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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. 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.


  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.


  1. 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.

  2. 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.

  3. 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.


  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.


  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.


  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.


  1. 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.

  2. Genome-wide compendium and functional assessment of in vivo heart enhancers

    Dickel, Diane E.; Barozzi, Iros; Zhu, Yiwen; Fukuda-Yuzawa, Yoko; Osterwalder, Marco; Mannion, Brandon J.; May, Dalit; Spurrell, Cailyn H.; Plajzer-Frick, Ingrid; Pickle, Catherine S.; Lee, Elizabeth; Garvin, Tyler H.; Kato, Momoe; Akiyama, Jennifer A.; Afzal, Veena; Lee, Ah Young; Gorkin, David U.; Ren, Bing; Rubin, Edward M.; Visel, Axel; Pennacchio, Len A.. Nature Communications. vol. 7, 12923. October 2016.

    Whole-genome sequencing is identifying growing numbers of non-coding variants in human disease studies, but the lack of accurate functional annotations prevents their interpretation. We describe the genome-wide landscape of distant-acting enhancers active in the developing and adult human heart, an organ whose impairment is a predominant cause of mortality and morbidity. Using integrative analysis of \textgreater35 epigenomic data sets from mouse and human pre- and postnatal hearts we created a comprehensive reference of \textgreater80,000 putative human heart enhancers. To illustrate the importance of enhancers in the regulation of genes involved in heart disease, we deleted the mouse orthologs of two human enhancers near cardiac myosin genes. In both cases, we observe in vivo expression changes and cardiac phenotypes consistent with human heart disease. Our study provides a comprehensive catalogue of human heart enhancers for use in clinical whole-genome sequencing studies and highlights the importance of enhancers for cardiac function.

  3. 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.

  4. Progressive Loss of Function in a Limb Enhancer during Snake Evolution

    Kvon, Evgeny Z.; Kamneva, Olga K.; Melo, Uirá S.; Barozzi, Iros; Osterwalder, Marco; Mannion, Brandon J.; Tissières, Virginie; Pickle, Catherine S.; Plajzer-Frick, Ingrid; Lee, Elizabeth A.; Kