shox2-controlled osteogenesis

    Ye, Wenduo; Song, Yingnan; Huang, Zhen; Osterwalder, Marco; Ljubojevic, Anja; Xu, Jue; Bobick, Brent; Abassah-Oppong, Samuel; Ruan, Ningsheng; Shamby, Ross; Yu, Diankun; Zhang, Lu; Cai, Chen-Leng; Visel, Axel; Zhang, Yanding; Cobb, John; Chen, YiPing. Development (Cambridge, England). vol. 143(14), 2548–2560. July 2016.

    Vertebrate appendage patterning is programmed by Hox-TALE factor-bound regulatory elements. However, it remains unclear which cell lineages are commissioned by Hox-TALE factors to generate regional specific patterns and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clustering around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.

  116. A distal 594 bp ECR specifies Hmx1 expression in pinna and lateral facial morphogenesis and is regulated by the Hox-Pbx-Meis complex

    Rosin, Jessica M.; Li, Wenjie; Cox, Liza L.; Rolfe, Sara M.; Latorre, Victor; Akiyama, Jennifer A.; Visel, Axel; Kuramoto, Takashi; Bobola, Nicoletta; Turner, Eric E.; Cox, Timothy C.. Development (Cambridge, England). vol. 143(14), 2582–2592. July 2016.

    Hmx1 encodes a homeodomain transcription factor expressed in the developing lateral craniofacial mesenchyme, retina and sensory ganglia. Mutation or mis-regulation of Hmx1 underlies malformations of the eye and external ear in multiple species. Deletion or insertional duplication of an evolutionarily conserved region (ECR) downstream of Hmx1 has recently been described in rat and cow, respectively. Here, we demonstrate that the impact of Hmx1 loss is greater than previously appreciated, with a variety of lateral cranioskeletal defects, auriculofacial nerve deficits, and duplication of the caudal region of the external ear. Using a transgenic approach, we demonstrate that a 594 bp sequence encompassing the ECR recapitulates specific aspects of the endogenous Hmx1 lateral facial expression pattern. Moreover, we show that Hoxa2, Meis and Pbx proteins act cooperatively on the ECR, via a core 32 bp sequence, to regulate Hmx1 expression. These studies highlight the conserved role for Hmx1 in BA2-derived tissues and provide an entry point for improved understanding of the causes of the frequent lateral facial birth defects in humans.

  117. Osterix/Sp7 limits cranial bone initiation sites and is required for formation of sutures

    Kague, Erika; Roy, Paula; Asselin, Garrett; Hu, Gui; Simonet, Jacqueline; Stanley, Alexandra; Albertson, Craig; Fisher, Shannon. Developmental Biology. vol. 413(2), 160–172. May 2016.

    During growth, individual skull bones overlap at sutures, where osteoblast differentiation and bone deposition occur. Mutations causing skull malformations have revealed some required genes, but many aspects of suture regulation remain poorly understood. We describe a zebrafish mutation in osterix/sp7, which causes a generalized delay in osteoblast maturation. While most of the skeleton is patterned normally, mutants have specific defects in the anterior skull and upper jaw, and the top of the skull comprises a random mosaic of bones derived from individual initiation sites. Osteoblasts at the edges of the bones are highly proliferative and fail to differentiate, consistent with global changes in gene expression. We propose that signals from the bone itself are required for orderly recruitment of precursor cells and growth along the edges. The delay in bone maturation caused by loss of Sp7 leads to unregulated bone formation, revealing a new mechanism for patterning the skull and sutures.

  118. From frames to OWL2: Converting the Foundational Model of Anatomy

    Detwiler, Landon T.; Mejino, Jose L. V.; Brinkley, James F.. Artificial Intelligence in Medicine. vol. 69, 12–21. May 2016.

    OBJECTIVE: The Foundational Model of Anatomy (FMA) [Rosse C, Mejino JLV. A reference ontology for bioinformatics: the Foundational Model of Anatomy. J. Biomed. Inform. 2003;36:478-500] is an ontology that represents canonical anatomy at levels ranging from the entire body to biological macromolecules, and has rapidly become the primary reference ontology for human anatomy, and a template for model organisms. Prior to this work, the FMA was developed in a knowledge modeling language known as Protégé Frames. Frames is an intuitive representational language, but is no longer the industry standard. Recognizing the need for an official version of the FMA in the more modern semantic web language OWL2 (hereafter referred to as OWL), the objective of this work was to create a generalizable Frames-to-OWL conversion tool, to use the tool to convert the FMA to OWL, to "clean up" the converted FMA so that it classifies under an EL reasoner, and then to do all further development in OWL. METHODS: The conversion tool is a Java application that uses the Protégé knowledge representation API for interacting with the initial Frames ontology, and uses the OWL-API for producing new statements (axioms, etc.) in OWL. The converter is relation centric. The conversion is configurable, on a property-by-property basis, via user-specifiable XML configuration files. The best conversion, for each property, was determined in conjunction with the FMA knowledge author. The convertor is potentially generalizable, which we partially demonstrate by using it to convert our Ontology of Craniofacial Development and Malformation as well as the FMA. Post-conversion cleanup involved using the Explain feature of Protégé to trace classification errors under the ELK reasoner in Protégé, fixing the errors, then re-running the reasoner. RESULTS: We are currently doing all our development in the converted and cleaned-up version of the FMA. The FMA (updated every 3 months) is available via our FMA web page, which also provides access to mailing lists, an issue tracker, a SPARQL endpoint (updated every week), and an online browser. The converted OCDM is available at The conversion code is open source, and available at Prior to the post-conversion cleanup 73% of the more than 100,000 classes were unsatisfiable. After correction of six types of errors no classes remained unsatisfiable. CONCLUSION: Because our FMA conversion captures all or most of the information in the Frames version, is the only complete OWL version that classifies under an EL reasoner, and is maintained by the FMA authors themselves, we propose that this version should be the only official release version of the FMA in OWL, supplanting all other versions. Although several issues remain to be resolved post-conversion, release of a single, standardized version of the FMA in OWL will greatly facilitate its use in informatics research and in the development of a global knowledge base within the semantic web. Because of the fundamental nature of anatomy in both understanding and organizing biomedical information, and because of the importance of the FMA in particular in representing human anatomy, the FMA in OWL should greatly accelerate the development of an anatomically based structural information framework for organizing and linking a large amount of biomedical information.

  119. Actin capping protein CAPZB regulates cell morphology, differentiation, and neural crest migration in craniofacial morphogenesis†

    Mukherjee, Kusumika; Ishii, Kana; Pillalamarri, Vamsee; Kammin, Tammy; Atkin, Joan F.; Hickey, Scott E.; Xi, Qiongchao J.; Zepeda, Cinthya J.; Gusella, James F.; Talkowski, Michael E.; Morton, Cynthia C.; Maas, Richard L.; Liao, Eric C.. Human Molecular Genetics. vol. 25(7), 1255–1270. April 2016.

    CAPZB is an actin-capping protein that caps the growing end of F-actin and modulates the cytoskeleton and tethers actin filaments to the Z-line of the sarcomere in muscles. Whole-genome sequencing was performed on a subject with micrognathia, cleft palate and hypotonia that harbored a de novo, balanced chromosomal translocation that disrupts the CAPZB gene. The function of capzb was analyzed in the zebrafish model. capzb(-/-) mutants exhibit both craniofacial and muscle defects that recapitulate the phenotypes observed in the human subject. Loss of capzb affects cell morphology, differentiation and neural crest migration. Differentiation of both myogenic stem cells and neural crest cells requires capzb. During palate morphogenesis, defective cranial neural crest cell migration in capzb(-/-) mutants results in loss of the median cell population, creating a cleft phenotype. capzb is also required for trunk neural crest migration, as evident from melanophores disorganization in capzb(-/-) mutants. In addition, capzb over-expression results in embryonic lethality. Therefore, proper capzb dosage is important during embryogenesis, and regulates both cell behavior and tissue morphogenesis.

  120. A Genome-wide Association Study of Nonsyndromic Cleft Palate Identifies an Etiologic Missense Variant in GRHL3

    Leslie, Elizabeth J.; Liu, Huan; Carlson, Jenna C.; Shaffer, John R.; Feingold, Eleanor; Wehby, George; Laurie, Cecelia A.; Jain, Deepti; Laurie, Cathy C.; Doheny, Kimberly F.; McHenry, Toby; Resick, Judith; Sanchez, Carla; Jacobs, Jennifer; Emanuele, Beth; Vieira, Alexandre R.; Neiswanger, Katherine; Standley, Jennifer; Czeizel, Andrew E.; Deleyiannis, Frederic; Christensen, Kaare; Munger, Ronald G.; Lie, Rolv T.; Wilcox, Allen; Romitti, Paul A.; Field, L. Leigh; Padilla, Carmencita D.; Cutiongco-de la Paz, Eva Maria C.; Lidral, Andrew C.; Valencia-Ramirez, Luz Consuelo; Lopez-Palacio, Ana Maria; Valencia, Dora Rivera; Arcos-Burgos, Mauricio; Castilla, Eduardo E.; Mereb, Juan C.; Poletta, Fernando A.; Orioli, Iêda M.; Carvalho, Flavia M.; Hecht, Jacqueline T.; Blanton, Susan H.; Buxó, Carmen J.; Butali, Azeez; Mossey, Peter A.; Adeyemo, Wasiu L.; James, Olutayo; Braimah, Ramat O.; Aregbesola, Babatunde S.; Eshete, Mekonen A.; Deribew, Milliard; Koruyucu, Mine; Seymen, Figen; Ma, Lian; de Salamanca, Javier Enríquez; Weinberg, Seth M.; Moreno, Lina; Cornell, Robert A.; Murray, Jeffrey C.; Marazita, Mary L.. American Journal of Human Genetics. vol. 98(4), 744–754. April 2016.

    Cleft palate (CP) is a common birth defect occurring in 1 in 2,500 live births. Approximately half of infants with CP have a syndromic form, exhibiting other physical and cognitive disabilities. The other half have nonsyndromic CP, and to date, few genes associated with risk for nonsyndromic CP have been characterized. To identify such risk factors, we performed a genome-wide association study of this disorder. We discovered a genome-wide significant association with a missense variant in GRHL3 (p.Thr454Met [c.1361C\textgreaterT]; rs41268753; p = 4.08 × 10(-9)) and replicated the result in an independent sample of case and control subjects. In both the discovery and replication samples, rs41268753 conferred increased risk for CP (OR = 8.3, 95% CI 4.1-16.8; OR = 2.16, 95% CI 1.43-3.27, respectively). In luciferase transactivation assays, p.Thr454Met had about one-third of the activity of wild-type GRHL3, and in zebrafish embryos, perturbed periderm development. We conclude that this mutation is an etiologic variant for nonsyndromic CP and is one of few functional variants identified to date for nonsyndromic orofacial clefting. This finding advances our understanding of the genetic basis of craniofacial development and might ultimately lead to improvements in recurrence risk prediction, treatment, and prognosis.

  121. An integrated miRNA functional screening and target validation method for organ morphogenesis

    Rebustini, Ivan T.; Vlahos, Maryann; Packer, Trevor; Kukuruzinska, Maria A.; Maas, Richard L.. Scientific Reports. vol. 6, 23215. March 2016.

    The relative ease of identifying microRNAs and their increasing recognition as important regulators of organogenesis motivate the development of methods to efficiently assess microRNA function during organ morphogenesis. In this context, embryonic organ explants provide a reliable and reproducible system that recapitulates some of the important early morphogenetic processes during organ development. Here we present a method to target microRNA function in explanted mouse embryonic organs. Our method combines the use of peptide-based nanoparticles to transfect specific microRNA inhibitors or activators into embryonic organ explants, with a microRNA pulldown assay that allows direct identification of microRNA targets. This method provides effective assessment of microRNA function during organ morphogenesis, allows prioritization of multiple microRNAs in parallel for subsequent genetic approaches, and can be applied to a variety of embryonic organs.

  122. Genetics of murine craniofacial morphology: diallel analysis of the eight founders of the Collaborative Cross

    Percival, Christopher J.; Liberton, Denise K.; Pardo-Manuel de Villena, Fernando; Spritz, Richard; Marcucio, Ralph; Hallgrímsson, Benedikt. Journal of Anatomy. vol. 228(1), 96–112. January 2016.

    Using eight inbred founder strains of the mouse Collaborative Cross (CC) project and their reciprocal F1 hybrids, we quantified variation in craniofacial morphology across mouse strains, explored genetic contributions to craniofacial variation that distinguish the founder strains, and tested whether specific or summary measures of craniofacial shape display stronger additive genetic contributions. This study thus provides critical information about phenotypic diversity among CC founder strains and about the genetic contributions to this phenotypic diversity, which is relevant to understanding the basis of variation in standard laboratory strains and natural populations. Craniofacial shape was quantified as a series of size-adjusted linear dimensions (RDs) and by principal components (PC) analysis of morphological landmarks captured from computed tomography images from 62 of the 64 reciprocal crosses of the CC founder strains. We first identified aspects of skull morphology that vary between these phenotypically ’normal’ founder strains and that are defining characteristics of these strains. We estimated the contributions of additive and various non-additive genetic factors to phenotypic variation using diallel analyses of a subset of these strongly differing RDs and the first eight PCs of skull shape variation. We find little difference in the genetic contributions to RD measures and PC scores, suggesting fundamental similarities in the magnitude of genetic contributions to both specific and summary measures of craniofacial phenotypes. Our results indicate that there are stronger additive genetic effects associated with defining phenotypic characteristics of specific founder strains, suggesting these distinguishing measures are good candidates for use in genotype-phenotype association studies of CC mice. Our results add significantly to understanding of genotype-phenotype associations in the skull, which serve as a foundation for modeling the origins of medically and evolutionarily relevant variation.

  123. From Bench to Bedside and Back: Improving Diagnosis and Treatment of Craniofacial Malformations Utilizing Animal Models

    Goodwin, Alice F.; Kim, Rebecca; Bush, Jeffrey O.; Klein, Ophir D.. Current Topics in Developmental Biology. vol. 115, 459–492. 2015.

    Craniofacial anomalies are among the most common birth defects and are associated with increased mortality and, in many cases, the need for lifelong treatment. Over the past few decades, dramatic advances in the surgical and medical care of these patients have led to marked improvements in patient outcomes. However, none of the treatments currently in clinical use address the underlying molecular causes of these disorders. Fortunately, the field of craniofacial developmental biology provides a strong foundation for improved diagnosis and for therapies that target the genetic causes of birth defects. In this chapter, we discuss recent advances in our understanding of the embryology of craniofacial conditions, and we focus on the use of animal models to guide rational therapies anchored in genetics and biochemistry.

  124. Molecular Anatomy of Palate Development

    Potter, Andrew S.; Potter, S. Steven. PloS One. vol. 10(7), e0132662. 2015.

    The NIH FACEBASE consortium was established in part to create a central resource for craniofacial researchers. One purpose is to provide a molecular anatomy of craniofacial development. To this end we have used a combination of laser capture microdissection and RNA-Seq to define the gene expression programs driving development of the murine palate. We focused on the E14.5 palate, soon after medial fusion of the two palatal shelves. The palate was divided into multiple compartments, including both medial and lateral, as well as oral and nasal, for both the anterior and posterior domains. A total of 25 RNA-Seq datasets were generated. The results provide a comprehensive view of the region specific expression of all transcription factors, growth factors and receptors. Paracrine interactions can be inferred from flanking compartment growth factor/receptor expression patterns. The results are validated primarily through very high concordance with extensive previously published gene expression data for the developing palate. In addition selected immunostain validations were carried out. In conclusion, this report provides an RNA-Seq based atlas of gene expression patterns driving palate development at microanatomic resolution. This FACEBASE resource is designed to promote discovery by the craniofacial research community.

  125. Morphometrics, 3D Imaging, and Craniofacial Development

    Hallgrimsson, Benedikt; Percival, Christopher J.; Green, Rebecca; Young, Nathan M.; Mio, Washington; Marcucio, Ralph. Current Topics in Developmental Biology. vol. 115, 561–597. 2015.

    Recent studies have shown how volumetric imaging and morphometrics can add significantly to our understanding of morphogenesis, the developmental basis for variation, and the etiology of structural birth defects. On the other hand, the complex questions and diverse imaging data in developmental biology present morphometrics with more complex challenges than applications in virtually any other field. Meeting these challenges is necessary in order to understand the mechanistic basis for variation in complex morphologies. This chapter reviews the methods and theory that enable the application of modern landmark-based morphometrics to developmental biology and craniofacial development, in particular. We discuss the theoretical foundations of morphometrics as applied to development and review the basic approaches to the quantification of morphology. Focusing on geometric morphometrics, we discuss the principal statistical methods for quantifying and comparing morphological variation and covariation structure within and among groups. Finally, we discuss the future directions for morphometrics in developmental biology that will be required for approaches that enable quantitative integration across the genotype-phenotype map.

  126. Disruption of the ERK/MAPK pathway in neural crest cells as a potential cause of Pierre Robin sequence

    Parada, Carolina; Han, Dong; Grimaldi, Alexandre; Sarrión, Patricia; Park, Shery S.; Pelikan, Richard; Sanchez-Lara, Pedro A.; Chai, Yang. Development (Cambridge, England). vol. 142(21), 3734–3745. November 2015.

    Disrupted ERK1/2 signaling is associated with several developmental syndromes in humans. To understand the function of ERK2 (MAPK1) in the postmigratory neural crest populating the craniofacial region, we studied two mouse models: Wnt1-Cre;Erk2(fl/fl) and Osr2-Cre;Erk2(fl/fl). Wnt1-Cre;Erk2(fl/fl) mice exhibited cleft palate, malformed tongue, micrognathia and mandibular asymmetry. Cleft palate in these mice was associated with delay/failure of palatal shelf elevation caused by tongue malposition and micrognathia. Osr2-Cre;Erk2(fl/fl) mice, in which the Erk2 deletion is restricted to the palatal mesenchyme, did not display cleft palate, suggesting that palatal clefting in Wnt1-Cre;Erk2(fl/fl) mice is a secondary defect. Tongues in Wnt1-Cre;Erk2(fl/fl) mice exhibited microglossia, malposition, disruption of the muscle patterning and compromised tendon development. The tongue phenotype was extensively rescued after culture in isolation, indicating that it might also be a secondary defect. The primary malformations in Wnt1-Cre;Erk2(fl/fl) mice, namely micrognathia and mandibular asymmetry, are linked to an early osteogenic differentiation defect. Collectively, our study demonstrates that mutation of Erk2 in neural crest derivatives phenocopies the human Pierre Robin sequence and highlights the interconnection of palate, tongue and mandible development. Because the ERK pathway serves as a crucial point of convergence for multiple signaling pathways, our study will facilitate a better understanding of the molecular regulatory mechanisms of craniofacial development.

  127. Stem Cells in Teeth and Craniofacial Bones

    Zhao, H.; Chai, Y.. Journal of Dental Research. vol. 94(11), 1495–1501. November 2015.

    Stem cells are remarkable, and stem cell-based tissue engineering is an emerging field of biomedical science aiming to restore damaged tissue or organs. In dentistry and reconstructive facial surgery, it is of great interest to restore lost teeth or craniofacial bone defects using stem cell-mediated therapy. In the craniofacial region, various stem cell populations have been identified with regeneration potential. In this review, we provide an overview of the current knowledge concerning the various types of tooth- and craniofacial bone-related stem cells and discuss their in vivo identities and regulating mechanisms.

  128. miRNA Nomenclature: A View Incorporating Genetic Origins, Biosynthetic Pathways, and Sequence Variants

    Desvignes, T.; Batzel, P.; Berezikov, E.; Eilbeck, K.; Eppig, J. T.; McAndrews, M. S.; Singer, A.; Postlethwait, J. H.. Trends in genetics: TIG. vol. 31(11), 613–626. November 2015.

    High-throughput sequencing of miRNAs has revealed the diversity and variability of mature and functional short noncoding RNAs, including their genomic origins, biogenesis pathways, sequence variability, and newly identified products such as miRNA-offset RNAs (moRs). Here we review known cases of alternative mature miRNA-like RNA fragments and propose a revised definition of miRNAs to encompass this diversity. We then review nomenclature guidelines for miRNAs and propose to extend nomenclature conventions to align with those for protein-coding genes established by international consortia. Finally, we suggest a system to encompass the full complexity of sequence variations (i.e., isomiRs) in the analysis of small RNA sequencing experiments.

  129. An Nfic-hedgehog signaling cascade regulates tooth root development

    Liu, Yang; Feng, Jifan; Li, Jingyuan; Zhao, Hu; Ho, Thach-Vu; Chai, Yang. Development (Cambridge, England). vol. 142(19), 3374–3382. October 2015.

    Coordination between the Hertwig’s epithelial root sheath (HERS) and apical papilla (AP) is crucial for proper tooth root development. The hedgehog (Hh) signaling pathway and Nfic are both involved in tooth root development; however, their relationship has yet to be elucidated. Here, we establish a timecourse of mouse molar root development by histological staining of sections, and we demonstrate that Hh signaling is active before and during root development in the AP and HERS using Gli1 reporter mice. The proper pattern of Hh signaling activity in the AP is crucial for the proliferation of dental mesenchymal cells, because either inhibition with Hh inhibitors or constitutive activation of Hh signaling activity in transgenic mice leads to decreased proliferation in the AP and shorter roots. Moreover, Hh activity is elevated in Nfic(-/-) mice, a root defect model, whereas RNA sequencing and in situ hybridization show that the Hh attenuator Hhip is downregulated. ChIP and RNAscope analyses suggest that Nfic binds to the promoter region of Hhip. Treatment of Nfic(-/-) mice with Hh inhibitor partially restores cell proliferation, AP growth and root development. Taken together, our results demonstrate that an Nfic-Hhip-Hh signaling pathway is crucial for apical papilla growth and proper root formation. This discovery provides insight into the molecular mechanisms regulating tooth root development.

  130. Identification of a Recognizable Progressive Skeletal Dysplasia Caused by RSPRY1 Mutations

    Faden, Maha; AlZahrani, Fatema; Mendoza-Londono, Roberto; Dupuis, Lucie; Hartley, Taila; Kannu, Peter; Raiman, Julian A.; Howard, Andrew; Qin, Wen; Tetreault, Martine; Xi, Joan Qiongchao; Al-Thamer, Imadeddin; Care4Rare Canada Consortium; Maas, Richard L.; Boycott, Kym; Alkuraya, Fowzan S.. American Journal of Human Genetics. vol. 97(4), 608–615. October 2015.

    Skeletal dysplasias are highly variable Mendelian phenotypes. Molecular diagnosis of skeletal dysplasias is complicated by their extreme clinical and genetic heterogeneity. We describe a clinically recognizable autosomal-recessive disorder in four affected siblings from a consanguineous Saudi family, comprising progressive spondyloepimetaphyseal dysplasia, short stature, facial dysmorphism, short fourth metatarsals, and intellectual disability. Combined autozygome/exome analysis identified a homozygous frameshift mutation in RSPRY1 with resulting nonsense-mediated decay. Using a gene-centric "matchmaking" system, we were able to identify a Peruvian simplex case subject whose phenotype is strikingly similar to the original Saudi family and whose exome sequencing had revealed a likely pathogenic homozygous missense variant in the same gene. RSPRY1 encodes a hypothetical RING and SPRY domain-containing protein of unknown physiological function. However, we detect strong RSPRY1 protein localization in murine embryonic osteoblasts and periosteal cells during primary endochondral ossification, consistent with a role in bone development. This study highlights the role of gene-centric matchmaking tools to establish causal links to genes, especially for rare or previously undescribed clinical entities.

  131. Enhancer divergence and cis-regulatory evolution in the human and chimp neural crest

    Prescott, Sara L.; Srinivasan, Rajini; Marchetto, Maria Carolina; Grishina, Irina; Narvaiza, Iñigo; Selleri, Licia; Gage, Fred H.; Swigut, Tomek; Wysocka, Joanna. Cell. vol. 163(1), 68–83. September 2015.

    cis-regulatory changes play a central role in morphological divergence, yet the regulatory principles underlying emergence of human traits remain poorly understood. Here, we use epigenomic profiling from human and chimpanzee cranial neural crest cells to systematically and quantitatively annotate divergence of craniofacial cis-regulatory landscapes. Epigenomic divergence is often attributable to genetic variation within TF motifs at orthologous enhancers, with a novel motif being most predictive of activity biases. We explore properties of this cis-regulatory change, revealing the role of particular retroelements, uncovering broad clusters of species-biased enhancers near genes associated with human facial variation, and demonstrating that cis-regulatory divergence is linked to quantitative expression differences of crucial neural crest regulators. Our work provides a wealth of candidates for future evolutionary studies and demonstrates the value of "cellular anthropology," a strategy of using in-vitro-derived embryonic cell types to elucidate both fundamental and evolving mechanisms underlying morphological variation in higher primates.

  132. A transgenic quail model that enables dynamic imaging of amniote embryogenesis

    Huss, David; Benazeraf, Bertrand; Wallingford, Allison; Filla, Michael; Yang, Jennifer; Fraser, Scott E.; Lansford, Rusty. Development (Cambridge, England). vol. 142(16), 2850–2859. August 2015.

    Embryogenesis is the coordinated assembly of tissues during morphogenesis through changes in individual cell behaviors and collective cell movements. Dynamic imaging, combined with quantitative analysis, is ideal for investigating fundamental questions in developmental biology involving cellular differentiation, growth control and morphogenesis. However, a reliable amniote model system that is amenable to the rigors of extended, high-resolution imaging and cell tracking has been lacking. To address this shortcoming, we produced a novel transgenic quail that ubiquitously expresses nuclear localized monomer cherry fluorescent protein (chFP). We characterize the expression pattern of chFP and provide concrete examples of how Tg(PGK1:H2B-chFP) quail can be used to dynamically image and analyze key morphogenetic events during embryonic stages X to 11.

  133. A single nucleotide polymorphism associated with isolated cleft lip and palate, thyroid cancer and hypothyroidism alters the activity of an oral epithelium and thyroid enhancer near FOXE1

    Lidral, Andrew C.; Liu, Huan; Bullard, Steven A.; Bonde, Greg; Machida, Junichiro; Visel, Axel; Uribe, Lina M. Moreno; Li, Xiao; Amendt, Brad; Cornell, Robert A.. Human Molecular Genetics. vol. 24(14), 3895–3907. July 2015.

    Three common diseases, isolated cleft lip and cleft palate (CLP), hypothyroidism and thyroid cancer all map to the FOXE1 locus, but causative variants have yet to be identified. In patients with CLP, the frequency of coding mutations in FOXE1 fails to account for the risk attributable to this locus, suggesting that the common risk alleles reside in nearby regulatory elements. Using a combination of zebrafish and mouse transgenesis, we screened 15 conserved non-coding sequences for enhancer activity, identifying three that regulate expression in a tissue specific pattern consistent with endogenous foxe1 expression. These three, located -82.4, -67.7 and +22.6 kb from the FOXE1 start codon, are all active in the oral epithelium or branchial arches. The -67.7 and +22.6 kb elements are also active in the developing heart, and the -67.7 kb element uniquely directs expression in the developing thyroid. Within the -67.7 kb element is the SNP rs7850258 that is associated with all three diseases. Quantitative reporter assays in oral epithelial and thyroid cell lines show that the rs7850258 allele (G) associated with CLP and hypothyroidism has significantly greater enhancer activity than the allele associated with thyroid cancer (A). Moreover, consistent with predicted transcription factor binding differences, the -67.7 kb element containing rs7850258 allele G is significantly more responsive to both MYC and ARNT than allele A. By demonstrating that this common non-coding variant alters FOXE1 expression, we have identified at least in part the functional basis for the genetic risk of these seemingly disparate disorders.

  134. Exome sequencing reveals pathogenic mutations in 91 strains of mice with Mendelian disorders

    Fairfield, Heather; Srivastava, Anuj; Ananda, Guruprasad; Liu, Rangjiao; Kircher, Martin; Lakshminarayana, Anuradha; Harris, Belinda S.; Karst, Son Yong; Dionne, Louise A.; Kane, Coleen C.; Curtain, Michelle; Berry, Melissa L.; Ward-Bailey, Patricia F.; Greenstein, Ian; Byers, Candice; Czechanski, Anne; Sharp, Jocelyn; Palmer, Kristina; Gudis, Polyxeni; Martin, Whitney; Tadenev, Abby; Bogdanik, Laurent; Pratt, C. Herbert; Chang, Bo; Schroeder, David G.; Cox, Gregory A.; Cliften, Paul; Milbrandt, Jeffrey; Murray, Stephen; Burgess, Robert; Bergstrom, David E.; Donahue, Leah Rae; Hamamy, Hanan; Masri, Amira; Santoni, Federico A.; Makrythanasis, Periklis; Antonarakis, Stylianos E.; Shendure, Jay; Reinholdt, Laura G.. Genome Research. vol. 25(7), 948–957. July 2015.

    Spontaneously arising mouse mutations have served as the foundation for understanding gene function for more than 100 years. We have used exome sequencing in an effort to identify the causative mutations for 172 distinct, spontaneously arising mouse models of Mendelian disorders, including a broad range of clinically relevant phenotypes. To analyze the resulting data, we developed an analytics pipeline that is optimized for mouse exome data and a variation database that allows for reproducible, user-defined data mining as well as nomination of mutation candidates through knowledge-based integration of sample and variant data. Using these new tools, putative pathogenic mutations were identified for 91 (53%) of the strains in our study. Despite the increased power offered by potentially unlimited pedigrees and controlled breeding, about half of our exome cases remained unsolved. Using a combination of manual analyses of exome alignments and whole-genome sequencing, we provide evidence that a large fraction of unsolved exome cases have underlying structural mutations. This result directly informs efforts to investigate the similar proportion of apparently Mendelian human phenotypes that are recalcitrant to exome sequencing.

  135. Automated Detection of 3D Landmarks for the Elimination of Non-Biological Variation in Geometric Morphometric Analyses

    Aneja, D.; Vora, S. R.; Camci, E. D.; Shapiro, L. G.; Cox, T. C.. Proceedings. IEEE International Symposium on Computer-Based Medical Systems. vol. 2015, 78–83. June 2015.

    Landmark-based morphometric analyses are used by anthropologists, developmental and evolutionary biologists to understand shape and size differences (eg. in the cranioskeleton) between groups of specimens. The standard, labor intensive approach is for researchers to manually place landmarks on 3D image datasets. As landmark recognition is subject to inaccuracies of human perception, digitization of landmark coordinates is typically repeated (often by more than one person) and the mean coordinates are used. In an attempt to improve efficiency and reproducibility between researchers, we have developed an algorithm to locate landmarks on CT mouse hemi-mandible data. The method is evaluated on 3D meshes of 28-day old mice, and results compared to landmarks manually identified by experts. Quantitative shape comparison between two inbred mouse strains demonstrate that data obtained using our algorithm also has enhanced statistical power when compared to data obtained by manual landmarking.

  136. Replication of 13q31.1 association in nonsyndromic cleft lip with cleft palate in Europeans

    Jia, Zhonglin; Leslie, Elizabeth J.; Cooper, Margaret E.; Butali, Azeez; Standley, Jennifer; Rigdon, Jennifer; Suzuki, Satoshi; Gongorjav, Ayana; Shonkhuuz, T. Enkhtur; Natsume, Nagato; Shi, Bing; Marazita, Mary L.; Murray, Jeffrey C.. American Journal of Medical Genetics. Part A. vol. 167A(5), 1054–1060. May 2015.

    Genome wide association (GWA) studies have successfully identified at least a dozen loci associated with orofacial clefts. However, these signals may be unique to specific populations and require replication to validate and extend findings as a prelude to etiologic SNP discovery. We attempted to replicate the findings of a recent meta-analysis of orofacial cleft GWA studies using four different ancestral populations. We studied 946 pedigrees (3,436 persons) of European (US white and Danish) and Asian (Japanese and Mongolian) origin. We genotyped six SNPs that represented the most significant P-value associations identified in published studies: rs742071 (1p36), rs7590268 (2p21), rs7632427 (3p11.1), rs12543318 (8q21.3), rs8001641 (13q31.1), and rs7179658 (15q22.2). We directly sequenced three non-coding conserved regions 200 kb downstream of SPRY2 in 713 cases, 438 controls, and 485 trios from the US, Mongolia, and the Philippines. We found rs8001641 to be significantly associated with nonsyndromic cleft lip with cleft palate (NSCLP) in Europeans (P-value = 4 × 10(-5), ORtransmission = 1.86 with 95% confidence interval: 1.38-2.52). We also found several novel sequence variants in the conserved regions in Asian and European samples, which may help to localize common variants contributing directly to the risk for NSCLP. This study confirms the prior association between rs8001641 and NSCLP in European populations.

  137. Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions

    Lupiáñez, Darío G.; Kraft, Katerina; Heinrich, Verena; Krawitz, Peter; Brancati, Francesco; Klopocki, Eva; Horn, Denise; Kayserili, Hülya; Opitz, John M.; Laxova, Renata; Santos-Simarro, Fernando; Gilbert-Dussardier, Brigitte; Wittler, Lars; Borschiwer, Marina; Haas, Stefan A.; Osterwalder, Marco; Franke, Martin; Timmermann, Bernd; Hecht, Jochen; Spielmann, Malte; Visel, Axel; Mundlos, Stefan. Cell. vol. 161(5), 1012–1025. May 2015.

    Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory architecture and result in pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. This rewiring occurred only if the variant disrupted a CTCF-associated boundary domain. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome.

  138. A novel allele of Alx4 results in reduced Fgf10 expression and failure of eyelid fusion in mice

    Curtain, Michelle; Heffner, Caleb S.; Maddox, Dennis M.; Gudis, Polyxeni; Donahue, Leah Rae; Murray, Stephen A.. Mammalian Genome: Official Journal of the International Mammalian Genome Society. vol. 26(3-4), 173–180. April 2015.

    Normal fusion of developing eyelids requires coordination of inductive signals from the eyelid mesenchyme with migration of the periderm cell layer and constriction of the eyelids across the eye. Failure of this process results in an eyelids open at birth (EOB) phenotype in mice. We have identified a novel spontaneous allele of Alx4 that displays EOB, in addition to polydactyly and cranial malformations. Alx4 is expressed in the eyelid mesenchyme prior to and during eyelid fusion in a domain overlapping the expression of genes that also play a role in normal eyelid development. We show that Alx4 mutant mice have reduced expression of Fgf10, a key factor expressed in the mesenchyme that is required for initiation of eyelid fusion by the periderm. This is accompanied by a reduced number of periderm cells expressing phosphorylated c-Jun, consistent with the incomplete ablation of Fgf10 expression. Together, these data demonstrate that eyelid fusion in mice requires the expression of Alx4, accompanied by the loss of normal expression of essential components of the eyelid fusion pathway.

  139. Integration of comprehensive 3D microCT and signaling analysis reveals differential regulatory mechanisms of craniofacial bone development

    Ho, Thach-Vu; Iwata, Junichi; Ho, Hoang Anh; Grimes, Weston C.; Park, Shery; Sanchez-Lara, Pedro A.; Chai, Yang. Developmental Biology. vol. 400(2), 180–190. April 2015.

    Growth factor signaling regulates tissue-tissue interactions to control organogenesis and tissue homeostasis. Specifically, transforming growth factor beta (TGFβ) signaling plays a crucial role in the development of cranial neural crest (CNC) cell-derived bone, and loss of Tgfbr2 in CNC cells results in craniofacial skeletal malformations. Our recent studies indicate that non-canonical TGFβ signaling is activated whereas canonical TGFβ signaling is compromised in the absence of Tgfbr2 (in Tgfbr2(fl/fl);Wnt1-Cre mice). A haploinsufficiency of Tgfbr1 (aka Alk5) (Tgfbr2(fl/fl);Wnt1-Cre;Alk5(fl/+)) largely rescues craniofacial deformities in Tgfbr2 mutant mice by reducing ectopic non-canonical TGFβ signaling. However, the relative involvement of canonical and non-canonical TGFβ signaling in regulating specific craniofacial bone formation remains unclear. We compared the size and volume of CNC-derived craniofacial bones (frontal bone, premaxilla, maxilla, palatine bone, and mandible) from E18.5 control, Tgfbr2(fl/fl);Wnt1-Cre, and Tgfbr2(fl/fl);Wnt1-Cre;Alk5(fl/+)mice. By analyzing three dimensional (3D) micro-computed tomography (microCT) images, we found that different craniofacial bones were restored to different degrees in Tgfbr2(fl/fl);Wnt1-Cre;Alk5(fl/+) mice. Our study provides comprehensive information on anatomical landmarks and the size and volume of each craniofacial bone, as well as insights into the extent that canonical and non-canonical TGFβ signaling cascades contribute to the formation of each CNC-derived bone. Our data will serve as an important resource for developmental biologists who are interested in craniofacial morphogenesis.

  140. The suture provides a niche for mesenchymal stem cells of craniofacial bones

    Zhao, Hu; Feng, Jifan; Ho, Thach-Vu; Grimes, Weston; Urata, Mark; Chai, Yang. Nature Cell Biology. vol. 17(4), 386–396. April 2015.

    Bone tissue undergoes constant turnover supported by stem cells. Recent studies showed that perivascular mesenchymal stem cells (MSCs) contribute to the turnover of long bones. Craniofacial bones are flat bones derived from a different embryonic origin than the long bones. The identity and regulating niche for craniofacial-bone MSCs remain unknown. Here, we identify Gli1+ cells within the suture mesenchyme as the main MSC population for craniofacial bones. They are not associated with vasculature, give rise to all craniofacial bones in the adult and are activated during injury repair. Gli1+ cells are typical MSCs in vitro. Ablation of Gli1+ cells leads to craniosynostosis and arrest of skull growth, indicating that these cells are an indispensable stem cell population. Twist1(+/-) mice with craniosynostosis show reduced Gli1+ MSCs in sutures, suggesting that craniosynostosis may result from diminished suture stem cells. Our study indicates that craniofacial sutures provide a unique niche for MSCs for craniofacial bone homeostasis and repair.

  141. A genome-wide study of inherited deletions identified two regions associated with nonsyndromic isolated oral clefts

    Younkin, Samuel G.; Scharpf, Robert B.; Schwender, Holger; Parker, Margaret M.; Scott, Alan F.; Marazita, Mary L.; Beaty, Terri H.; Ruczinski, Ingo. Birth Defects Research. Part A, Clinical and Molecular Teratology. vol. 103(4), 276–283. April 2015.

    BACKGROUND: DNA copy number variants play an important part in the development of common birth defects such as oral clefts. Individual patients with multiple birth defects (including oral clefts) have been shown to carry small and large chromosomal deletions. METHODS: We investigated the role of polymorphic copy number deletions by comparing transmission rates of deletions from parents to offspring in case-parent trios of European ancestry ascertained through a cleft proband with trios ascertained through a normal offspring. DNA copy numbers in trios were called using the joint hidden Markov model in the freely available PennCNV software. All statistical analyses were performed using Bioconductor tools in the open source environment R. RESULTS: We identified a 67 kb region in the gene MGAM on chromosome 7q34, and a 206 kb region overlapping genes ADAM3A and ADAM5 on chromosome 8p11, where deletions are more frequently transmitted to cleft offspring than control offspring. CONCLUSIONS: These genes or nearby regulatory elements may be involved in the etiology of oral clefts.

  142. BMP-SHH signaling network controls epithelial stem cell fate via regulation of its niche in the developing tooth

    Li, Jingyuan; Feng, Jifan; Liu, Yang; Ho, Thach-Vu; Grimes, Weston; Ho, Hoang Anh; Park, Shery; Wang, Songlin; Chai, Yang. Developmental Cell. vol. 33(2), 125–135. April 2015.

    During embryogenesis, ectodermal stem cells adopt different fates and form diverse ectodermal organs, such as teeth, hair follicles, mammary glands, and salivary glands. Interestingly, these ectodermal organs differ in their tissue homeostasis, which leads to differential abilities for continuous growth postnatally. Mouse molars lose the ability to grow continuously, whereas incisors retain this ability. In this study, we found that a BMP-Smad4-SHH-Gli1 signaling network may provide a niche supporting transient Sox2+ dental epithelial stem cells in mouse molars. This mechanism also plays a role in continuously growing mouse incisors. The differential fate of epithelial stem cells in mouse molars and incisors is controlled by this BMP/SHH signaling network, which partially accounts for the different postnatal growth potential of molars and incisors. Collectively, our study highlights the importance of crosstalk between two signaling pathways, BMP and SHH, in regulating the fate of epithelial stem cells during organogenesis.

  143. Expanding the genetic and phenotypic spectrum of popliteal pterygium disorders

    Leslie, Elizabeth J.; O’Sullivan, James; Cunningham, Michael L.; Singh, Ankur; Goudy, Steven L.; Ababneh, Faroug; Alsubaie, Lamia; Ch’ng, Gaik-Siew; van der Laar, Ingrid M. B. H.; Hoogeboom, A. Jeannette M.; Dunnwald, Martine; Kapoor, Seema; Jiramongkolchai, Pawina; Standley, Jennifer; Manak, J. Robert; Murray, Jeffrey C.; Dixon, Michael J.. American Journal of Medical Genetics. Part A. vol. 167A(3), 545–552. March 2015.

    The popliteal pterygia syndromes are a distinct subset of the hundreds of Mendelian orofacial clefting syndromes. Popliteal pterygia syndromes have considerable variability in severity and in the associated phenotypic features but are all characterized by cutaneous webbing across one or more major joints, cleft lip and/or palate, syndactyly, and genital malformations. Heterozygous mutations in IRF6 cause popliteal pterygium syndrome (PPS) while homozygous mutations in RIPK4 or CHUK (IKKA) cause the more severe Bartsocas-Papas syndrome (BPS) and Cocoon syndrome, respectively. In this study, we report mutations in six pedigrees with children affected with PPS or BPS. Using a combination of Sanger and exome sequencing, we report the first case of an autosomal recessive popliteal pterygium syndrome caused by homozygous mutation of IRF6 and the first case of uniparental disomy of chromosome 21 leading to a recessive disorder. We also demonstrate that mutations in RIPK4 can cause features with a range of severity along the PPS-BPS spectrum and that mutations in IKKA can cause a range of features along the BPS-Cocoon spectrum. Our findings have clinical implications for genetic counseling of families with pterygia syndromes and further implicate IRF6, RIPK4, and CHUK (IKKA) in potentially interconnected pathways governing epidermal and craniofacial development.

  144. Prenatal sex hormones, digit ratio, and face shape in adult males

    Weinberg, S. M.; Parsons, T. E.; Raffensperger, Z. D.; Marazita, M. L.. Orthodontics & Craniofacial Research. vol. 18(1), 21–26. February 2015.

    OBJECTIVES: Several reports have demonstrated a relationship between second to fourth digit ratio (2D:4D) and facial shape, suggesting that prenatal sex hormones play a role in the development of the craniofacial complex. Using 3D surface imaging and geometric morphometrics, we test the hypothesis that decreased digit ratio (indicative of increased prenatal androgen exposure) is associated with a more masculine facial phenotype. METHODS: 3D facial surface images and digit measures were collected on a sample of 151 adult males. Facial landmarks collected from the images were aligned by Procrustes superimposition and the resulting shape coordinates regressed on 2D:4D. Variations in facial shape related to 2D:4D were visualized with deformable surface warps. RESULTS: A significant statistical relationship was observed between facial shape variation and 2D:4D (p = 0.0084). Lower 2D:4D ratio in adult males was associated with increased facial width relative to height, increased mandibular prognathism, greater nasal projection, and increased upper and lower lip projection. CONCLUSIONS: A statistical relationship between 2D:4D and facial shape in adult males was observed. Faces tended to look more masculine as 2D:4D decreased, suggesting a biologically plausible link between prenatal androgen exposure and the development of male facial characteristics.

  145. The UCSC Genome Browser database: 2015 update

    Rosenbloom, Kate R.; Armstrong, Joel; Barber, Galt P.; Casper, Jonathan; Clawson, Hiram; Diekhans, Mark; Dreszer, Timothy R.; Fujita, Pauline A.; Guruvadoo, Luvina; Haeussler, Maximilian; Harte, Rachel A.; Heitner, Steve; Hickey, Glenn; Hinrichs, Angie S.; Hubley, Robert; Karolchik, Donna; Learned, Katrina; Lee, Brian T.; Li, Chin H.; Miga, Karen H.; Nguyen, Ngan; Paten, Benedict; Raney, Brian J.; Smit, Arian F. A.; Speir, Matthew L.; Zweig, Ann S.; Haussler, David; Kuhn, Robert M.; Kent, W. James. Nucleic Acids Research. vol. 43(Database issue), D670–681. January 2015.

    Launched in 2001 to showcase the draft human genome assembly, the UCSC Genome Browser database ( and associated tools continue to grow, providing a comprehensive resource of genome assemblies and annotations to scientists and students worldwide. Highlights of the past year include the release of a browser for the first new human genome reference assembly in 4 years in December 2013 (GRCh38, UCSC hg38), a watershed comparative genomics annotation (100-species multiple alignment and conservation) and a novel distribution mechanism for the browser (GBiB: Genome Browser in a Box). We created browsers for new species (Chinese hamster, elephant shark, minke whale), ’mined the web’ for DNA sequences and expanded the browser display with stacked color graphs and region highlighting. As our user community increasingly adopts the UCSC track hub and assembly hub representations for sharing large-scale genomic annotation data sets and genome sequencing projects, our menu of public data hubs has tripled.

  146. BCL11B expression in intramembranous osteogenesis during murine craniofacial suture development

    Holmes, Greg; van Bakel, Harm; Zhou, Xueyan; Losic, Bojan; Jabs, Ethylin Wang. Gene expression patterns: GEP. vol. 17(1), 16–25. January 2015.

    Sutures, where neighboring craniofacial bones are separated by undifferentiated mesenchyme, are major growth sites during craniofacial development. Pathologic fusion of bones within sutures occurs in a wide variety of craniosynostosis conditions and can result in dysmorphic craniofacial growth and secondary neurologic deficits. Our knowledge of the genes involved in suture formation is poor. Here we describe the novel expression pattern of the BCL11B transcription factor protein during murine embryonic craniofacial bone formation. We examined BCL11B protein expression at E14.5, E16.5, and E18.5 in 14 major craniofacial sutures of C57BL/6J mice. We found BCL11B expression to be associated with all intramembranous craniofacial bones examined. The most striking aspects of BCL11B expression were its high levels in suture mesenchyme and increasingly complementary expression with RUNX2 in differentiating osteoblasts during development. BCL11B was also expressed in mesenchyme at the non-sutural edges of intramembranous bones. No expression was seen in osteoblasts involved in endochondral ossification of the cartilaginous cranial base. BCL11B is expressed to potentially regulate the transition of mesenchymal differentiation and suture formation within craniofacial intramembranous bone.

  147. Tfap2a-dependent changes in mouse facial morphology result in clefting that can be ameliorated by a reduction in Fgf8 gene dosage

    Green, Rebecca M.; Feng, Weiguo; Phang, Tzulip; Fish, Jennifer L.; Li, Hong; Spritz, Richard A.; Marcucio, Ralph S.; Hooper, Joan; Jamniczky, Heather; Hallgrímsson, Benedikt; Williams, Trevor. Disease Models & Mechanisms. vol. 8(1), 31–43. January 2015.

    Failure of facial prominence fusion causes cleft lip and palate (CL/P), a common human birth defect. Several potential mechanisms can be envisioned that would result in CL/P, including failure of prominence growth and/or alignment as well as a failure of fusion of the juxtaposed epithelial seams. Here, using geometric morphometrics, we analyzed facial outgrowth and shape change over time in a novel mouse model exhibiting fully penetrant bilateral CL/P. This robust model is based upon mutations in Tfap2a, the gene encoding transcription factor AP-2α, which has been implicated in both syndromic and non-syndromic human CL/P. Our findings indicate that aberrant morphology and subsequent misalignment of the facial prominences underlies the inability of the mutant prominences to fuse. Exencephaly also occured in some of the Tfap2a mutants and we observed additional morphometric differences that indicate an influence of neural tube closure defects on facial shape. Molecular analysis of the CL/P model indicates that Fgf signaling is misregulated in the face, and that reducing Fgf8 gene dosage can attenuate the clefting pathology by generating compensatory changes. Furthermore, mutations in either Tfap2a or Fgf8 increase variance in facial shape, but the combination of these mutations restores variance to normal levels. The alterations in variance provide a potential mechanistic link between clefting and the evolution and diversity of facial morphology. Overall, our findings suggest that CL/P can result from small gene-expression changes that alter the shape of the facial prominences and uncouple their coordinated morphogenesis, which is necessary for normal fusion.

  148. Automated face extraction and normalization of 3D Mesh Data

    Wu, Jia; Tse, Raymond; Shapiro, Linda G.. Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference. vol. 2014, 750–753. 2014.

    3D stereophotography is rapidly being adopted by medical researchers for analysis of facial forms and features. An essential step for many applications using 3D face data is to first crop the head and face from the raw images. The goal of this paper is to develop a reliable automatic methodology for extracting the face from raw data with texture acquired from a stereo imaging system, based on the medical researchers’ specific requirements. We present an automated process, including eye and nose estimation, face detection, Procrustes analysis and final noise removal to crop out the faces and normalize them. The proposed method shows very reliable results on several datasets, including a normal adult dataset and a very challenging dataset consisting of infants with cleft lip and palate.

  149. Quantification of skull deformity for craniofacial research

    Lam, Irma; Cunningham, Michael; Birgfeld, Craig; Speltz, Matthew; Shapiro, Linda. Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference. vol. 2014, 758–761. 2014.

    Craniosynostosis, a disorder in which one or more fibrous joints of the skull fuse prematurely, causes skull malformation and may be associated with increased intracranial pressure and developmental delays. In order to perform medical research studies that relate phenotypic abnormalities to outcomes such as cognitive ability or results of surgery, biomedical researchers need an automated methodology for quantifying the degree of abnormality of the disorder. This paper addresses that need by proposing a set of features derived from CT scans of the skull that can be used for this purpose. A thorough set of experiments is used to evaluate the features as compared to two human craniofacial experts in a ranking evaluation.

  150. Evolution of the miR199-214 cluster and vertebrate skeletal development

    Desvignes, Thomas; Contreras, Adam; Postlethwait, John H.. RNA biology. vol. 11(4), 281–294. 2014.

    MicroRNA (miRs) are short non-coding RNAs that fine-tune the regulation of gene expression to coordinate a wide range of biological processes. MicroRNAs are transcribed from miR genes and primary miR transcripts are processed to approximately 22 nucleotide single strand mature forms that function as repressors of transcript translation when bound to the 3’UTR of protein coding transcripts in association with the RISC. Because of their role in the regulation of gene expression, miRs are essential players in development by acting on cell fate determination and progression toward cell differentiation. The miR199 and miR214 genes occupy an intronic cluster located on the opposite strand of the Dynamin3 gene. These miRNAs play major roles in a broad variety of developmental processes and diseases, including skeletal development and several types of cancer. In the work reported here, we first deciphered the origin of the miR199 and miR214 families by following evolution of miR paralogs and their host Dynamin paralogs. We then examined the expression patterns of miR199 and miR214 in developing zebrafish embryos and demonstrated their regulation through a common primary transcript. Results suggest an evolutionarily conserved regulation across vertebrate lineages. Our expression study showed predominant expression patterns for both miR in tissues surrounding developing craniofacial skeletal elements consistent with expression data in mouse and human, thus indicating a conserved role of miR199 and miR214 in vertebrate skeletogenesis.

  151. Katanin p80 regulates human cortical development by limiting centriole and cilia number

    Hu, Wen F.; Pomp, Oz; Ben-Omran, Tawfeg; Kodani, Andrew; Henke, Katrin; Mochida, Ganeshwaran H.; Yu, Timothy W.; Woodworth, Mollie B.; Bonnard, Carine; Raj, Grace Selva; Tan, Thong Teck; Hamamy, Hanan; Masri, Amira; Shboul, Mohammad; Al Saffar, Muna; Partlow, Jennifer N.; Al-Dosari, Mohammed; Alazami, Anas; Alowain, Mohammed; Alkuraya, Fowzan S.; Reiter, Jeremy F.; Harris, Matthew P.; Reversade, Bruno; Walsh, Christopher A.. Neuron. vol. 84(6), 1240–1257. December 2014.

    Katanin is a microtubule-severing complex whose catalytic activities are well characterized, but whose in vivo functions are incompletely understood. Human mutations in KATNB1, which encodes the noncatalytic regulatory p80 subunit of katanin, cause severe microlissencephaly. Loss of Katnb1 in mice confirms essential roles in neurogenesis and cell survival, while loss of zebrafish katnb1 reveals specific roles for katnin p80 in early and late developmental stages. Surprisingly, Katnb1 null mutant mouse embryos display hallmarks of aberrant Sonic hedgehog signaling, including holoprosencephaly. KATNB1-deficient human cells show defective proliferation and spindle structure, while Katnb1 null fibroblasts also demonstrate a remarkable excess of centrioles, with supernumerary cilia but deficient Hedgehog signaling. Our results reveal unexpected functions for KATNB1 in regulating overall centriole, mother centriole, and cilia number, and as an essential gene for normal Hedgehog signaling during neocortical development.

  152. Let’s face it–complex traits are just not that simple

    Hallgrimsson, Benedikt; Mio, Washington; Marcucio, Ralph S.; Spritz, Richard. PLoS genetics. vol. 10(11), e1004724. November 2014.

  153. HAND2 targets define a network of transcriptional regulators that compartmentalize the early limb bud mesenchyme

    Osterwalder, Marco; Speziale, Dario; Shoukry, Malak; Mohan, Rajiv; Ivanek, Robert; Kohler, Manuel; Beisel, Christian; Wen, Xiaohui; Scales, Suzie J.; Christoffels, Vincent M.; Visel, Axel; Lopez-Rios, Javier; Zeller, Rolf. Developmental Cell. vol. 31(3), 345–357. November 2014.

    The genetic networks that govern vertebrate development are well studied, but how the interactions of trans-acting factors with cis-regulatory modules (CRMs) are integrated into spatiotemporal regulation of gene expression is not clear. The transcriptional regulator HAND2 is required during limb, heart, and branchial arch development. Here, we identify the genomic regions enriched in HAND2 chromatin complexes from mouse embryos and limb buds. Then we analyze the HAND2 target CRMs in the genomic landscapes encoding transcriptional regulators required in early limb buds. HAND2 controls the expression of genes functioning in the proximal limb bud and orchestrates the establishment of anterior and posterior polarity of the nascent limb bud mesenchyme by impacting Gli3 and Tbx3 expression. TBX3 is required downstream of HAND2 to refine the posterior Gli3 expression boundary. Our analysis uncovers the transcriptional circuits that function in establishing distinct mesenchymal compartments downstream of HAND2 and upstream of SHH signaling.

  154. Principles of regulatory information conservation between mouse and human

    Cheng, Yong; Ma, Zhihai; Kim, Bong-Hyun; Wu, Weisheng; Cayting, Philip; Boyle, Alan P.; Sundaram, Vasavi; Xing, Xiaoyun; Dogan, Nergiz; Li, Jingjing; Euskirchen, Ghia; Lin, Shin; Lin, Yiing; Visel, Axel; Kawli, Trupti; Yang, Xinqiong; Patacsil, Dorrelyn; Keller, Cheryl A.; Giardine, Belinda; mouse ENCODE Consortium; Kundaje, Anshul; Wang, Ting; Pennacchio, Len A.; Weng, Zhiping; Hardison, Ross C.; Snyder, Michael P.. Nature. vol. 515(7527), 371–375. November 2014.

    To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human-mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here we deduce several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF-occupied sequences. However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and with genomic location: binding at promoters is more highly conserved than binding at distal elements. Notably, occupancy-conserved TF-occupied sequences tend to be pleiotropic; they function in several tissues and also co-associate with many TFs. Single nucleotide variants at sites with potential regulatory functions are enriched in occupancy-conserved TF-occupied sequences.

  155. Airway branching has conserved needs for local parasympathetic innervation but not neurotransmission

    Bower, Danielle V.; Lee, Hyung-Kook; Lansford, Rusty; Zinn, Kai; Warburton, David; Fraser, Scott E.; Jesudason, Edwin C.. BMC biology. vol. 12, 92. November 2014.

    BACKGROUND: Parasympathetic signaling has been inferred to regulate epithelial branching as well as organ regeneration and tumor development. However, the relative contribution of local nerve contact versus secreted signals remains unclear. Here, we show a conserved (vertebrates to invertebrates) requirement for intact local nerves in airway branching, persisting even when cholinergic neurotransmission is blocked. RESULTS: In the vertebrate lung, deleting enhanced green fluorescent protein (eGFP)-labeled intrinsic neurons using a two-photon laser leaves adjacent cells intact, but abolishes branching. Branching is unaffected by similar laser power delivered to the immediately adjacent non-neural mesodermal tissue, by blocking cholinergic receptors or by blocking synaptic transmission with botulinum toxin A. Because adjacent vasculature and epithelial proliferation also contribute to branching in the vertebrate lung, the direct dependence on nerves for airway branching was tested by deleting neurons in Drosophila embryos. A specific deletion of neurons in the Drosophila embryo by driving cell-autonomous RicinA under the pan-neuronal elav enhancer perturbed Drosophila airway development. This system confirmed that even in the absence of a vasculature or epithelial proliferation, airway branching is still disrupted by neural lesioning. CONCLUSIONS: Together, this shows that airway morphogenesis requires local innervation in vertebrates and invertebrates, yet neurotransmission is dispensable. The need for innervation persists in the fly, wherein adjacent vasculature and epithelial proliferation are absent. Our novel, targeted laser ablation technique permitted the local function of parasympathetic innervation to be distinguished from neurotransmission.

  156. Functional analysis of SPECC1L in craniofacial development and oblique facial cleft pathogenesis

    Gfrerer, Lisa; Shubinets, Valeriy; Hoyos, Tatiana; Kong, Yawei; Nguyen, Christina; Pietschmann, Peter; Morton, Cynthia C.; Maas, Richard L.; Liao, Eric C.. Plastic and Reconstructive Surgery. vol. 134(4), 748–759. October 2014.

    BACKGROUND: Oblique facial clefts, also known as Tessier clefts, are severe orofacial clefts, the genetic basis of which is poorly understood. Human genetics studies revealed that disruption in SPECC1L resulted in oblique facial clefts, demonstrating that oblique facial cleft malformation has a genetic basis. An important step toward innovation in treatment of oblique facial clefts would be improved understanding of its genetic pathogenesis. The authors exploit the zebrafish model to elucidate the function of SPECC1L by studying its homolog, specc1lb. METHODS: Gene and protein expression analysis was carried out by reverse-transcriptase polymerase chain reaction and immunohistochemistry staining. Morpholino knockdown, mRNA rescue, lineage tracing and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assays were performed for functional analysis. RESULTS: Expression of specc1lb was detected in epithelia juxtaposed to chondrocytes. Knockdown of specc1lb resulted in bilateral clefts between median and lateral elements of the ethmoid plate, structures analogous to the frontonasal process and the paired maxillary processes. Lineage tracing analysis revealed that cranial neural crest cells contributing to the frontonasal prominence failed to integrate with the maxillary prominence populations. Cells contributing to lower jaw structures were able to migrate to their destined pharyngeal segment but failed to converge to form mandibular elements. CONCLUSIONS: These results demonstrate that specc1lb is required for integration of frontonasal and maxillary elements and convergence of mandibular prominences. The authors confirm the role of SPECC1L in orofacial cleft pathogenesis in the first animal model of Tessier cleft, providing morphogenetic insight into the mechanisms of normal craniofacial development and oblique facial cleft pathogenesis.

  157. Craniofacial morphometric analysis of individuals with X-linked hypohidrotic ectodermal dysplasia

    Goodwin, Alice F.; Larson, Jacinda R.; Jones, Kyle B.; Liberton, Denise K.; Landan, Maya; Wang, Zhifeng; Boekelheide, Anne; Langham, Margaret; Mushegyan, Vagan; Oberoi, Snehlata; Brao, Rosalie; Wen, Timothy; Johnson, Ramsey; Huttner, Kenneth; Grange, Dorothy K.; Spritz, Richard A.; Hallgrímsson, Benedikt; Jheon, Andrew H.; Klein, Ophir D.. Molecular Genetics & Genomic Medicine. vol. 2(5), 422–429. September 2014.

    Hypohidrotic ectodermal dysplasia (HED) is the most prevalent type of ectodermal dysplasia (ED). ED is an umbrella term for a group of syndromes characterized by missing or malformed ectodermal structures, including skin, hair, sweat glands, and teeth. The X-linked recessive (XL), autosomal recessive (AR), and autosomal dominant (AD) types of HED are caused by mutations in the genes encoding ectodysplasin (EDA1), EDA receptor (EDAR), or EDAR-associated death domain (EDARADD). Patients with HED have a distinctive facial appearance, yet a quantitative analysis of the HED craniofacial phenotype using advanced three-dimensional (3D) technologies has not been reported. In this study, we characterized craniofacial morphology in subjects with X-linked hypohidrotic ectodermal dysplasia (XLHED) by use of 3D imaging and geometric morphometrics (GM), a technique that uses defined landmarks to quantify size and shape in complex craniofacial morphologies. We found that the XLHED craniofacial phenotype differed significantly from controls. Patients had a smaller and shorter face with a proportionally longer chin and midface, prominent midfacial hypoplasia, a more protrusive chin and mandible, a narrower and more pointed nose, shorter philtrum, a narrower mouth, and a fuller and more rounded lower lip. Our findings refine the phenotype of XLHED and may be useful both for clinical diagnosis of XLHED and to extend understanding of the role of EDA in craniofacial development.

  158. Tissue-specific RNA expression marks distant-acting developmental enhancers

    Wu, Han; Nord, Alex S.; Akiyama, Jennifer A.; Shoukry, Malak; Afzal, Veena; Rubin, Edward M.; Pennacchio, Len A.; Visel, Axel. PLoS genetics. vol. 10(9), e1004610. September 2014.

    Short non-coding transcripts can be transcribed from distant-acting transcriptional enhancer loci, but the prevalence of such enhancer RNAs (eRNAs) within the transcriptome, and the association of eRNA expression with tissue-specific enhancer activity in vivo remain poorly understood. Here, we investigated the expression dynamics of tissue-specific non-coding RNAs in embryonic mouse tissues via deep RNA sequencing. Overall, approximately 80% of validated in vivo enhancers show tissue-specific RNA expression that correlates with tissue-specific enhancer activity. Globally, we identified thousands of tissue-specifically transcribed non-coding regions (TSTRs) displaying various genomic hallmarks of bona fide enhancers. In transgenic mouse reporter assays, over half of tested TSTRs functioned as enhancers with reproducible activity in the predicted tissue. Together, our results demonstrate that tissue-specific eRNA expression is a common feature of in vivo enhancers, as well as a major source of extragenic transcription, and that eRNA expression signatures can be used to predict tissue-specific enhancers independent of known epigenomic enhancer marks.

  159. Expanding the annotation of zebrafish microRNAs based on small RNA sequencing

    Desvignes, Thomas; Beam, Michael J.; Batzel, Peter; Sydes, Jason; Postlethwait, John H.. Gene. vol. 546(2), 386–389. August 2014.

    MicroRNAs (miRs) are short non-coding RNAs that fine-tune the regulation of gene expression to coordinate a wide range of biological processes. Because of their role in the regulation of gene expression, miRs are essential players in development by acting on cell fate determination and progression towards cell differentiation and are increasingly relevant to human health and disease. Although the zebrafish Danio rerio is a major model for studies of development, genetics, physiology, evolution, and human biology, the annotation of zebrafish miR-producing genes remains limited. In the present work, we report deep sequencing data of zebrafish small RNAs from brain, heart, testis, and ovary. Results provide evidence for the expression of 56 un-annotated mir genes and 248 un-annotated mature strands, increasing the number of zebrafish mir genes over those already deposited in miRBase by 16% and the number of mature sequences by 63%. We also describe the existence of three pairs of mirror-mir genes and two mirtron genes, genetic features previously undescribed in non-mammalian vertebrates. This report provides information that substantially increases our knowledge of the zebrafish miRNome and will benefit the entire miR community.

  160. Identification of novel craniofacial regulatory domains located far upstream of SOX9 and disrupted in Pierre Robin sequence

    Gordon, Christopher T.; Attanasio, Catia; Bhatia, Shipra; Benko, Sabina; Ansari, Morad; Tan, Tiong Y.; Munnich, Arnold; Pennacchio, Len A.; Abadie, Véronique; Temple, I. Karen; Goldenberg, Alice; van Heyningen, Veronica; Amiel, Jeanne; FitzPatrick, David; Kleinjan, Dirk A.; Visel, Axel; Lyonnet, Stanislas. Human Mutation. vol. 35(8), 1011–1020. August 2014.

    Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions, and duplications within a ∼2 Mb region upstream of SOX9 can recapitulate the CD-DSD phenotype fully or partially, suggesting the existence of an unusually large cis-regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at ∼1.2-1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harboring candidate craniofacial regulatory elements. In parallel, ChIP-Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple noncoding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS.

  161. Inferring rare disease risk variants based on exact probabilities of sharing by multiple affected relatives

    Bureau, Alexandre; Younkin, Samuel G.; Parker, Margaret M.; Bailey-Wilson, Joan E.; Marazita, Mary L.; Murray, Jeffrey C.; Mangold, Elisabeth; Albacha-Hejazi, Hasan; Beaty, Terri H.; Ruczinski, Ingo. Bioinformatics (Oxford, England). vol. 30(15), 2189–2196. August 2014.

    MOTIVATION: Family-based designs are regaining popularity for genomic sequencing studies because they provide a way to test cosegregation with disease of variants that are too rare in the population to be tested individually in a conventional case-control study. RESULTS: Where only a few affected subjects per family are sequenced, the probability that any variant would be shared by all affected relatives-given it occurred in any one family member-provides evidence against the null hypothesis of a complete absence of linkage and association. A P-value can be obtained as the sum of the probabilities of sharing events as (or more) extreme in one or more families. We generalize an existing closed-form expression for exact sharing probabilities to more than two relatives per family. When pedigree founders are related, we show that an approximation of sharing probabilities based on empirical estimates of kinship among founders obtained from genome-wide marker data is accurate for low levels of kinship. We also propose a more generally applicable approach based on Monte Carlo simulations. We applied this method to a study of 55 multiplex families with apparent non-syndromic forms of oral clefts from four distinct populations, with whole exome sequences available for two or three affected members per family. The rare single nucleotide variant rs149253049 in ADAMTS9 shared by affected relatives in three Indian families achieved significance after correcting for multiple comparisons ([Formula: see text]). AVAILABILITY AND IMPLEMENTATION: Source code and binaries of the R package RVsharing are freely available for download at CONTACT: or SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  162. Learning to Rank the Severity of Unrepaired Cleft Lip Nasal Deformity on 3D Mesh Data

    Wu, Jia; Tse, Raymond; Shapiro, Linda G.. Proceedings of the ... IAPR International Conference on Pattern Recognition. International Conference on Pattern Recognition. vol. 2014, 460–464. August 2014.

    Cleft lip is a birth defect that results in deformity of the upper lip and nose. Its severity is widely variable and the results of treatment are influenced by the initial deformity. Objective assessment of severity would help to guide prognosis and treatment. However, most assessments are subjective. The purpose of this study is to develop and test quantitative computer-based methods of measuring cleft lip severity. In this paper, a grid-patch based measurement of symmetry is introduced, with which a computer program learns to rank the severity of cleft lip on 3D meshes of human infant faces. Three computer-based methods to define the midfacial reference plane were compared to two manual methods. Four different symmetry features were calculated based upon these reference planes, and evaluated. The result shows that the rankings predicted by the proposed features were highly correlated with the ranking orders provided by experts that were used as the ground truth.

  163. ToppMiR: ranking microRNAs and their mRNA targets based on biological functions and context

    Wu, Chao; Bardes, Eric E.; Jegga, Anil G.; Aronow, Bruce J.. Nucleic Acids Research. vol. 42(Web Server issue), W107–113. July 2014.

    Identifying functionally significant microRNAs (miRs) and their correspondingly most important messenger RNA targets (mRNAs) in specific biological contexts is a critical task to improve our understanding of molecular mechanisms underlying organismal development, physiology and disease. However, current miR-mRNA target prediction platforms rank miR targets based on estimated strength of physical interactions and lack the ability to rank interactants as a function of their potential to impact a given biological system. To address this, we have developed ToppMiR (, a web-based analytical workbench that allows miRs and mRNAs to be co-analyzed via biologically centered approaches in which gene function associated annotations are used to train a machine learning-based analysis engine. ToppMiR learns about biological contexts based on gene associated information from expression data or from a user-specified set of genes that relate to context-relevant knowledge or hypotheses. Within the biological framework established by the genes in the training set, its associated information content is then used to calculate a features association matrix composed of biological functions, protein interactions and other features. This scoring matrix is then used to jointly rank both the test/candidate miRs and mRNAs. Results of these analyses are provided as downloadable tables or network file formats usable in Cytoscape.

  164. Facial morphometrics of children with non-syndromic orofacial clefts in Tanzania

    Manyama, Mange; Larson, Jacinda R.; Liberton, Denise K.; Rolian, Campbell; Smith, Francis J.; Kimwaga, Emmanuel; Gilyoma, Japhet; Lukowiak, Kenneth D.; Spritz, Richard A.; Hallgrimsson, Benedikt. BMC oral health. vol. 14, 93. July 2014.

    BACKGROUND: Orofacial clefts (cleft lip/palate; CL/P) are among the most common congenital anomalies, with prevalence that varies among different ethnic groups. Craniofacial shape differences between individuals with CL/P and healthy controls have been widely reported in non-African populations. Knowledge of craniofacial shape among individuals with non-syndromic CL/P in African populations will provide further understanding of the ethnic and phenotypic variation present in non-syndromic orofacial clefts. METHODS: A descriptive cross-sectional study was carried out at Bugando Medical Centre, Tanzania, comparing individuals with unrepaired non-syndromic CL/P and normal individuals without orofacial clefts. Three-dimensional (3D) facial surfaces were captured using a non-invasive 3D camera. The corresponding 3D coordinates for 26 soft tissue landmarks were used to characterize facial shape. Facial shape variation within and between groups, based on Procrustes superimposed data, was studied using geometric morphometric methods. RESULTS: Facial shape of children with cleft lip differed significantly from the control group, beyond the cleft itself. The CL/P group exhibited increased nasal and mouth width, increased interorbital distance, and more prognathic premaxillary region. Within the CL/P group, PCA showed that facial shape variation is associated with facial height, nasal cavity width, interorbital distance and midfacial prognathism. The isolated cleft lip (CL) and combined cleft lip and palate (CLP) groups did not differ significantly from one another (Procrustes distance = 0.0416, p = 0.50). Procrustes distance permutation tests within the CL/P group showed a significant shape difference between unilateral clefts and bilateral clefts (Procrustes distance = 0.0728, p = 0.0001). Our findings indicate the morphological variation is similar to those of studies of CL/P patients and their unaffected close relatives in non-African populations. CONCLUSION: The mean facial shape in African children with non-syndromic CL/P differs significantly from children without orofacial clefts. The main differences involve interorbital width, facial width and midface prognathism. The axes of facial shape differences we observed are similar to the patterns seen in Caucasian populations, despite apparent differences in cleft prevalence and cleft type distribution. Similar facial morphology in individuals with CL/P in African and Caucasian populations suggests a similar aetiology.

  165. A gene expression atlas of early craniofacial development

    Brunskill, Eric W.; Potter, Andrew S.; Distasio, Andrew; Dexheimer, Phillip; Plassard, Andrew; Aronow, Bruce J.; Potter, S. Steven. Developmental Biology. vol. 391(2), 133–146. July 2014.

    We present a gene expression atlas of early mouse craniofacial development. Laser capture microdissection (LCM) was used to isolate cells from the principal critical microregions, whose development, differentiation and signaling interactions are responsible for the construction of the mammalian face. At E8.5, as migrating neural crest cells begin to exit the neural fold/epidermal ectoderm boundary, we examined the cranial mesenchyme, composed of mixed neural crest and paraxial mesoderm cells, as well as cells from adjacent neuroepithelium. At E9.5 cells from the cranial mesenchyme, overlying olfactory placode/epidermal ectoderm, and underlying neuroepithelium, as well as the emerging mandibular and maxillary arches were sampled. At E10.5, as the facial prominences form, cells from the medial and lateral prominences, the olfactory pit, multiple discrete regions of underlying neuroepithelium, the mandibular and maxillary arches, including both their mesenchymal and ectodermal components, as well as Rathke’s pouch, were similarly sampled and profiled using both microarray and RNA-seq technologies. Further, we performed single cell studies to better define the gene expression states of the early E8.5 pioneer neural crest cells and paraxial mesoderm. Taken together, and analyzable by a variety of biological network approaches, these data provide a complementing and cross validating resource capable of fueling discovery of novel compartment specific markers and signatures whose combinatorial interactions of transcription factors and growth factors/receptors are responsible for providing the master genetic blueprint for craniofacial development.

  166. Tissue-specific SMARCA4 binding at active and repressed regulatory elements during embryogenesis

    Attanasio, Catia; Nord, Alex S.; Zhu, Yiwen; Blow, Matthew J.; Biddie, Simon C.; Mendenhall, Eric M.; Dixon, Jesse; Wright, Crystal; Hosseini, Roya; Akiyama, Jennifer A.; Holt, Amy; Plajzer-Frick, Ingrid; Shoukry, Malak; Afzal, Veena; Ren, Bing; Bernstein, Bradley E.; Rubin, Edward M.; Visel, Axel; Pennacchio, Len A.. Genome Research. vol. 24(6), 920–929. June 2014.

    The SMARCA4 (also known as BRG1 in humans) chromatin remodeling factor is critical for establishing lineage-specific chromatin states during early mammalian development. However, the role of SMARCA4 in tissue-specific gene regulation during embryogenesis remains poorly defined. To investigate the genome-wide binding landscape of SMARCA4 in differentiating tissues, we engineered a Smarca4(FLAG) knock-in mouse line. Using ChIP-seq, we identified ∼51,000 SMARCA4-associated regions across six embryonic mouse tissues (forebrain, hindbrain, neural tube, heart, limb, and face) at mid-gestation (E11.5). The majority of these regions was distal from promoters and showed dynamic occupancy, with most distal SMARCA4 sites (73%) confined to a single or limited subset of tissues. To further characterize these regions, we profiled active and repressive histone marks in the same tissues and examined the intersection of informative chromatin states and SMARCA4 binding. This revealed distinct classes of distal SMARCA4-associated elements characterized by activating and repressive chromatin signatures that were associated with tissue-specific up- or down-regulation of gene expression and relevant active/repressed biological pathways. We further demonstrate the predicted active regulatory properties of SMARCA4-associated elements by retrospective analysis of tissue-specific enhancers and direct testing of SMARCA4-bound regions in transgenic mouse assays. Our results indicate a dual active/repressive function of SMARCA4 at distal regulatory sequences in vivo and support its role in tissue-specific gene regulation during embryonic development.

  167. Classifying Craniosynostosis with a 3D Projection-Based Feature Extraction System

    Lam, Irma; Cunningham, Michael; Speltz, Matthew; Shapiro, Linda. Proceedings. IEEE International Symposium on Computer-Based Medical Systems. vol. 2014, 215–220. May 2014.

    Craniosynostosis, a disorder in which one or more fibrous joints of the skull fuse prematurely, causes skull deformity and is associated with increased intracranial pressure and developmental delays. Although clinicians can easily diagnose craniosynostosis and can classify its type, being able to quantify the condition is an important problem in craniofacial research. While several papers have attempted this quantification through statistical models, the methods have not been intuitive to biomedical researchers and clinicians who want to use them. The goal of this work was to develop a general platform upon which new quantification measures could be developed and tested. The features reported in this paper were developed as basic shape measures, both single-valued and vector-valued, that are extracted from a single plane projection of the 3D skull. This technique allows us to process images that would otherwise be eliminated in previous systems due to poor resolution, noise or imperfections on their CT scans. We test our new features on classification tasks and also compare their performance to previous research. In spite of its simplicity, the classification accuracy of our new features is significantly higher than previous results on head CT scan data from the same research studies.

  168. An etiologic regulatory mutation in IRF6 with loss- and gain-of-function effects

    Fakhouri, Walid D.; Rahimov, Fedik; Attanasio, Catia; Kouwenhoven, Evelyn N.; Ferreira De Lima, Renata L.; Felix, Temis Maria; Nitschke, Larissa; Huver, David; Barrons, Julie; Kousa, Youssef A.; Leslie, Elizabeth; Pennacchio, Len A.; Van Bokhoven, Hans; Visel, Axel; Zhou, Huiqing; Murray, Jeffrey C.; Schutte, Brian C.. Human Molecular Genetics. vol. 23(10), 2711–2720. May 2014.

    DNA variation in Interferon Regulatory Factor 6 (IRF6) causes Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate (CLP). However, an etiologic variant in IRF6 has been found in only 70% of VWS families. To test whether DNA variants in regulatory elements cause VWS, we sequenced three conserved elements near IRF6 in 70 VWS families that lack an etiologic mutation within IRF6 exons. A rare mutation (350dupA) was found in a conserved IRF6 enhancer element (MCS9.7) in a Brazilian family. The 350dupA mutation abrogated the binding of p63 and E47 transcription factors to cis-overlapping motifs, and significantly disrupted enhancer activity in human cell cultures. Moreover, using a transgenic assay in mice, the 350dupA mutation disrupted the activation of MCS9.7 enhancer element and led to failure of lacZ expression in all head and neck pharyngeal arches. Interestingly, disruption of the p63 Motif1 and/or E47 binding sites by nucleotide substitution did not fully recapitulate the effect of the 350dupA mutation. Rather, we recognized that the 350dupA created a CAAAGT motif, a binding site for Lef1 protein. We showed that Lef1 binds to the mutated site and that overexpression of Lef1/β-Catenin chimeric protein repressed MCS9.7-350dupA enhancer activity. In conclusion, our data strongly suggest that 350dupA variant is an etiologic mutation in VWS patients and disrupts enhancer activity by a loss- and gain-of-function mechanism, and thus support the rationale for additional screening for regulatory mutations in patients with CLP.

  169. Function-based identification of mammalian enhancers using site-specific integration

    Dickel, Diane E.; Zhu, Yiwen; Nord, Alex S.; Wylie, John N.; Akiyama, Jennifer A.; Afzal, Veena; Plajzer-Frick, Ingrid; Kirkpatrick, Aileen; Göttgens, Berthold; Bruneau, Benoit G.; Visel, Axel; Pennacchio, Len A.. Nature Methods. vol. 11(5), 566–571. May 2014.

    The accurate and comprehensive identification of functional regulatory sequences in mammalian genomes remains a major challenge. Here we describe site-specific integration fluorescence-activated cell sorting followed by sequencing (SIF-seq), an unbiased, medium-throughput functional assay for the discovery of distant-acting enhancers. Targeted single-copy genomic integration into pluripotent cells, reporter assays and flow cytometry are coupled with high-throughput DNA sequencing to enable parallel screening of large numbers of DNA sequences. By functionally interrogating \textgreater500 kilobases (kb) of mouse and human sequence in mouse embryonic stem cells for enhancer activity we identified enhancers at pluripotency loci including NANOG. In in vitro-differentiated cardiomyocytes and neural progenitor cells, we identified cardiac enhancers and neuronal enhancers, respectively. SIF-seq is a powerful and flexible method for de novo functional identification of mammalian enhancers in a potentially wide variety of cell types.

  170. TGFβ regulates epithelial-mesenchymal interactions through WNT signaling activity to control muscle development in the soft palate

    Iwata, Jun-ichi; Suzuki, Akiko; Yokota, Toshiaki; Ho, Thach-Vu; Pelikan, Richard; Urata, Mark; Sanchez-Lara, Pedro A.; Chai, Yang. Development (Cambridge, England). vol. 141(4), 909–917. February 2014.

    Clefting of the soft palate occurs as a congenital defect in humans and adversely affects the physiological function of the palate. However, the molecular and cellular mechanism of clefting of the soft palate remains unclear because few animal models exhibit an isolated cleft in the soft palate. Using three-dimensional microCT images and histological reconstruction, we found that loss of TGFβ signaling in the palatal epithelium led to soft palate muscle defects in Tgfbr2(fl/fl);K14-Cre mice. Specifically, muscle mass was decreased in the soft palates of Tgfbr2 mutant mice, following defects in cell proliferation and differentiation. Gene expression of Dickkopf (Dkk1 and Dkk4), negative regulators of WNT-β-catenin signaling, is upregulated in the soft palate of Tgfbr2(fl/fl);K14-Cre mice, and WNT-β-catenin signaling is disrupted in the palatal mesenchyme. Importantly, blocking the function of DKK1 and DKK4 rescued the cell proliferation and differentiation defects in the soft palate of Tgfbr2(fl/fl);K14-Cre mice. Thus, our findings indicate that loss of TGFβ signaling in epithelial cells compromises activation of WNT signaling and proper muscle development in the soft palate through tissue-tissue interactions, resulting in a cleft soft palate. This information has important implications for prevention and non-surgical correction of cleft soft palate.

  171. A genome-wide study of de novo deletions identifies a candidate locus for non-syndromic isolated cleft lip/palate risk

    Younkin, Samuel G.; Scharpf, Robert B.; Schwender, Holger; Parker, Margaret M.; Scott, Alan F.; Marazita, Mary L.; Beaty, Terri H.; Ruczinski, Ingo. BMC genetics. vol. 15, 24. February 2014.

    BACKGROUND: Copy number variants (CNVs) may play an important part in the development of common birth defects such as oral clefts, and individual patients with multiple birth defects (including clefts) have been shown to carry small and large chromosomal deletions. In this paper we investigate de novo deletions defined as DNA segments missing in an oral cleft proband but present in both unaffected parents. We compare de novo deletion frequencies in children of European ancestry with an isolated, non-syndromic oral cleft to frequencies in children of European ancestry from randomly sampled trios. RESULTS: We identified a genome-wide significant 62 kilo base (kb) non-coding region on chromosome 7p14.1 where de novo deletions occur more frequently among oral cleft cases than controls. We also observed wider de novo deletions among cleft lip and palate (CLP) cases than seen among cleft palate (CP) and cleft lip (CL) cases. CONCLUSIONS: This study presents a region where de novo deletions appear to be involved in the etiology of oral clefts, although the underlying biological mechanisms are still unknown. Larger de novo deletions are more likely to interfere with normal craniofacial development and may result in more severe clefts. Study protocol and sample DNA source can severely affect estimates of de novo deletion frequencies. Follow-up studies are needed to further validate these findings and to potentially identify additional structural variants underlying oral clefts.

  172. Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor

    Zhao, Hu; Feng, Jifan; Seidel, Kerstin; Shi, Songtao; Klein, Ophir; Sharpe, Paul; Chai, Yang. Cell Stem Cell. vol. 14(2), 160–173. February 2014.

    Mesenchymal stem cells (MSCs) are typically defined by their in vitro characteristics, and as a consequence the in vivo identity of MSCs and their niches are poorly understood. To address this issue, we used lineage tracing in a mouse incisor model and identified the neurovascular bundle (NVB) as an MSC niche. We found that NVB sensory nerves secrete Shh protein, which activates Gli1 expression in periarterial cells that contribute to all mesenchymal derivatives. These periarterial cells do not express classical MSC markers used to define MSCs in vitro. In contrast, NG2(+) pericytes represent an MSC subpopulation derived from Gli1+ cells; they express classical MSC markers and contribute little to homeostasis but are actively involved in injury repair. Likewise, incisor Gli1(+) cells, but not NG2(+) cells, exhibit typical MSC characteristics in vitro. Collectively, we demonstrate that MSCs originate from periarterial cells and are regulated by Shh secretion from an NVB.

  173. The UCSC Genome Browser database: 2014 update

    Karolchik, Donna; Barber, Galt P.; Casper, Jonathan; Clawson, Hiram; Cline, Melissa S.; Diekhans, Mark; Dreszer, Timothy R.; Fujita, Pauline A.; Guruvadoo, Luvina; Haeussler, Maximilian; Harte, Rachel A.; Heitner, Steve; Hinrichs, Angie S.; Learned, Katrina; Lee, Brian T.; Li, Chin H.; Raney, Brian J.; Rhead, Brooke; Rosenbloom, Kate R.; Sloan, Cricket A.; Speir, Matthew L.; Zweig, Ann S.; Haussler, David; Kuhn, Robert M.; Kent, W. James. Nucleic Acids Research. vol. 42(Database issue), D764–770. January 2014.

    The University of California Santa Cruz (UCSC) Genome Browser ( offers online public access to a growing database of genomic sequence and annotations for a large collection of organisms, primarily vertebrates, with an emphasis on the human and mouse genomes. The Browser’s web-based tools provide an integrated environment for visualizing, comparing, analysing and sharing both publicly available and user-generated genomic data sets. As of September 2013, the database contained genomic sequence and a basic set of annotation ’tracks’ for ∼90 organisms. Significant new annotations include a 60-species multiple alignment conservation track on the mouse, updated UCSC Genes tracks for human and mouse, and several new sets of variation and ENCODE data. New software tools include a Variant Annotation Integrator that returns predicted functional effects of a set of variants uploaded as a custom track, an extension to UCSC Genes that displays haplotype alleles for protein-coding genes and an expansion of data hubs that includes the capability to display remotely hosted user-provided assembly sequence in addition to annotation data. To improve European access, we have added a Genome Browser mirror ( hosted at Bielefeld University in Germany.

  174. Modulation of lipid metabolic defects rescues cleft palate in Tgfbr2 mutant mice

    Iwata, Junichi; Suzuki, Akiko; Pelikan, Richard C.; Ho, Thach-Vu; Sanchez-Lara, Pedro A.; Chai, Yang. Human Molecular Genetics. vol. 23(1), 182–193. January 2014.

    Mutations in transforming growth factor beta (TGFβ) receptor type II (TGFBR2) cause Loeys-Dietz syndrome, characterized by craniofacial and cardiovascular abnormalities. Mice with a deletion of Tgfbr2 in cranial neural crest cells (Tgfbr2(fl/fl);Wnt1-Cre mice) develop cleft palate as the result of abnormal TGFβ signaling activation. However, little is known about metabolic processes downstream of TGFβ signaling during palatogenesis. Here, we show that Tgfbr2 mutant palatal mesenchymal cells spontaneously accumulate lipid droplets, resulting from reduced lipolysis activity. Tgfbr2 mutant palatal mesenchymal cells failed to respond to the cell proliferation stimulator sonic hedgehog, derived from the palatal epithelium. Treatment with p38 mitogen-activated protein kinase (MAPK) inhibitor or telmisartan, a modulator of p38 MAPK activation and lipid metabolism, blocked abnormal TGFβ-mediated p38 MAPK activation, restoring lipid metabolism and cell proliferation activity both in vitro and in vivo. Our results highlight the influence of alternative TGFβ signaling on lipid metabolic activities, as well as how lipid metabolic defects can affect cell proliferation and adversely impact palatogenesis. This discovery has broader implications for the understanding of metabolic defects and potential prevention of congenital birth defects.

  175. FishFace: interactive atlas of zebrafish craniofacial development at cellular resolution

    Eames, B; DeLaurier, April; Ullmann, Bonnie; Huycke, Tyler R; Nichols, James T; Dowd, John; McFadden, Marcie; Sasaki, Mark M; Kimmel, Charles B. BMC Developmental Biology. vol. 13(1), 23. 2013.

  176. A new tool for quantifying and characterizing asymmetry in bilaterally paired structures

    Rolfe, S. M.; Camci, E. D.; Mercan, E.; Shapiro, L. G.; Cox, T. C.. Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference. vol. 2013, 2364–2367. 2013.

    This paper introduces a new tool to quantify and characterize asymmetry in bilaterally paired structures. This method uses deformable registration to produce a dense vector field describing the point correspondences between two images of bilaterally paired structures. The deformation vector field properties are clustered to detect and describe regions of relevant asymmetry. Three methods are provided to analyze the asymmetries: the global asymmetry score uses cluster features to quantify overall asymmetry, the local asymmetry score quantifies asymmetry in user-defined regions of interest, and the asymmetry similarity measure quantifies pairwise similarity of individual asymmetry. The scores and image distances generated by this tool are shown to correlate highly with asymmetry ratings assigned by an expert.

  177. The use of pseudo-landmarks for craniofacial analysis: a comparative study with L₁-regularized logistic regression

    Mercan, Ezgi; Shapiro, Linda G.; Weinberg, Seth M.; Lee, Su-In. Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference. vol. 2013, 6083–6086. 2013.

    Morphometrics, the quantitative analysis of shape, is used by craniofacial researchers to study abnormalities in human face shapes. Most of the work in craniofacial morphometrics uses landmark points that are manually marked on 3D face data and processed via a generalized Procrustes analysis. For large data sets this manual process is very time-consuming. Dense sets of pseudo-landmarks have also been proposed and successfully used for classification and clustering, but the two main methods in the literature are very computationally intensive. We have developed a computationally simple method that can compute pseudo-landmark points at different resolutions from 3D meshes of human faces. In this paper, we perform a comparative study employing L1-regularized logistic regression to train a classifier that predicts the sex of 500 normal adult face meshes in order to compare our method to two alternative pseudo-landmark methods and a distance matrix approach.Our results show that our method, which is fully automatic, achieved similar results to the best-scoring methods with no manual landmarking and with much lower computation time. Use of the distance matrix did not improve classification results.

  178. Improved detection of landmarks on 3D human face data

    Liang, Shu; Wu, Jia; Weinberg, Seth M.; Shapiro, Linda G.. Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference. vol. 2013, 6482–6485. 2013.

    Craniofacial researchers make heavy use of established facial landmarks in their morphometric analyses. For studies on very large facial image datasets, the standard approach of manual landmarking is very labor intensive. With the goal of producing 20 established landmarks, we have developed a geometric methodology that can automatically locate 10 established landmark points and 7 other supporting points on human 3D facial scans. Then, to improve accuracy and produce all 20 landmarks, a deformable matching procedure establishes a dense correspondence from a template 3D mesh with a full set of 20 landmarks to each individual 3D mesh. The 17 geometrically-determined points on the individual 3D mesh are used for the initial correspondence required by the deformable matching. The method is evaluated on 115 3D facial meshes of normal adults, and results are compared to landmarks manually identified by medical experts. Our results show a marked improvement to prior results in the recent literature.

  179. Functional anatomy of distant-acting mammalian enhancers

    Dickel, D. E.; Visel, A.; Pennacchio, L. A.. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. vol. 368(1620), 20120359. 2013.

    Transcriptional enhancers are a major class of functional element embedded in the vast non-coding portion of the human genome. Acting over large genomic distances, enhancers play critical roles in the tissue and cell type-specific regulation of genes, and there is mounting evidence that they contribute to the aetiology of many human diseases. Methods for genome-wide mapping of enhancer regions are now available, but the functional architecture contained within human enhancer elements remains unclear. Here, we review recent approaches aimed at understanding the functional anatomy of individual enhancer elements, using systematic qualitative and quantitative assessments of mammalian enhancer variants in cultured cells and in vivo. These studies provide direct insight into common architectural characteristics of enhancers including the presence of multiple transcription factor-binding sites and the mixture of both transcriptionally activating and repressing domains within the same enhancer. Despite such progress in understanding the functional composition of enhancers, the inherent complexities of enhancer anatomy continue to limit our ability to predict the impact of sequence changes on in vivo enhancer function. While providing an initial glimpse into the mutability of mammalian enhancers, these observations highlight the continued need for experimental enhancer assessment as genome sequencing becomes routine in the clinic.

  180. Skull Retrieval for Craniosynostosis Using Sparse Logistic Regression Models

    Yang, Shulin; Shapiro, Linda; Cunningham, Michael; Speltz, Matthew; Birgfeld, Craig; Atmosukarto, Indriyati; Lee, Su-In. Medical image computing and computer-assisted intervention: MICCAI ... International Conference on Medical Image Computing and Computer-Assisted Intervention. vol. 7723, 33–44. 2013.

    Craniosynostosis is the premature fusion of the bones of the calvaria resulting in abnormal skull shapes that can be associated with increased intracranial pressure. While craniosynostoses of multiple different types can be easily diagnosed, quantifying the severity of the abnormality is much more subjective and not a standard part of clinical practice. For this purpose we have developed a severity-based retrieval system that uses a logistic regression approach to quantify the severity of the abnormality of each of three types of craniosynostoses. We compare several different sparse feature selection techniques: L1 regularized logistic regression, fused lasso, and clustering lasso (cLasso). We evaluate our methodology in three ways: 1) for classification of normal vs. abnormal skulls, 2) for comparing pre-operative to post-operative skulls, and 3) for retrieving skulls in order of abnormality severity as compared with the ordering of a craniofacial expert.

  181. Rapid and pervasive changes in genome-wide enhancer usage during mammalian development

    Nord, Alex S.; Blow, Matthew J.; Attanasio, Catia; Akiyama, Jennifer A.; Holt, Amy; Hosseini, Roya; Phouanenavong, Sengthavy; Plajzer-Frick, Ingrid; Shoukry, Malak; Afzal, Veena; Rubenstein, John L. R.; Rubin, Edward M.; Pennacchio, Len A.; Visel, Axel. Cell. vol. 155(7), 1521–1531. December 2013.

    Enhancers are distal regulatory elements that can activate tissue-specific gene expression and are abundant throughout mammalian genomes. Although substantial progress has been made toward genome-wide annotation of mammalian enhancers, their temporal activity patterns and global contributions in the context of developmental in vivo processes remain poorly explored. Here we used epigenomic profiling for H3K27ac, a mark of active enhancers, coupled to transgenic mouse assays to examine the genome-wide utilization of enhancers in three different mouse tissues across seven developmental stages. The majority of the ∼90,000 enhancers identified exhibited tightly temporally restricted predicted activity windows and were associated with stage-specific biological functions and regulatory pathways in individual tissues. Comparative genomic analysis revealed that evolutionary conservation of enhancers decreases following midgestation across all tissues examined. The dynamic enhancer activities uncovered in this study illuminate rapid and pervasive temporal in vivo changes in enhancer usage that underlie processes central to development and disease.

  182. Congenital heart defects in patients with deletions upstream of SOX9

    Sanchez-Castro, Marta; Gordon, Christopher T.; Petit, Florence; Nord, Alex S.; Callier, Patrick; Andrieux, Joris; Guérin, Patrice; Pichon, Olivier; David, Albert; Abadie, Véronique; Bonnet, Damien; Visel, Axel; Pennacchio, Len A.; Amiel, Jeanne; Lyonnet, Stanislas; Le Caignec, Cédric. Human Mutation. vol. 34(12), 1628–1631. December 2013.

    Heterozygous loss-of-function coding-sequence mutations of the transcription factor SOX9 cause campomelic dysplasia, a rare skeletal dysplasia with congenital bowing of long bones (campomelia), hypoplastic scapulae, a missing pair of ribs, pelvic, and vertebral malformations, clubbed feet, Pierre Robin sequence (PRS), facial dysmorphia, and disorders of sex development. We report here two unrelated families that include patients with isolated PRS, isolated congenital heart defect (CHD), or both anomalies. Patients from both families carried a very similar ∼1 Mb deletion upstream of SOX9. Analysis of ChIP-Seq from mouse cardiac tissue for H3K27ac, a marker of active regulatory elements, led us to identify several putative cardiac enhancers within the deleted region. One of these elements is known to interact with Nkx2.5 and Gata4, two transcription factors responsible for CHDs. Altogether, these data suggest that disruption of cardiac enhancers located upstream of SOX9 may be responsible for CHDs in humans.

  183. Evaluating rare coding variants as contributing causes to non-syndromic cleft lip and palate

    Leslie, E. J.; Murray, J. C.. Clinical Genetics. vol. 84(5), 496–500. November 2013.

    Rare coding variants are a current focus in studies of complex disease. Previously, at least 68 rare coding variants were reported from candidate gene sequencing studies in non-syndromic cleft lip and palate (NSCL/P), a common birth defect. Advances in sequencing technology have now resulted in thousands of sequenced exomes, providing a large resource for comparative genetic studies. We collated rare coding variants reported to contribute to NSCL/P and compared them to variants identified from control exome databases to determine if some might be rare but benign variants. Seventy-one percentage of the variants described as etiologic for NSCL/P were not present in the exome data, suggesting that many likely contribute to disease. Our results strongly support a role for rare variants previously reported in the majority of NSCL/P candidate genes but diminish support for variants in others. However, because clefting is a complex trait it is not possible to be definitive about the role of any particular variant for its risk for NSCL/P.

  184. The ontology of craniofacial development and malformation for translational craniofacial research

    Brinkley, J. F.; Borromeo, C.; Clarkson, M.; Cox, T. C.; Cunningham, M. J.; Detwiler, L. T.; Heike, C. L.; Hochheiser, H.; Mejino, J. L. V.; Travillian, R. S.; Shapiro, L. G.. American Journal of Medical Genetics. Part C, Seminars in Medical Genetics. vol. 163C(4), 232–245. November 2013.

    We introduce the Ontology of Craniofacial Development and Malformation (OCDM) as a mechanism for representing knowledge about craniofacial development and malformation, and for using that knowledge to facilitate integrating craniofacial data obtained via multiple techniques from multiple labs and at multiple levels of granularity. The OCDM is a project of the NIDCR-sponsored FaceBase Consortium, whose goal is to promote and enable research into the genetic and epigenetic causes of specific craniofacial abnormalities through the provision of publicly accessible, integrated craniofacial data. However, the OCDM should be usable for integrating any web-accessible craniofacial data, not just those data available through FaceBase. The OCDM is based on the Foundational Model of Anatomy (FMA), our comprehensive ontology of canonical human adult anatomy, and includes modules to represent adult and developmental craniofacial anatomy in both human and mouse, mappings between homologous structures in human and mouse, and associated malformations. We describe these modules, as well as prototype uses of the OCDM for integrating craniofacial data. By using the terms from the OCDM to annotate data, and by combining queries over the ontology with those over annotated data, it becomes possible to create "intelligent" queries that can, for example, find gene expression data obtained from mouse structures that are precursors to homologous human structures involved in malformations such as cleft lip. We suggest that the OCDM can be useful not only for integrating craniofacial data, but also for expressing new knowledge gained from analyzing the integrated data.

  185. Understanding the basis of auriculocondylar syndrome: Insights from human, mouse and zebrafish genetic studies

    Clouthier, David E.; Passos-Bueno, Maria Rita; Tavares, Andre L. P.; Lyonnet, Stanislas; Amiel, Jeanne; Gordon, Christopher T.. American Journal of Medical Genetics. Part C, Seminars in Medical Genetics. vol. 163C(4), 306–317. November 2013.

    Among human birth defect syndromes, malformations affecting the face are perhaps the most striking due to cultural and psychological expectations of facial shape. One such syndrome is auriculocondylar syndrome (ACS), in which patients present with defects in ear and mandible development. Affected structures arise from cranial neural crest cells, a population of cells in the embryo that reside in the pharyngeal arches and give rise to most of the bone, cartilage and connective tissue of the face. Recent studies have found that most cases of ACS arise from defects in signaling molecules associated with the endothelin signaling pathway. Disruption of this signaling pathway in both mouse and zebrafish results in loss of identity of neural crest cells of the mandibular portion of the first pharyngeal arch and the subsequent repatterning of these cells, leading to homeosis of lower jaw structures into more maxillary-like structures. These findings illustrate the importance of endothelin signaling in normal human craniofacial development and illustrate how clinical and basic science approaches can coalesce to improve our understanding of the genetic basis of human birth defect syndromes. Further, understanding the genetic basis for ACS that lies outside of known endothelin signaling components may help elucidate unknown aspects critical to the establishment of neural crest cell patterning during facial morphogenesis.

  186. Genetics of cleft lip and cleft palate

    Leslie, Elizabeth J.; Marazita, Mary L.. American Journal of Medical Genetics. Part C, Seminars in Medical Genetics. vol. 163C(4), 246–258. November 2013.

    Orofacial clefts are common birth defects and can occur as isolated, nonsyndromic events or as part of Mendelian syndromes. There is substantial phenotypic diversity in individuals with these birth defects and their family members: from subclinical phenotypes to associated syndromic features that is mirrored by the many genes that contribute to the etiology of these disorders. Identification of these genes and loci has been the result of decades of research using multiple genetic approaches. Significant progress has been made recently due to advances in sequencing and genotyping technologies, primarily through the use of whole exome sequencing and genome-wide association studies. Future progress will hinge on identifying functional variants, investigation of pathway and other interactions, and inclusion of phenotypic and ethnic diversity in studies.

  187. Noncanonical transforming growth factor β (TGFβ) signaling in cranial neural crest cells causes tongue muscle developmental defects

    Iwata, Jun-ichi; Suzuki, Akiko; Pelikan, Richard C.; Ho, Thach-Vu; Chai, Yang. The Journal of Biological Chemistry. vol. 288(41), 29760–29770. October 2013.

    Microglossia is a congenital birth defect in humans and adversely impacts quality of life. In vertebrates, tongue muscle derives from the cranial mesoderm, whereas tendons and connective tissues in the craniofacial region originate from cranial neural crest (CNC) cells. Loss of transforming growth factor β (TGFβ) type II receptor in CNC cells in mice (Tgfbr2(fl/fl);Wnt1-Cre) causes microglossia due to a failure of cell-cell communication between cranial mesoderm and CNC cells during tongue development. However, it is still unclear how TGFβ signaling in CNC cells regulates the fate of mesoderm-derived myoblasts during tongue development. Here we show that activation of the cytoplasmic and nuclear tyrosine kinase 1 (ABL1) cascade in Tgfbr2(fl/fl);Wnt1-Cre mice results in a failure of CNC-derived cell differentiation followed by a disruption of TGFβ-mediated induction of growth factors and reduction of myogenic cell proliferation and differentiation activities. Among the affected growth factors, the addition of fibroblast growth factor 4 (FGF4) and neutralizing antibody for follistatin (FST; an antagonist of bone morphogenetic protein (BMP)) could most efficiently restore cell proliferation, differentiation, and organization of muscle cells in the tongue of Tgfbr2(fl/fl);Wnt1-Cre mice. Thus, our data indicate that CNC-derived fibroblasts regulate the fate of mesoderm-derived myoblasts through TGFβ-mediated regulation of FGF and BMP signaling during tongue development.

  188. Search for genetic modifiers of IRF6 and genotype-phenotype correlations in Van der Woude and popliteal pterygium syndromes

    Leslie, Elizabeth J.; Mancuso, Jennifer L.; Schutte, Brian C.; Cooper, Margaret E.; Durda, Kate M.; L’Heureux, Jamie; Zucchero, Theresa M.; Marazita, Mary L.; Murray, Jeffrey C.. American Journal of Medical Genetics. Part A. vol. 161A(10), 2535–2544. October 2013.

    Van der Woude syndrome is the most common form of syndromic orofacial clefting, accounting for 1-2% of all patients with cleft lip and/or cleft palate. Van der Woude and popliteal pterygium syndromes are caused by mutations in IRF6, but phenotypic variability within and among families with either syndrome suggests that other genetic factors contribute to the phenotypes. The aim of this study was to identify common variants acting as genetic modifiers of IRF6 as well as genotype-phenotype correlations based on mutation type and location. We identified an association between mutations in the DNA-binding domain of IRF6 and limb defects (including pterygia). Although we did not detect formally significant associations with the genes tested, borderline associations suggest several genes that could modify the VWS phenotype, including FOXE1, TGFB3, and TFAP2A. Some of these genes are hypothesized to be part of the IRF6 gene regulatory network and may suggest additional genes for future study when larger sample sizes are also available. We also show that families with the Van de Woude phenotype but in whom no mutations have been identified have a lower frequency of cleft lip, suggesting there may be locus and/or mutation class differences in Van de Woude syndrome.

  189. Fine tuning of craniofacial morphology by distant-acting enhancers

    Attanasio, Catia; Nord, Alex S.; Zhu, Yiwen; Blow, Matthew J.; Li, Zirong; Liberton, Denise K.; Morrison, Harris; Plajzer-Frick, Ingrid; Holt, Amy; Hosseini, Roya; Phouanenavong, Sengthavy; Akiyama, Jennifer A.; Shoukry, Malak; Afzal, Veena; Rubin, Edward M.; FitzPatrick, David R.; Ren, Bing; Hallgrímsson, Benedikt; Pennacchio, Len A.; Visel, Axel. Science (New York, N.Y.). vol. 342(6157), 1241006. October 2013.

    The shape of the human face and skull is largely genetically determined. However, the genomic basis of craniofacial morphology is incompletely understood and hypothesized to involve protein-coding genes, as well as gene regulatory sequences. We used a combination of epigenomic profiling, in vivo characterization of candidate enhancer sequences in transgenic mice, and targeted deletion experiments to examine the role of distant-acting enhancers in craniofacial development. We identified complex regulatory landscapes consisting of enhancers that drive spatially complex developmental expression patterns. Analysis of mouse lines in which individual craniofacial enhancers had been deleted revealed significant alterations of craniofacial shape, demonstrating the functional importance of enhancers in defining face and skull morphology. These results demonstrate that enhancers are involved in craniofacial development and suggest that enhancer sequence variation contributes to the diversity of human facial morphology.

  190. CTGF mediates Smad-dependent transforming growth factor β signaling to regulate mesenchymal cell proliferation during palate development

    Parada, Carolina; Li, Jingyuan; Iwata, Junichi; Suzuki, Akiko; Chai, Yang. Molecular and Cellular Biology. vol. 33(17), 3482–3493. September 2013.

    Transforming growth factor β (TGF-β) signaling plays crucial functions in the regulation of craniofacial development, including palatogenesis. Here, we have identified connective tissue growth factor (Ctgf) as a downstream target of the TGF-β signaling pathway in palatogenesis. The pattern of Ctgf expression in wild-type embryos suggests that it may be involved in key processes during palate development. We found that Ctgf expression is downregulated in both Wnt1-Cre; Tgfbr2(fl/fl) and Osr2-Cre; Smad4(fl/fl) palates. In Tgfbr2 mutant embryos, downregulation of Ctgf expression is associated with p38 mitogen-activated protein kinase (MAPK) overactivation, whereas loss of function of Smad4 itself leads to downregulation of Ctgf expression. We also found that CTGF regulates its own expression via TGF-β signaling. Osr2-Cre; Smad4(fl/fl) mice exhibit a defect in cell proliferation similar to that of Tgfbr2 mutant mice, as well as cleft palate. We detected no alteration in bone morphogenetic protein (BMP) downstream targets in Smad4 mutant palates, suggesting that the reduction in cell proliferation is due to defective transduction of TGF-β signaling via decreased Ctgf expression. Significantly, an exogenous source of CTGF was able to rescue the cell proliferation defect in both Tgfbr2 and Smad4 mutant palates. Collectively, our data suggest that CTGF regulates proliferation as a mediator of the canonical pathway of TGF-β signaling during palatogenesis.

  191. SP8 regulates signaling centers during craniofacial development

    Kasberg, Abigail D.; Brunskill, Eric W.; Steven Potter, S.. Developmental Biology. vol. 381(2), 312–323. September 2013.

    Much of the bone, cartilage and smooth muscle of the vertebrate face is derived from neural crest (NC) cells. During craniofacial development, the anterior neural ridge (ANR) and olfactory pit (OP) signaling centers are responsible for driving the outgrowth, survival, and differentiation of NC populated facial prominences, primarily via FGF. While much is known about the functional importance of signaling centers, relatively little is understood of how these signaling centers are made and maintained. In this report we describe a dramatic craniofacial malformation in mice mutant for the zinc finger transcription factor gene Sp8. At E14.5 they show facial prominences that are reduced in size and underdeveloped, giving an almost faceless phenotype. At later times they show severe midline defects, excencephaly, hyperterlorism, cleft palate, and a striking loss of many NC and paraxial mesoderm derived cranial bones. Sp8 expression was primarily restricted to the ANR and OP regions during craniofacial development. Analysis of an extensive series of conditional Sp8 mutants confirmed the critical role of Sp8 in signaling centers, and not directly in the NC and paraxial mesoderm cells. The NC cells of the Sp8 mutants showed increased levels of apoptosis and decreased cell proliferation, thereby explaining the reduced sizes of the facial prominences. Perturbed gene expression in the Sp8 mutants was examined by laser capture microdissection coupled with microarrays, as well as in situ hybridization and immunostaining. The most dramatic differences included striking reductions in Fgf8 and Fgf17 expression in the ANR and OP signaling centers. We were also able to achieve genetic and pharmaceutical partial rescue of the Sp8 mutant phenotype by reducing Sonic Hedgehog (SHH) signaling. These results show that Sp8 primarily functions to promote Fgf expression in the ANR and OP signaling centers that drive the survival, proliferation, and differentiation of the NC and paraxial mesoderm that make the face.

  192. Human Development Domain of the Ontology of Craniofacial Development and Malformation

    Mejino, Jose Lv; Travillian, Ravensara S.; Cox, Timothy C.; Shapiro, Linda G.; Brinkley, James F.. CEUR workshop proceedings. vol. 1060, 74–77. July 2013.

    In this paper we describe an ontological scheme for representing anatomical entities undergoing morphological transformation and changes in phenotype during prenatal development. This is a proposed component of the Anatomical Transformation Abstraction (ATA) of the Foundational Model of Anatomy (FMA) Ontology that was created to provide an ontological framework for capturing knowledge about human development from the zygote to postnatal life. It is designed to initially describe the structural properties of the anatomical entities that participate in human development and then enhance their description with developmental properties, such as temporal attributes and developmental processes. This approach facilitates the correlation and integration of the classical but static representation of embryology with the evolving novel concepts of developmental biology, which primarily deals with the experimental data on the mechanisms of embryogenesis and organogenesis. This is important for describing and understanding the underlying processes involved in structural malformations. In this study we focused on the development of the lips and the palate in conjunction with our work on the pathogenesis and classification of cleft lip and palate (CL/P) in the FaceBase program. Our aim here is to create the Craniofacial Human Development Ontology (CHDO) to support the Ontology of Craniofacial Development and Malformation (OCDM), which provides the infrastructure for integrating multiple and disparate craniofacial data generated by FaceBase researchers.

  193. 3D object retrieval using salient views

    Atmosukarto, Indriyati; Shapiro, Linda G.. International Journal of Multimedia Information Retrieval. vol. 2(2), 103–115. June 2013.

    This paper presents a method for selecting salient 2D views to describe 3D objects for the purpose of retrieval. The views are obtained by first identifying salient points via a learning approach that uses shape characteristics of the 3D points (Atmosukarto and Shapiro in International workshop on structural, syntactic, and statistical pattern recognition, 2008; Atmosukarto and Shapiro in ACM multimedia information retrieval, 2008). The salient views are selected by choosing views with multiple salient points on the silhouette of the object. Silhouette-based similarity measures from Chen et al. (Comput Graph Forum 22(3):223-232, 2003) are then used to calculate the similarity between two 3D objects. Retrieval experiments were performed on three datasets: the Heads dataset, the SHREC2008 dataset, and the Princeton dataset. Experimental results show that the retrieval results using the salient views are comparable to the existing light field descriptor method (Chen et al. in Comput Graph Forum 22(3):223-232, 2003), and our method achieves a 15-fold speedup in the feature extraction computation time.

  194. Replication of genome wide association identified candidate genes confirm the role of common and rare variants in PAX7 and VAX1 in the etiology of nonsyndromic CL(P)

    Butali, Azeez; Suzuki, Satoshi; Cooper, Margaret E.; Mansilla, Adela M.; Cuenco, Karen; Leslie, Elizabeth J.; Suzuki, Yasushi; Niimi, Teruyuki; Yamamoto, Masahiko; Ayanga, Gongorjav; Erkhembaatar, Tudevdorj; Furukawa, Hiroo; Fujiwawa, Kumiko; Imura, Hideto; Petrin, Aline L.; Natsume, Nagato; Beaty, Terri H.; Marazita, Mary L.; Murray, Jeffery C.. American Journal of Medical Genetics. Part A. vol. 161A(5), 965–972. May 2013.

    Following recent genome wide association studies (GWAS), significant genetic associations have been identified for several genes with nonsyndromic cleft lip with or without cleft palate (CL(P)). To replicate two of these GWAS signals, we investigated the role of common and rare variants in the PAX7 and VAX1 genes. TaqMan genotyping was carried out for SNPs in VAX1 and PAX7 and transmission disequilibrium test (TDT) was performed to test for linkage and association in each population. Direct sequencing in and around the PAX7 and VAX1 genes in 1,326 individuals of European and Asian ancestry was done. The TDT analysis showed strong associations with markers in VAX1 (rs7078160, P = 2.7E-06 and rs475202, P = 0.0002) in a combined sample of Mongolian and Japanese CL(P) case-parent triads. Analyses using parent-of-origin effects showed significant excess transmission of the minor allele from both parents with the effect in the mothers (P = 6.5E-05, OR (transmission) = 1.91) more striking than in the fathers (P = 0.004, OR (transmission) = 1.67) for VAX1 marker rs7078160 in the combined Mongolian and Japanese samples when all cleft types were combined. The rs6659735 trinucleotide marker in PAX7 was significantly associated with all the US cleft groups combined (P = 0.007 in all clefts and P = 0.02 in CL(P)). Eight rare missense mutations found in PAX7 and two rare missense mutations in VAX1. Our study replicated previous GWAS findings for markers in VAX1 in the Asian population, and identified rare variants in PAX7 and VAX1 that may contribute to the etiology of CL(P). Determining the role of rare variants clearly warrants further investigation.

  195. Comparative analysis of IRF6 variants in families with Van der Woude syndrome and popliteal pterygium syndrome using public whole-exome databases

    Leslie, Elizabeth J.; Standley, Jennifer; Compton, John; Bale, Sherri; Schutte, Brian C.; Murray, Jeffrey C.. Genetics in Medicine: Official Journal of the American College of Medical Genetics. vol. 15(5), 338–344. May 2013.

    PURPOSE: Mutations in the transcription factor IRF6 cause allelic autosomal dominant clefting syndromes, Van der Woude syndrome, and popliteal pterygium syndrome. We compared the distribution of IRF6 coding and splice-site mutations from 549 families with Van der Woude syndrome or popliteal pterygium syndrome with that of variants from the 1000 Genomes and National Heart, Lung, and Blood Institute Exome Sequencing Projects. METHODS: We compiled all published pathogenic IRF6 mutations and performed direct sequencing of IRF6 in families with Van der Woude syndrome or popliteal pterygium syndrome. RESULTS: Although mutations causing Van der Woude syndrome or popliteal pterygium syndrome were nonrandomly distributed with significantly increased frequencies in the DNA-binding domain (P = 0.0001), variants found in controls were rare and evenly distributed in IRF6. Of 194 different missense or nonsense variants described as potentially pathogenic, we identified only two in more than 6,000 controls. PolyPhen and SIFT (sorting intolerant from tolerant) reported 5.9% of missense mutations in patients as benign, suggesting that use of current in silico prediction models to determine function can have significant false negatives. CONCLUSION: Mutation of IRF6 occurs infrequently in controls, suggesting that for IRF6 there is a high probability that disruption of the coding sequence, particularly the DNA-binding domain, will result in syndromic features. Prior associations of coding sequence variants in IRF6 with clefting syndromes have had few false positives.

  196. Identification of candidate downstream targets of TGFβ signaling during palate development by genome-wide transcript profiling

    Pelikan, Richard C.; Iwata, Junichi; Suzuki, Akiko; Chai, Yang; Hacia, Joseph G.. Journal of Cellular Biochemistry. vol. 114(4), 796–807. April 2013.

    Nonsyndromic orofacial clefts are common birth defects whose etiology is influenced by complex genetic and environmental factors and gene-environment interactions. Although these risk factors are not yet fully elucidated, it is known that alterations in transforming growth factor-beta (TGFβ) signaling can cause craniofacial abnormalities, including cleft palate, in mammals. To elucidate the downstream targets of TGFβ signaling in palatogenesis, we analyzed the gene expression profiles of Tgfbr2(fl/fl) ;Wnt1-Cre mouse embryos with cleft palate and other craniofacial deformities resulting from the targeted inactivation of the Tgfbr2 gene in their cranial neural crest (CNC) cells. Relative to controls, palatal tissues obtained from Tgfbr2(fl/fl) ;Wnt1-Cre mouse embryos at embryonic day 14.5 (E14.5) of gestation have a robust gene expression signature reflective of known defects in CNC-derived mesenchymal cell proliferation. Groups of differentially expressed genes (DEGs) were involved in diverse cellular processes and components associated with orofacial clefting, including the extracellular matrix, cholesterol metabolism, ciliogenesis, and multiple signaling pathways. A subset of the DEGs are known or suspected to be associated with an increased risk of orofacial clefting in humans and/or genetically engineered mice. Based on bioinformatics analyses, we highlight the functional relationships among differentially expressed transcriptional regulators of palatogenesis as well as transcriptional factors not previously associated with this process. We suggest that gene expression profiling studies of mice with TGFβ signaling defects provide a valuable approach for identifying candidate mechanisms by which this pathway controls cell fate during palatogenesis and its role in the etiology of human craniofacial abnormalities.

  197. Effects of growth hormone on the ontogenetic allometry of craniofacial bones

    Gonzalez, Paula N.; Kristensen, Erika; Morck, Douglas W.; Boyd, Steven; Hallgrímsson, Benedikt. Evolution & Development. vol. 15(2), 133–145. April 2013.

    Organism size is controlled by interactions between genetic and environmental factors mediated by hormones with systemic and local effects. As changes in size are usually not isometric, a considerable diversity in shape can be generated through modifications in the patterns of ontogenetic allometry. In this study we evaluated the role of timing and dose of growth hormone (GH) release on growth and correlated shape changes in craniofacial bones. Using a longitudinal study design, we analyzed GH deficient mice treated with GH supplementation commencing pre- and post-puberty. We obtained 3D in vivo micro-CT images of the skull between 21 and 60 days of age and used geometric morphometrics to analyze size and shape changes among control and GH deficient treated and non-treated mice. The variable levels of circulating GH altered the size and shape of the adult skull, and influenced the cranial base, vault, and face differently. While cranial base synchondroses and facial sutures were susceptible to either the direct or indirect effect of GH supplementation, its effect was negligible on the vault. Such different responses support the role of intrinsic growth trajectories of skeletal components in controlling the modifications induced by systemic factors. Contrary to the expected, the timing of GH treatment did not have an effect on catch-up growth. GH levels also altered the ontogenetic trajectories by inducing changes in their location and extension in the shape space, indicating that differences arose before 21 days and were further accentuated by a truncation of the ontogenetic trajectories in GHD groups.

  198. The UCSC genome browser and associated tools

    Kuhn, Robert M.; Haussler, David; Kent, W. James. Briefings in Bioinformatics. vol. 14(2), 144–161. March 2013.

    The UCSC Genome Browser ( is a graphical viewer for genomic data now in its 13th year. Since the early days of the Human Genome Project, it has presented an integrated view of genomic data of many kinds. Now home to assemblies for 58 organisms, the Browser presents visualization of annotations mapped to genomic coordinates. The ability to juxtapose annotations of many types facilitates inquiry-driven data mining. Gene predictions, mRNA alignments, epigenomic data from the ENCODE project, conservation scores from vertebrate whole-genome alignments and variation data may be viewed at any scale from a single base to an entire chromosome. The Browser also includes many other widely used tools, including BLAT, which is useful for alignments from high-throughput sequencing experiments. Private data uploaded as Custom Tracks and Data Hubs in many formats may be displayed alongside the rich compendium of precomputed data in the UCSC database. The Table Browser is a full-featured graphical interface, which allows querying, filtering and intersection of data tables. The Saved Session feature allows users to store and share customized views, enhancing the utility of the system for organizing multiple trains of thought. Binary Alignment/Map (BAM), Variant Call Format and the Personal Genome Single Nucleotide Polymorphisms (SNPs) data formats are useful for visualizing a large sequencing experiment (whole-genome or whole-exome), where the differences between the data set and the reference assembly may be displayed graphically. Support for high-throughput sequencing extends to compact, indexed data formats, such as BAM, bigBed and bigWig, allowing rapid visualization of large datasets from RNA-seq and ChIP-seq experiments via local hosting.

  199. Smad4-Irf6 genetic interaction and TGFβ-mediated IRF6 signaling cascade are crucial for palatal fusion in mice

    Iwata, Jun-ichi; Suzuki, Akiko; Pelikan, Richard C.; Ho, Thach-Vu; Sanchez-Lara, Pedro A.; Urata, Mark; Dixon, Michael J.; Chai, Yang. Development (Cambridge, England). vol. 140(6), 1220–1230. March 2013.

    Cleft palate is one of the most common human birth defects and is associated with multiple genetic and environmental risk factors. Although mutations in the genes encoding transforming growth factor beta (TGFβ) signaling molecules and interferon regulatory factor 6 (Irf6) have been identified as genetic risk factors for cleft palate, little is known about the relationship between TGFβ signaling and IRF6 activity during palate formation. Here, we show that TGFβ signaling regulates expression of Irf6 and the fate of the medial edge epithelium (MEE) during palatal fusion in mice. Haploinsufficiency of Irf6 in mice with basal epithelial-specific deletion of the TGFβ signaling mediator Smad4 (Smad4(fl/fl);K14-Cre;Irf6(+/R84C)) results in compromised p21 expression and MEE persistence, similar to observations in Tgfbr2(fl/fl);K14-Cre mice, although the secondary palate of Irf6(+/R84C) and Smad4(fl/fl);K14-Cre mice form normally. Furthermore, Smad4(fl/fl);K14-Cre;Irf6(+/R84C) mice show extra digits that are consistent with abnormal toe and nail phenotypes in individuals with Van der Woude and popliteal pterygium syndromes, suggesting that the TGFβ/SMAD4/IRF6 signaling cascade might be a well-conserved mechanism in regulating multiple organogenesis. Strikingly, overexpression of Irf6 rescued p21 expression and MEE degeneration in Tgfbr2(fl/fl);K14-Cre mice. Thus, IRF6 and SMAD4 synergistically regulate the fate of the MEE, and TGFβ-mediated Irf6 activity is responsible for MEE degeneration during palatal fusion in mice.

  200. The UCSC Genome Browser database: extensions and updates 2013

    Meyer, Laurence R.; Zweig, Ann S.; Hinrichs, Angie S.; Karolchik, Donna; Kuhn, Robert M.; Wong, Matthew; Sloan, Cricket A.; Rosenbloom, Kate R.; Roe, Greg; Rhead, Brooke; Raney, Brian J.; Pohl, Andy; Malladi, Venkat S.; Li, Chin H.; Lee, Brian T.; Learned, Katrina; Kirkup, Vanessa; Hsu, Fan; Heitner, Steve; Harte, Rachel A.; Haeussler, Maximilian; Guruvadoo, Luvina; Goldman, Mary; Giardine, Belinda M.; Fujita, Pauline A.; Dreszer, Timothy R.; Diekhans, Mark; Cline, Melissa S.; Clawson, Hiram; Barber, Galt P.; Haussler, David; Kent, W. James. Nucleic Acids Research. vol. 41(Database issue), D64–69. January 2013.

    The University of California Santa Cruz (UCSC) Genome Browser ( offers online public access to a growing database of genomic sequence and annotations for a wide variety of organisms. The Browser is an integrated tool set for visualizing, comparing, analysing and sharing both publicly available and user-generated genomic datasets. As of September 2012, genomic sequence and a basic set of annotation ’tracks’ are provided for 63 organisms, including 26 mammals, 13 non-mammal vertebrates, 3 invertebrate deuterostomes, 13 insects, 6 worms, yeast and sea hare. In the past year 19 new genome assemblies have been added, and we anticipate releasing another 28 in early 2013. Further, a large number of annotation tracks have been either added, updated by contributors or remapped to the latest human reference genome. Among these are an updated UCSC Genes track for human and mouse assemblies. We have also introduced several features to improve usability, including new navigation menus. This article provides an update to the UCSC Genome Browser database, which has been previously featured in the Database issue of this journal.

  201. Transgenic quail as a model for research in the avian nervous system: a comparative study of the auditory brainstem

    Seidl, Armin H.; Sanchez, Jason Tait; Schecterson, Leslayann; Tabor, Kathryn M.; Wang, Yuan; Kashima, Daniel T.; Poynter, Greg; Huss, David; Fraser, Scott E.; Lansford, Rusty; Rubel, Edwin W.. The Journal of Comparative Neurology. vol. 521(1), 5–23. January 2013.

    Research performed on transgenic animals has led to numerous advances in biological research. However, using traditional retroviral methods to generate transgenic avian research models has proved problematic. As a result, experiments aimed at genetic manipulations on birds have remained difficult for this popular research tool. Recently, lentiviral methods have allowed the production of transgenic birds, including a transgenic Japanese quail (Coturnix coturnix japonica) line showing neuronal specificity and stable expression of enhanced green fluorescent protein (eGFP) across generations (termed here GFP quail). To test whether the GFP quail may serve as a viable alternative to the popular chicken model system, with the additional benefit of genetic manipulation, we compared the development, organization, structure, and function of a specific neuronal circuit in chicken (Gallus gallus domesticus) with that of the GFP quail. This study focuses on a well-defined avian brain region, the principal nuclei of the sound localization circuit in the auditory brainstem, nucleus magnocellularis (NM), and nucleus laminaris (NL). Our results demonstrate that structural and functional properties of NM and NL neurons in the GFP quail, as well as their dynamic properties in response to changes in the environment, are nearly identical to those in chickens. These similarities demonstrate that the GFP quail, as well as other transgenic quail lines, can serve as an attractive avian model system, with the advantage of being able to build on the wealth of information already available from the chicken.

  202. 3D shape isometric correspondence by spectral assignment

    Pan, Xiang; Shapiro, Linda. Proceedings of the ... IAPR International Conference on Pattern Recognition. International Conference on Pattern Recognition. vol. 2012, 2210–2213. 2012.

    Finding correspondences between two 3D shapes is common both in computer vision and computer graphics. In this paper, we propose a general framework that shows how to build correspondences by utilizing the isometric property. We show that the problem of finding such correspondences can be reduced to the problem of spectral assignment, which can be solved by finding the principal eigenvector of the pairwise correspondence matrix. The proposed framework consists of four main steps. First, it obtains initial candidate pairs by performing a preliminary matching using local shape features. Second, it constructs a pairwise correspondence matrix using geodesic distance and these initial pairs. Next, the principal eigenvector of the matrix is computed. Finally, the final correspondence is obtained from the maximal elements of the principal eigenvector. In our experiments, we show that the proposed method is robust under a variety of poses. Furthermore, our results show a great improvement over the best related method in the literature.

  203. The evolution of human genetic studies of cleft lip and cleft palate

    Marazita, Mary L.. Annual Review of Genomics and Human Genetics. vol. 13, 263–283. 2012.

    Orofacial clefts (OFCs)–primarily cleft lip and cleft palate–are among the most common birth defects in all populations worldwide, and have notable population, ethnicity, and gender differences in birth prevalence. Interest in these birth defects goes back centuries, as does formal scientific interest; scientists often used OFCs as examples or evidence during paradigm shifts in human genetics, and have also used virtually every new method of human genetic analysis to deepen our understanding of OFC. This review traces the evolution of human genetic investigations of OFC, highlights the specific insights gained about OFC through the years, and culminates in a review of recent key OFC genetic findings resulting from the powerful tools of the genomics era. Notably, OFC represents a major success for genome-wide approaches, and the field is poised for further breakthroughs in the near future.

  204. Roles of BMP signaling pathway in lip and palate development

    Parada, Carolina; Chai, Yang. Frontiers of Oral Biology. vol. 16, 60–70. 2012.

    Cleft lip with or without cleft palate (CLP) and cleft palate only (CP) are severe disruptions affecting orofacial structures. Patients with orofacial clefts require complex interdisciplinary care, which includes nursing, plastic surgery, maxillofacial surgery, otolaryngology, speech therapy, audiology, psychological and genetic counseling, orthodontics and dental treatment, among others. Overall, treatment of clefts of the lip and palate entails a significant economic burden for families and society. Therefore, prevention is the ultimate objective and this will be facilitated by a complete understanding of the etiology of this condition. Here we review the current concepts regarding the genetic and environmental factors contributing to orofacial clefts and emphasize on the roles of BMP signaling pathway components in the normal and aberrant development of the lip and palate.

  205. Laser capture

    Potter, S. Steven; Brunskill, Eric W.. Methods in Molecular Biology (Clifton, N.J.). vol. 886, 211–221. 2012.

    This chapter describes detailed methods used for laser capture microdissection (LCM) of discrete subpopulations of cells. Topics covered include preparing tissue blocks, cryostat sectioning, processing slides, performing the LCM, and purification of RNA from LCM samples. Notes describe the fine points of each operation, which can often mean the difference between success and failure.

  206. Expression and mutation analyses implicate ARHGAP29 as the etiologic gene for the cleft lip with or without cleft palate locus identified by genome-wide association on chromosome 1p22

    Leslie, Elizabeth J.; Mansilla, M. Adela; Biggs, Leah C.; Schuette, Kristi; Bullard, Steve; Cooper, Margaret; Dunnwald, Martine; Lidral, Andrew C.; Marazita, Mary L.; Beaty, Terri H.; Murray, Jeffrey C.. Birth Defects Research. Part A, Clinical and Molecular Teratology. vol. 94(11), 934–942. November 2012.

    BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect with complex etiology reflecting the action of multiple genetic and environmental factors. Genome-wide association studies have successfully identified five novel loci associated with NSCL/P, including a locus on 1p22.1 near the ABCA4 gene. Because neither expression analysis nor mutation screening support a role for ABCA4 in NSCL/P, we investigated the adjacent gene ARHGAP29. METHODS: Mutation screening for ARHGAP29 protein coding exons was conducted in 180 individuals with NSCL/P and controls from the United States and the Philippines. Nine exons with variants in ARHGAP29 were then screened in an independent set of 872 cases and 802 controls. Arhgap29 expression was evaluated using in situ hybridization in murine embryos. RESULTS: Sequencing of ARHGAP29 revealed eight potentially deleterious variants in cases including a frameshift and a nonsense variant. Arhgap29 showed craniofacial expression and was reduced in a mouse deficient for Irf6, a gene previously shown to have a critical role in craniofacial development. CONCLUSION: The combination of genome-wide association, rare coding sequence variants, craniofacial specific expression, and interactions with IRF6 support a role for ARHGAP29 in NSCL/P and as the etiologic gene at the 1p22 genome-wide association study locus for NSCL/P. This work suggests a novel pathway in which the IRF6 gene regulatory network interacts with the Rho pathway via ARHGAP29. Birth Defects Research (Part A) 2012. © 2012 Wiley Periodicals, Inc.

  207. Beyond knockouts: cre resources for conditional mutagenesis

    Murray, Stephen A.; Eppig, Janan T.; Smedley, Damian; Simpson, Elizabeth M.; Rosenthal, Nadia. Mammalian Genome: Official Journal of the International Mammalian Genome Society. vol. 23(9-10), 587–599. October 2012.

    With the effort of the International Phenotyping Consortium to produce thousands of strains with conditional potential gathering steam, there is growing recognition that it must be supported by a rich toolbox of cre driver strains. The approaches to build cre strains have evolved in both sophistication and reliability, replacing first-generation strains with tools that can target individual cell populations with incredible precision and specificity. The modest set of cre drivers generated by individual labs over the past 15+ years is now growing rapidly, thanks to a number of large-scale projects to produce new cre strains for the community. The power of this growing resource, however, depends upon the proper deep characterization of strain function, as even the best designed strain can display a variety of undesirable features that must be considered in experimental design. This must be coupled with the parallel development of informatics tools to provide functional data to the user and facilitated access to the strains through public repositories. We discuss the current progress on all of these fronts and the challenges that remain to ensure the scientific community can capitalize on the tremendous number of mouse resources at their disposal.

  208. fras1 shapes endodermal pouch 1 and stabilizes zebrafish pharyngeal skeletal development

    Talbot, Jared Coffin; Walker, Macie B.; Carney, Thomas J.; Huycke, Tyler R.; Yan, Yi-Lin; BreMiller, Ruth A.; Gai, Linda; Delaurier, April; Postlethwait, John H.; Hammerschmidt, Matthias; Kimmel, Charles B.. Development (Cambridge, England). vol. 139(15), 2804–2813. August 2012.

    Lesions in the epithelially expressed human gene FRAS1 cause Fraser syndrome, a complex disease with variable symptoms, including facial deformities and conductive hearing loss. The developmental basis of facial defects in Fraser syndrome has not been elucidated. Here we show that zebrafish fras1 mutants exhibit defects in facial epithelia and facial skeleton. Specifically, fras1 mutants fail to generate a late-forming portion of pharyngeal pouch 1 (termed late-p1) and skeletal elements adjacent to late-p1 are disrupted. Transplantation studies indicate that fras1 acts in endoderm to ensure normal morphology of both skeleton and endoderm, consistent with well-established epithelial expression of fras1. Late-p1 formation is concurrent with facial skeletal morphogenesis, and some skeletal defects in fras1 mutants arise during late-p1 morphogenesis, indicating a temporal connection between late-p1 and skeletal morphogenesis. Furthermore, fras1 mutants often show prominent second arch skeletal fusions through space occupied by late-p1 in wild type. Whereas every fras1 mutant shows defects in late-p1 formation, skeletal defects are less penetrant and often vary in severity, even between the left and right sides of the same individual. We interpret the fluctuating asymmetry in fras1 mutant skeleton and the changes in fras1 mutant skeletal defects through time as indicators that skeletal formation is destabilized. We propose a model wherein fras1 prompts late-p1 formation and thereby stabilizes skeletal formation during zebrafish facial development. Similar mechanisms of stochastic developmental instability might also account for the high phenotypic variation observed in human FRAS1 patients.

  209. Shape-based classification of 3D facial data to support 22q11.2DS craniofacial research

    Wilamowska, Katarzyna; Wu, Jia; Heike, Carrie; Shapiro, Linda. Journal of Digital Imaging. vol. 25(3), 400–408. June 2012.

    3D imaging systems are used to construct high-resolution meshes of patient’s heads that can be analyzed by computer algorithms. Our work starts with such 3D head meshes and produces both global and local descriptors of 3D shape. Since these descriptors are numeric feature vectors, they can be used in both classification and quantification of various different abnormalities. In this paper, we define these descriptors, describe our methodology for constructing them from 3D head meshes, and show through a set of classification experiments involving cases and controls for a genetic disorder called 22q11.2 deletion syndrome that they are suitable for use in craniofacial research studies. The main contributions of this work include: automatic generation of novel global and local data representations, robust automatic placement of anthropometric landmarks, generation of local descriptors for nasal and oral facial features from landmarks, use of local descriptors for predicting various local facial features, and use of global features for 22q11.2DS classification, showing their potential use as descriptors in craniofacial research.

  210. Histone deacetylase-4 is required during early cranial neural crest development for generation of the zebrafish palatal skeleton

    DeLaurier, April; Nakamura, Yukio; Braasch, Ingo; Khanna, Vishesh; Kato, Hiroyuki; Wakitani, Shigeyuki; Postlethwait, John H.; Kimmel, Charles B.. BMC developmental biology. vol. 12, 16. June 2012.

    BACKGROUND: Histone deacetylase-4 (Hdac4) is a class II histone deacetylase that inhibits the activity of transcription factors. In humans, HDAC4 deficiency is associated with non-syndromic oral clefts and brachydactyly mental retardation syndrome (BDMR) with craniofacial abnormalities. RESULTS: We identify hdac4 in zebrafish and characterize its function in craniofacial morphogenesis. The gene is present as a single copy, and the deduced Hdac4 protein sequence shares all known functional domains with human HDAC4. The zebrafish hdac4 transcript is widely present in migratory cranial neural crest (CNC) cells of the embryo, including populations migrating around the eye, which previously have been shown to contribute to the formation of the palatal skeleton of the early larva. Embryos injected with hdac4 morpholinos (MO) have reduced or absent CNC populations that normally migrate medial to the eye. CNC-derived palatal precursor cells do not recover at the post-migratory stage, and subsequently we found that defects in the developing cartilaginous palatal skeleton correlate with reduction or absence of early CNC cells. Palatal skeletal defects prominently include a shortened, clefted, or missing ethmoid plate, and are associated with a shortening of the face of young larvae. CONCLUSIONS: Our results demonstrate that Hdac4 is a regulator of CNC-derived palatal skeletal precursors during early embryogenesis. Cleft palate resulting from HDAC4 mutations in human patients may result from defects in a homologous CNC progenitor cell population.

  211. Examining markers in 8q24 to explain differences in evidence for association with cleft lip with/without cleft palate between Asians and Europeans

    Murray, Tanda; Taub, Margaret A.; Ruczinski, Ingo; Scott, Alan F.; Hetmanski, Jacqueline B.; Schwender, Holger; Patel, Poorav; Zhang, Tian Xiao; Munger, Ronald G.; Wilcox, Allen J.; Ye, Xiaoqian; Wang, Hong; Wu, Tao; Wu-Chou, Yah Huei; Shi, Bing; Jee, Sun Ha; Chong, Samuel; Yeow, Vincent; Murray, Jeffrey C.; Marazita, Mary L.; Beaty, Terri H.. Genetic Epidemiology. vol. 36(4), 392–399. May 2012.

    In a recent genome-wide association study (GWAS) from an international consortium, evidence of linkage and association in chr8q24 was much stronger among nonsyndromic cleft lip/palate (CL/P) case-parent trios of European ancestry than among trios of Asian ancestry. We examined marker information content and haplotype diversity across 13 recruitment sites (from Europe, United States, and Asia) separately, and conducted principal components analysis (PCA) on parents. As expected, PCA revealed large genetic distances between Europeans and Asians, and a north-south cline from Korea to Singapore in Asia, with Filipino parents forming a somewhat distinct Southeast Asian cluster. Hierarchical clustering of SNP heterozygosity revealed two major clades consistent with PCA results. All genotyped SNPs giving P \textless 10(-6) in the allelic transmission disequilibrium test (TDT) showed higher heterozygosity in Europeans than Asians. On average, European ancestry parents had higher haplotype diversity than Asians. Imputing additional variants across chr8q24 increased the strength of statistical evidence among Europeans and also revealed a significant signal among Asians (although it did not reach genome-wide significance). Tests for SNP-population interaction were negative, indicating the lack of strong signal for 8q24 in families of Asian ancestry was not due to any distinct genetic effect, but could simply reflect low power due to lower allele frequencies in Asians.

  212. A TGFβ-Smad4-Fgf6 signaling cascade controls myogenic differentiation and myoblast fusion during tongue development

    Han, Dong; Zhao, Hu; Parada, Carolina; Hacia, Joseph G.; Bringas, Pablo; Chai, Yang. Development (Cambridge, England). vol. 139(9), 1640–1650. May 2012.

    The tongue is a muscular organ and plays a crucial role in speech, deglutition and taste. Despite the important physiological functions of the tongue, little is known about the regulatory mechanisms of tongue muscle development. TGFβ family members play important roles in regulating myogenesis, but the functional significance of Smad-dependent TGFβ signaling in regulating tongue skeletal muscle development remains unclear. In this study, we have investigated Smad4-mediated TGFβ signaling in the development of occipital somite-derived myogenic progenitors during tongue morphogenesis through tissue-specific inactivation of Smad4 (using Myf5-Cre;Smad4(flox/flox) mice). During the initiation of tongue development, cranial neural crest (CNC) cells occupy the tongue buds before myogenic progenitors migrate into the tongue primordium, suggesting that CNC cells play an instructive role in guiding tongue muscle development. Moreover, ablation of Smad4 results in defects in myogenic terminal differentiation and myoblast fusion. Despite compromised muscle differentiation, tendon formation appears unaffected in the tongue of Myf5-Cre;Smad4(flox/flox) mice, suggesting that the differentiation and maintenance of CNC-derived tendon cells are independent of Smad4-mediated signaling in myogenic cells in the tongue. Furthermore, loss of Smad4 results in a significant reduction in expression of several members of the FGF family, including Fgf6 and Fgfr4. Exogenous Fgf6 partially rescues the tongue myoblast fusion defect of Myf5-Cre;Smad4(flox/flox) mice. Taken together, our study demonstrates that a TGFβ-Smad4-Fgf6 signaling cascade plays a crucial role in myogenic cell fate determination and lineage progression during tongue myogenesis.

  213. Modulation of noncanonical TGF-β signaling prevents cleft palate in Tgfbr2 mutant mice

    Iwata, Jun-ichi; Hacia, Joseph G.; Suzuki, Akiko; Sanchez-Lara, Pedro A.; Urata, Mark; Chai, Yang. The Journal of Clinical Investigation. vol. 122(3), 873–885. March 2012.

    Patients with mutations in either TGF-β receptor type I (TGFBR1) or TGF-β receptor type II (TGFBR2), such as those with Loeys-Dietz syndrome, have craniofacial defects and signs of elevated TGF-β signaling. Similarly, mutations in TGF-β receptor gene family members cause craniofacial deformities, such as cleft palate, in mice. However, it is unknown whether TGF-β ligands are able to elicit signals in Tgfbr2 mutant mice. Here, we show that loss of Tgfbr2 in mouse cranial neural crest cells results in elevated expression of TGF-β2 and TGF-β receptor type III (TβRIII); activation of a TβRI/TβRIII-mediated, SMAD-independent, TRAF6/TAK1/p38 signaling pathway; and defective cell proliferation in the palatal mesenchyme. Strikingly, Tgfb2, Tgfbr1 (also known as Alk5), or Tak1 haploinsufficiency disrupted TβRI/TβRIII-mediated signaling and rescued craniofacial deformities in Tgfbr2 mutant mice, indicating that activation of this noncanonical TGF-β signaling pathway was responsible for craniofacial malformations in Tgfbr2 mutant mice. Thus, modulation of TGF-β signaling may be beneficial for the prevention of congenital craniofacial birth defects.

  214. Evidence of gene-environment interaction for the RUNX2 gene and environmental tobacco smoke in controlling the risk of cleft lip with/without cleft palate

    Wu, Tao; Fallin, M. Daniele; Shi, Min; Ruczinski, Ingo; Liang, Kung Yee; Hetmanski, Jacqueline B.; Wang, Hong; Ingersoll, Roxann G.; Huang, Shangzhi; Ye, Xiaoqian; Wu-Chou, Yah-Huei; Chen, Philip K.; Jabs, Ethylin Wang; Shi, Bing; Redett, Richard; Scott, Alan F.; Murray, Jeffrey C.; Marazita, Mary L.; Munger, Ronald G.; Beaty, Terri H.. Birth Defects Research. Part A, Clinical and Molecular Teratology. vol. 94(2), 76–83. February 2012.

    This study examined the association between 49 markers in the Runt-related transcription factor 2 (RUNX2) gene and nonsyndromic cleft lip with/without cleft palate (CL/P) among 326 Chinese case-parent trios, while considering gene-environment (GxE) interaction and parent-of-origin effects. Five single-nucleotide polymorphisms (SNPs) showed significant evidence of linkage and association with CL/P and these results were replicated in an independent European sample of 825 case-parent trios. We also report compelling evidence for interaction between markers in RUNX2 and environmental tobacco smoke (ETS). Although most marginal SNP effects (i.e., ignoring maternal exposures) were not statistically significant, eight SNPs were significant when considering possible interaction with ETS when testing for gene (G) and GxE interaction simultaneously or when considering GxE alone. Independent samples from European populations showed consistent evidence of significant GxETS interaction at two SNPs (rs6904353 and rs7748231). Our results suggest genetic variation in RUNX2 may influence susceptibility to CL/P through interacting with ETS.

  215. The UCSC Genome Browser database: extensions and updates 2011

    Dreszer, Timothy R.; Karolchik, Donna; Zweig, Ann S.; Hinrichs, Angie S.; Raney, Brian J.; Kuhn, Robert M.; Meyer, Laurence R.; Wong, Mathew; Sloan, Cricket A.; Rosenbloom, Kate R.; Roe, Greg; Rhead, Brooke; Pohl, Andy; Malladi, Venkat S.; Li, Chin H.; Learned, Katrina; Kirkup, Vanessa; Hsu, Fan; Harte, Rachel A.; Guruvadoo, Luvina; Goldman, Mary; Giardine, Belinda M.; Fujita, Pauline A.; Diekhans, Mark; Cline, Melissa S.; Clawson, Hiram; Barber, Galt P.; Haussler, David; James Kent, W.. Nucleic Acids Research. vol. 40(Database issue), D918–923. January 2012.

    The University of California Santa Cruz Genome Browser ( offers online public access to a growing database of genomic sequence and annotations for a wide variety of organisms. The Browser is an integrated tool set for visualizing, comparing, analyzing and sharing both publicly available and user-generated genomic data sets. In the past year, the local database has been updated with four new species assemblies, and we anticipate another four will be released by the end of 2011. Further, a large number of annotation tracks have been either added, updated by contributors, or remapped to the latest human reference genome. Among these are new phenotype and disease annotations, UCSC genes, and a major dbSNP update, which required new visualization methods. Growing beyond the local database, this year we have introduced ’track data hubs’, which allow the Genome Browser to provide access to remotely located sets of annotations. This feature is designed to significantly extend the number and variety of annotation tracks that are publicly available for visualization and analysis from within our site. We have also introduced several usability features including track search and a context-sensitive menu of options available with a right-click anywhere on the Browser’s image.

  216. Learning to compute the plane of symmetry for human faces

    Wu, Jia; Tse, Raymond; Heike, Carrie L.; Shapiro, Linda G.. . , 471–474. 2011.

  217. Wwp2 is essential for palatogenesis mediated by the interaction between Sox9 and mediator subunit 25

    Nakamura, Yukio; Yamamoto, Koji; He, Xinjun; Otsuki, Bungo; Kim, Youngwoo; Murao, Hiroki; Soeda, Tsunemitsu; Tsumaki, Noriyuki; Deng, Jian Min; Zhang, Zhaoping; Behringer, Richard R.; Crombrugghe, Benoit de; Postlethwait, John H.; Warman, Matthew L.; Nakamura, Takashi; Akiyama, Haruhiko. Nature Communications. vol. 2, 251. 2011.

    Sox9 is a direct transcriptional activator of cartilage-specific extracellular matrix genes and has essential roles in chondrogenesis. Mutations in or around the SOX9 gene cause campomelic dysplasia or Pierre Robin Sequence. However, Sox9-dependent transcriptional control in chondrogenesis remains largely unknown. Here we identify Wwp2 as a direct target of Sox9. Wwp2 interacts physically with Sox9 and is associated with Sox9 transcriptional activity via its nuclear translocation. A yeast two-hybrid screen using a cDNA library reveals that Wwp2 interacts with Med25, a component of the Mediator complex. The positive regulation of Sox9 transcriptional activity by Wwp2 is mediated by the binding between Sox9 and Med25. In zebrafish, morpholino-mediated knockdown of either wwp2 or med25 induces palatal malformation, which is comparable to that in sox9 mutants. These results provide evidence that the regulatory interaction between Sox9, Wwp2 and Med25 defines the Sox9 transcriptional mechanisms of chondrogenesis in the forming palate.

  218. The mechanism of TGF-β signaling during palate development

    Iwata, J.; Parada, C.; Chai, Y.. Oral Diseases. vol. 17(8), 733–744. November 2011.

    Cleft palate, a malformation of the secondary palate development, is one of the most common human congenital birth defects. Palate formation is a complex process resulting in the separation of the oral and nasal cavities that involves multiple events, including palatal growth, elevation, and fusion. Recent findings show that transforming growth factor beta (TGF-β) signaling plays crucial roles in regulating palate development in both the palatal epithelium and mesenchyme. Here, we highlight recent advances in our understanding of TGF-β signaling during palate development.

  219. Epigenetic integration of the developing brain and face

    Parsons, Trish E.; Schmidt, Eric J.; Boughner, Julia C.; Jamniczky, Heather A.; Marcucio, Ralph S.; Hallgrímsson, Benedikt. Developmental Dynamics: An Official Publication of the American Association of Anatomists. vol. 240(10), 2233–2244. October 2011.

    The integration of the brain and face and to what extent this relationship constrains or enables evolutionary change in the craniofacial complex is an issue of long-standing interest in vertebrate evolution. To investigate brain-face integration, we studied the covariation between the forebrain and midface at gestational days 10-10.5 in four strains of laboratory mice. We found that phenotypic variation in the forebrain is highly correlated with that of the face during face formation such that variation in the size of the forebrain correlates with the degree of prognathism and orientation of the facial prominences. This suggests strongly that the integration of the brain and face is relevant to the etiology of midfacial malformations such as orofacial clefts. This axis of integration also has important implications for the evolutionary developmental biology of the mammalian craniofacial complex.

  220. Advances in multiphoton microscopy for imaging embryos

    Supatto, Willy; Truong, Thai V.; Débarre, Delphine; Beaurepaire, Emmanuel. Current Opinion in Genetics & Development. vol. 21(5), 538–548. October 2011.

    Multiphoton imaging is a promising approach for addressing current issues in systems biology and high-content investigation of embryonic development. Recent advances in multiphoton microscopy, including light-sheet illumination, optimized laser scanning, adaptive and label-free strategies, open new opportunities for embryo imaging. However, the literature is often unclear about which microscopy technique is most adapted for achieving specific experimental goals. In this review, we describe and discuss the key concepts of imaging speed, imaging depth, photodamage, and nonlinear contrast mechanisms in the context of recent advances in live embryo imaging. We illustrate the potentials of these new imaging approaches with a selection of recent applications in developmental biology.

  221. Evidence for gene-environment interaction in a genome wide study of nonsyndromic cleft palate

    Beaty, Terri H.; Ruczinski, Ingo; Murray, Jeffrey C.; Marazita, Mary L.; Munger, Ronald G.; Hetmanski, Jacqueline B.; Murray, Tanda; Redett, Richard J.; Fallin, M. Daniele; Liang, Kung Yee; Wu, Tao; Patel, Poorav J.; Jin, Sheng-Chih; Zhang, Tian Xiao; Schwender, Holger; Wu-Chou, Yah Huei; Chen, Philip K.; Chong, Samuel S.; Cheah, Felicia; Yeow, Vincent; Ye, Xiaoqian; Wang, Hong; Huang, Shangzhi; Jabs, Ethylin W.; Shi, Bing; Wilcox, Allen J.; Lie, Rolv T.; Jee, Sun Ha; Christensen, Kaare; Doheny, Kimberley F.; Pugh, Elizabeth W.; Ling, Hua; Scott, Alan F.. Genetic Epidemiology. vol. 35(6), 469–478. September 2011.

    Nonsyndromic cleft palate (CP) is a common birth defect with a complex and heterogeneous etiology involving both genetic and environmental risk factors. We conducted a genome-wide association study (GWAS) using 550 case-parent trios, ascertained through a CP case collected in an international consortium. Family-based association tests of single nucleotide polymorphisms (SNP) and three common maternal exposures (maternal smoking, alcohol consumption, and multivitamin supplementation) were used in a combined 2 df test for gene (G) and gene-environment (G × E) interaction simultaneously, plus a separate 1 df test for G × E interaction alone. Conditional logistic regression models were used to estimate effects on risk to exposed and unexposed children. While no SNP achieved genome-wide significance when considered alone, markers in several genes attained or approached genome-wide significance when G × E interaction was included. Among these, MLLT3 and SMC2 on chromosome 9 showed multiple SNPs resulting in an increased risk if the mother consumed alcohol during the peri-conceptual period (3 months prior to conception through the first trimester). TBK1 on chr. 12 and ZNF236 on chr. 18 showed multiple SNPs associated with higher risk of CP in the presence of maternal smoking. Additional evidence of reduced risk due to G × E interaction in the presence of multivitamin supplementation was observed for SNPs in BAALC on chr. 8. These results emphasize the need to consider G × E interaction when searching for genes influencing risk to complex and heterogeneous disorders, such as nonsyndromic CP.

  222. miR-196 regulates axial patterning and pectoral appendage initiation

    He, Xinjun; Yan, Yi-Lin; Eberhart, Johann K.; Herpin, Amaury; Wagner, Toni U.; Schartl, Manfred; Postlethwait, John H.. Developmental Biology. vol. 357(2), 463–477. September 2011.

    Vertebrate Hox clusters contain protein-coding genes that regulate body axis development and microRNA (miRNA) genes whose functions are not yet well understood. We overexpressed the Hox cluster microRNA miR-196 in zebrafish embryos and found four specific, viable phenotypes: failure of pectoral fin bud initiation, deletion of the 6th pharyngeal arch, homeotic aberration and loss of rostral vertebrae, and reduced number of ribs and somites. Reciprocally, miR-196 knockdown evoked an extra pharyngeal arch, extra ribs, and extra somites, confirming endogenous roles of miR-196. miR-196 injection altered expression of hox genes and the signaling of retinoic acid through the retinoic acid receptor gene rarab. Knocking down rarab mimicked the pectoral fin phenotype of miR-196 overexpression, and reporter constructs tested in tissue culture and in embryos showed that the rarab 3’UTR is a miR-196 target for pectoral fin bud initiation. These results show that a Hox cluster microRNA modulates development of axial patterning similar to nearby protein-coding Hox genes, and acts on appendicular patterning at least in part by modulating retinoic acid signaling.

  223. The FaceBase Consortium: a comprehensive program to facilitate craniofacial research

    Hochheiser, Harry; Aronow, Bruce J.; Artinger, Kristin; Beaty, Terri H.; Brinkley, James F.; Chai, Yang; Clouthier, David; Cunningham, Michael L.; Dixon, Michael; Donahue, Leah Rae; Fraser, Scott E.; Hallgrimsson, Benedikt; Iwata, Junichi; Klein, Ophir; Marazita, Mary L.; Murray, Jeffrey C.; Murray, Stephen; de Villena, Fernando Pardo-Manuel; Postlethwait, John; Potter, Steven; Shapiro, Linda; Spritz, Richard; Visel, Axel; Weinberg, Seth M.; Trainor, Paul A.. Developmental Biology. vol. 355(2), 175–182. July 2011.

    The FaceBase Consortium consists of ten interlinked research and technology projects whose goal is to generate craniofacial research data and technology for use by the research community through a central data management and integrated bioinformatics hub. Funded by the National Institute of Dental and Craniofacial Research (NIDCR) and currently focused on studying the development of the middle region of the face, the Consortium will produce comprehensive datasets of global gene expression patterns, regulatory elements and sequencing; will generate anatomical and molecular atlases; will provide human normative facial data and other phenotypes; conduct follow up studies of a completed genome-wide association study; generate independent data on the genetics of craniofacial development, build repositories of animal models and of human samples and data for community access and analysis; and will develop software tools and animal models for analyzing and functionally testing and integrating these data. The FaceBase website ( will serve as a web home for these efforts, providing interactive tools for exploring these datasets, together with discussion forums and other services to support and foster collaboration within the craniofacial research community.

  224. Dynamic lineage analysis of embryonic morphogenesis using transgenic quail and 4D multispectral imaging

    Bower, Danielle V.; Sato, Yuki; Lansford, Rusty. Genesis (New York, N.Y.: 2000). vol. 49(7), 619–643. July 2011.

    We describe the development of transgenic quail that express various fluorescent proteins in targeted manners and their use as a model system that integrates advanced imaging approaches with conventional and emerging molecular genetics technologies. We also review the progression and complications of past fate mapping techniques that led us to generate transgenic quail, which permit dynamic imaging of amniote embryogenesis with unprecedented subcellular resolution.

  225. SMAD4-mediated WNT signaling controls the fate of cranial neural crest cells during tooth morphogenesis

    Li, Jingyuan; Huang, Xiaofeng; Xu, Xun; Mayo, Julie; Bringas, Pablo; Jiang, Rulang; Wang, Songling; Chai, Yang. Development (Cambridge, England). vol. 138(10), 1977–1989. May 2011.

    TGFβ/BMP signaling regulates the fate of multipotential cranial neural crest (CNC) cells during tooth and jawbone formation as these cells differentiate into odontoblasts and osteoblasts, respectively. The functional significance of SMAD4, the common mediator of TGFβ/BMP signaling, in regulating the fate of CNC cells remains unclear. In this study, we investigated the mechanism of SMAD4 in regulating the fate of CNC-derived dental mesenchymal cells through tissue-specific inactivation of Smad4. Ablation of Smad4 results in defects in odontoblast differentiation and dentin formation. Moreover, ectopic bone-like structures replaced normal dentin in the teeth of Osr2-IresCre;Smad4(fl/fl) mice. Despite the lack of dentin, enamel formation appeared unaffected in Osr2-IresCre;Smad4(fl/fl) mice, challenging the paradigm that the initiation of enamel development depends on normal dentin formation. At the molecular level, loss of Smad4 results in downregulation of the WNT pathway inhibitors Dkk1 and Sfrp1 and in the upregulation of canonical WNT signaling, including increased β-catenin activity. More importantly, inhibition of the upregulated canonical WNT pathway in Osr2-IresCre;Smad4(fl/fl) dental mesenchyme in vitro partially rescued the CNC cell fate change. Taken together, our study demonstrates that SMAD4 plays a crucial role in regulating the interplay between TGFβ/BMP and WNT signaling to ensure the proper CNC cell fate decision during organogenesis.

  226. Mouse resources for craniofacial research

    Murray, Stephen A.. Genesis (New York, N.Y.: 2000). vol. 49(4), 190–199. April 2011.

    The mouse, as a genetically defined and easily manipulated model organism, has played a critical role in unraveling the mechanisms of craniofacial development and dysmorphology. While numerous gene knockout strains that display craniofacial abnormalities and essential recombinase tool strains with craniofacial-specific expression have been generated, many are absent from public repositories. Large-scale, international resource-generating initiatives promise to address this concern, providing a comprehensive set of targeted mutations and a suite of new Cre driver strains. In addition, panels of genetically defined strains provide tools to dissect the multigenic, complex nature of craniofacial development, adding to the foundation of information gained from single gene studies. Continued progress will require awareness and access to these essential mouse resources. In this review, current mouse resources, large-scale efforts, and potential future directions will be outlined and discussed.

  227. Cleft lip and palate: understanding genetic and environmental influences

    Dixon, Michael J.; Marazita, Mary L.; Beaty, Terri H.; Murray, Jeffrey C.. Nature Reviews. Genetics. vol. 12(3), 167–178. March 2011.

    Clefts of the lip and/or palate (CLP) are common birth defects of complex aetiology. CLP can occur in isolation or as part of a broad range of chromosomal, Mendelian or teratogenic syndromes. Although there has been marked progress in identifying genetic and environmental triggers for syndromic CLP, the aetiology of the more common non-syndromic (isolated) forms remains poorly characterized. Recently, using a combination of epidemiology, careful phenotyping, genome-wide association studies and analysis of animal models, several distinct genetic and environmental risk factors have been identified and confirmed for non-syndromic CLP. These findings have advanced our understanding of developmental biology and created new opportunities for clinical translational research.

  228. Observation of miRNA gene expression in zebrafish embryos by in situ hybridization to microRNA primary transcripts

    He, Xinjun; Yan, Yi-Lin; DeLaurier, April; Postlethwait, John H.. Zebrafish. vol. 8(1), 1–8. March 2011.

    MicroRNAs (miRNAs) add a previously unexpected layer to the post-transcriptional regulation of protein production. Although locked nucleic acids (LNAs) reveal the distribution of mature miRNAs by in situ hybridization (ISH) experiments in zebrafish and other organisms, high cost has restricted their use. Further, LNA probes designed to recognize mature miRNAs do not distinguish expression patterns of two miRNA genes that produce the same mature miRNA sequence. Riboprobes are substantially less expensive than LNAs, but have not been used to detect miRNA gene expression because they do not bind with high affinity to the short, 22-nucleotide-long mature miRNAs. To solve these problems, we capitalized on the fact that miRNAs are initially transcribed into long primary transcripts (pri-mRNAs). We show here that conventional digoxigenin-labeled riboprobes can bind to primary miRNA transcripts in zebrafish embryos. We tested intergenic and intronic miRNAs (miR-10d, miR-21, miR-27a, miR-126a, miR-126b, miR-138, miR-140, miR-144, miR-196a1, miR-196a2, miR-196a2b [miR-196c], miR-196b, miR-196b1b [miR-196d], miR-199, miR-214, miR-200, and miR-222) in whole mounts and some of these in histological sections. Results showed that pri-miRNA ISH provides an attractive and cost-effective tool to study miRNA expression by ISH. We use this method to show that miR-126a and miR-126b are transcribed in the caudal vasculature in the pattern of their neighboring gene ci116 or host gene egfl7, respectively, and that the chondrocyte miRNA mir-140 lies downstream of Sox9 in development of the craniofacial skeleton.

  229. The UCSC Genome Browser database: update 2011

    Fujita, Pauline A.; Rhead, Brooke; Zweig, Ann S.; Hinrichs, Angie S.; Karolchik, Donna; Cline, Melissa S.; Goldman, Mary; Barber, Galt P.; Clawson, Hiram; Coelho, Antonio; Diekhans, Mark; Dreszer, Timothy R.; Giardine, Belinda M.; Harte, Rachel A.; Hillman-Jackson, Jennifer; Hsu, Fan; Kirkup, Vanessa; Kuhn, Robert M.; Learned, Katrina; Li, Chin H.; Meyer, Laurence R.; Pohl, Andy; Raney, Brian J.; Rosenbloom, Kate R.; Smith, Kayla E.; Haussler, David; Kent, W. James. Nucleic Acids Research. vol. 39(Database issue), D876–882. January 2011.

    The University of California, Santa Cruz Genome Browser ( offers online access to a database of genomic sequence and annotation data for a wide variety of organisms. The Browser also has many tools for visualizing, comparing and analyzing both publicly available and user-generated genomic data sets, aligning sequences and uploading user data. Among the features released this year are a gene search tool and annotation track drag-reorder functionality as well as support for BAM and BigWig/BigBed file formats. New display enhancements include overlay of multiple wiggle tracks through use of transparent coloring, options for displaying transformed wiggle data, a ’mean+whiskers’ windowing function for display of wiggle data at high zoom levels, and more color schemes for microarray data. New data highlights include seven new genome assemblies, a Neandertal genome data portal, phenotype and disease association data, a human RNA editing track, and a zebrafish Conservation track. We also describe updates to existing tracks.

  230. Groupwise Pose Normalization for Craniofacial Applications

    Chen, Jiun-Hung; Shapiro, Linda G.. Proceedings. IEEE Workshop on Applications of Computer Vision. vol. 2011, 248–255. January 2011.

    A general framework is proposed for solving groupwise pose normalization problems and is analyzed in detail under different feature spaces. The analysis shows that using principal component analysis for pose normalization is a special case of using the proposed framework under a special feature space. The experimental results on two craniofacial datasets show the proposed method achieved promising results for solving groupwise pose normalization problems for craniofacial applications.

  231. The Use of Genetic Programming for Learning 3D Craniofacial Shape Quantifications

    Atmosukarto, Indriyati; Shapiro, Linda G.; Heike, Carrie. Proceedings of the ... IAPR International Conference on Pattern Recognition. International Conference on Pattern Recognition. vol. 2010, 2444–2447. 2010.

    Craniofacial disorders commonly result in various head shape dysmorphologies. The goal of this work is to quantify the various 3D shape variations that manifest in the different facial abnormalities in individuals with a craniofacial disorder called 22q11.2 Deletion Syndrome. Genetic programming (GP) is used to learn the different 3D shape quantifications. Experimental results show that the GP method achieves a higher classification rate than those of human experts and existing computer algorithms [1], [2].

  232. A landmark-free framework for the detection and description of shape differences in embryos

    Rolfe, S. M.; Shapiro, L. G.; Cox, T. C.; Maga, A. M.; Cox, L. L.. Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference. vol. 2011, 5153–5156. 2011.

    This paper introduces a new method to quantify and characterize shape changes during early facial development without the use of landmarks. Landmarks are traditionally used in morphometric analysis, but very few can be identified reliably across all stages of embryonic development. This method uses deformable registration to produce a dense vector field describing the point correspondences between two images. Low and mid-level features are extracted from the deformable vector field to find regions of organized differences that are biologically relevant. These methods are shown to detect regions of difference when evaluated on chick embryo images warped with small magnitude deformations in regions critical to midfacial development.

  233. 3D Point Correspondence by Minimum Description Length in Feature Space

    Chen, Jiun-Hung; Zheng, Ke Colin; Shapiro, Linda G.. Computer vision - ECCV ...: ... European Conference on Computer Vision: proceedings. European Conference on Computer Vision. vol. 6313, 621–634. 2010.

    Finding point correspondences plays an important role in automatically building statistical shape models from a training set of 3D surfaces. For the point correspondence problem, Davies et al. [1] proposed a minimum-description-length-based objective function to balance the training errors and generalization ability. A recent evaluation study [2] that compares several well-known 3D point correspondence methods for modeling purposes shows that the MDL-based approach [1] is the best method. We adapt the MDL-based objective function for a feature space that can exploit nonlinear properties in point correspondences, and propose an efficient optimization method to minimize the objective function directly in the feature space, given that the inner product of any vector pair can be computed in the feature space. We further employ a Mercer kernel [3] to define the feature space implicitly. A key aspect of our proposed framework is the generalization of the MDL-based objective function to kernel principal component analysis (KPCA) [4] spaces and the design of a gradient-descent approach to minimize such an objective function. We compare the generalized MDL objective function on KPCA spaces with the original one and evaluate their abilities in terms of reconstruction errors and specificity. From our experimental results on different sets of 3D shapes of human body organs, the proposed method performs significantly better than the original method.

  234. Three-dimensional head shape quantification for infants with and without deformational plagiocephaly

    Atmosukarto, I.; Shapiro, L. G.; Starr, J. R.; Heike, C. L.; Collett, B.; Cunningham, M. L.; Speltz, M. L.. The Cleft Palate-Craniofacial Journal: Official Publication of the American Cleft Palate-Craniofacial Association. vol. 47(4), 368–377. July 2010.

    OBJECTIVE: The authors developed and tested three-dimensional (3D) indices for quantifying the severity of deformational plagiocephaly (DP). DESIGN: The authors evaluated the extent to which infants with and without DP (as determined by clinic referral and two experts’ ratings) could be correctly classified. PARTICIPANTS: Infants aged 4 to 11 months, including 154 with diagnosed DP and 100 infants without a history of DP or other craniofacial condition. After excluding participants with discrepant expert ratings, data from 90 infants with DP and 50 infants without DP were retained. MEASUREMENTS: Two-dimensional (2D) histograms of surface normal vector angles were extracted from 3D mesh data and used to compute the severity scores. OUTCOME MEASURES: Left posterior flattening score (LPFS), right posterior flattening score (RPFS), asymmetry score (AS), absolute asymmetry score (AAS), and an approximation of a previously described 2D measure, the oblique cranial length ratio (aOCLR). Two-dimensional histograms localized the posterior flatness for each participant. ANALYSIS: The authors fit receiver operating characteristic curves and calculated the area under the curves (AUC) to evaluate the relative accuracy of DP classification using the above measures. RESULTS: The AUC statistics were AAS = 91%, LPFS = 97%, RPFS = 91%, AS = 99%, and aOCLR = 79%. CONCLUSION: Novel 3D-based plagiocephaly posterior severity scores provided better sensitivity and specificity in the discrimination of plagiocephalic and typical head shapes than the 2D measurements provided by a close approximation of OCLR. These indices will allow for more precise quantification of the DP phenotype in future studies on the prevalence of this condition, which may lead to improved clinical care.

  235. 3D digital stereophotogrammetry: a practical guide to facial image acquisition

    Heike, Carrie L.; Upson, Kristen; Stuhaug, Erik; Weinberg, Seth M.. Head & Face Medicine. vol. 6, 18. July 2010.

    The use of 3D surface imaging technology is becoming increasingly common in craniofacial clinics and research centers. Due to fast capture speeds and ease of use, 3D digital stereophotogrammetry is quickly becoming the preferred facial surface imaging modality. These systems can serve as an unparalleled tool for craniofacial surgeons, proving an objective digital archive of the patient’s face without exposure to radiation. Acquiring consistent high-quality 3D facial captures requires planning and knowledge of the limitations of these devices. Currently, there are few resources available to help new users of this technology with the challenges they will inevitably confront. To address this deficit, this report will highlight a number of common issues that can interfere with the 3D capture process and offer practical solutions to optimize image quality.

  236. PhenoHM: human-mouse comparative phenome-genome server

    Sardana, Divya; Vasa, Suresh; Vepachedu, Nishanth; Chen, Jing; Gudivada, Ranga Chandra; Aronow, Bruce J.; Jegga, Anil G.. Nucleic Acids Research. vol. 38(Web Server issue), W165–174. July 2010.

    PhenoHM is a human-mouse comparative phenome-genome server that facilitates cross-species identification of genes associated with orthologous phenotypes (; full open access, login not required). Combining and extrapolating the knowledge about the roles of individual gene functions in the determination of phenotype across multiple organisms improves our understanding of gene function in normal and perturbed states and offers the opportunity to complement biologically the rapidly expanding strategies in comparative genomics. The Mammalian Phenotype Ontology (MPO), a structured vocabulary of phenotype terms that leverages observations encompassing the consequences of mouse gene knockout studies, is a principal component of mouse phenotype knowledge source. On the other hand, the Unified Medical Language System (UMLS) is a composite collection of various human-centered biomedical terminologies. In the present study, we mapped terms reciprocally from the MPO to human disease concepts such as clinical findings from the UMLS and clinical phenotypes from the Online Mendelian Inheritance in Man knowledgebase. By cross-mapping mouse-human phenotype terms, extracting implicated genes and extrapolating phenotype-gene associations between species PhenoHM provides a resource that enables rapid identification of genes that trigger similar outcomes in human and mouse and facilitates identification of potentially novel disease causal genes. The PhenoHM server can be accessed freely at

  237. Limits of sequence and functional conservation

    Pennacchio, Len A.; Visel, Axel. Nature Genetics. vol. 42(7), 557–558. July 2010.

    Sequence conservation of noncoding DNA across species can indicate functional conservation. However, a new study demonstrates notable differences between human and mouse stem cell regulatory networks, suggesting caution in generalizing from sequence to functional conservation.

  238. ToppCluster: a multiple gene list feature analyzer for comparative enrichment clustering and network-based dissection of biological systems

    Kaimal, Vivek; Bardes, Eric E.; Tabar, Scott C.; Jegga, Anil G.; Aronow, Bruce J.. Nucleic Acids Research. vol. 38(Web Server issue), W96–102. July 2010.

    ToppCluster is a web server application that leverages a powerful enrichment analysis and underlying data environment for comparative analyses of multiple gene lists. It generates heatmaps or connectivity networks that reveal functional features shared or specific to multiple gene lists. ToppCluster uses hypergeometric tests to obtain list-specific feature enrichment P-values for currently 17 categories of annotations of human-ortholog genes, and provides user-selectable cutoffs and multiple testing correction methods to control false discovery. Each nameable gene list represents a column input to a resulting matrix whose rows are overrepresented features, and individual cells per-list P-values and corresponding genes per feature. ToppCluster provides users with choices of tabular outputs, hierarchical clustering and heatmap generation, or the ability to interactively select features from the functional enrichment matrix to be transformed into XGMML or GEXF network format documents for use in Cytoscape or Gephi applications, respectively. Here, as example, we demonstrate the ability of ToppCluster to enable identification of list-specific phenotypic and regulatory element features (both cis-elements and 3’UTR microRNA binding sites) among tissue-specific gene lists. ToppCluster’s functionalities enable the identification of specialized biological functions and regulatory networks and systems biology-based dissection of biological states. ToppCluster can be accessed freely at

  239. Smad4-Shh-Nfic signaling cascade-mediated epithelial-mesenchymal interaction is crucial in regulating tooth root development

    Huang, Xiaofeng; Xu, Xun; Bringas, Pablo; Hung, Yee Ping; Chai, Yang. Journal of Bone and Mineral Research: The Official Journal of the American Society for Bone and Mineral Research. vol. 25(5), 1167–1178. May 2010.

    Transforming growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) signaling is crucial for regulating epithelial-mesenchymal interaction during organogenesis, and the canonical Smad pathway-mediated TGF-beta/BMP signaling plays important roles during development and disease. During tooth development, dental epithelial cells, known as Hertwig’s epithelial root sheath (HERS), participate in root formation following crown development. However, the functional significance of HERS in regulating root development remains unknown. In this study we investigated the signaling mechanism of Smad4, the common Smad for TGF-beta/BMP signaling, in HERS in regulating root development. Tissue-specific inactivation of Smad4 in HERS results in abnormal enamel and dentin formation in K14-Cre;Smad4(fl/fl) mice. HERS enlarges but cannot elongate to guide root development without Smad4. At the molecular level, Smad4-mediated TGF-beta/BMP signaling is required for Shh expression in HERS and Nfic (nuclear factor Ic) expression in the cranial neural crest (CNC)-derived dental mesenchyme. Nfic is crucial for root development, and loss of Nfic results in a CNC-derived dentin defect similar to the one of K14-Cre;Smad4(fl/fl) mice. Significantly, we show that ectopic Shh induces Nfic expression in dental mesenchyme and partially rescues root development in K14-Cre;Smad4(fl/fl) mice. Taken together, our study has revealed an important signaling mechanism in which TGF-beta/BMP signaling relies on a Smad-dependent mechanism in regulating Nfic expression via Shh signaling to control root development. The interaction between HERS and the CNC-derived dental mesenchyme may guide the size, shape, and number of tooth roots.

  240. Transforming growth factor-beta regulates basal transcriptional regulatory machinery to control cell proliferation and differentiation in cranial neural crest-derived osteoprogenitor cells

    Iwata, Jun-ichi; Hosokawa, Ryoichi; Sanchez-Lara, Pedro A.; Urata, Mark; Slavkin, Harold; Chai, Yang. The Journal of Biological Chemistry. vol. 285(7), 4975–4982. February 2010.

    Transforming growth factor-beta (Tgf-beta) signaling is crucial for regulating craniofacial development. Loss of Tgf-beta signaling results in defects in cranial neural crest cells (CNCC), but the mechanism by which Tgf-beta signaling regulates bone formation in CNCC-derived osteogenic cells remains largely unknown. In this study, we discovered that Tgf-beta regulates the basal transcriptional regulatory machinery to control intramembranous bone development. Specifically, basal transcription factor Taf4b is down-regulated in the CNCC-derived intramembranous bone in Tgfbr2(fl/fl);Wnt1-Cre mice. Tgf-beta specifically induces Taf4b expression. Moreover, small interfering RNA knockdown of Taf4b results in decreased cell proliferation and altered osteogenic differentiation in primary mouse embryonic maxillary mesenchymal cells, as seen in Tgfbr2 mutant cells. In addition, we show that Taf1 is decreased at the osteogenic initiation stage in the maxilla of Tgfbr2 mutant mice. Furthermore, small interfering RNA knockdown of Taf4b and Taf1 together in primary mouse embryonic maxillary mesenchymal cells results in up-regulated osteogenic initiator Runx2 expression, with decreased cell proliferation and altered osteogenic differentiation. Our results indicate a critical function of Tgf-beta-mediated basal transcriptional factors in regulating osteogenic cell proliferation and differentiation in CNCC-derived osteoprogenitor cells during intramembranous bone formation.

  241. Shape-Based Classification of 3D Head Data

    Shapiro, Linda; Wilamowska, Katarzyna; Atmosukarto, Indriyati; Wu, Jia; Heike, Carrie; Speltz, Matthew; Cunningham, Michael. Proceedings of the ... International Conference on Image Analysis and Processing. International Conference on Image Analysis and Processing. vol. 5716, 692–700. 2009.

    Craniofacial disorders are one of the most common category of birth defects worldwide, and are an important topic of biomedical research. In order to better understand these disorders and correlate them with genetic patterns and life outcomes, researchers need to quantify the craniofacial anatomy. In this paper we introduce several different craniofacial descriptors that are being used in research studies for two craniofacial disorders: the 22q11.2 deletion syndrome (a genetic disorder) and plagiocephaly/brachycephaly, disorders caused by pressure on the head. Experimental results show that our descriptors show promise for quantifying craniofacial shape.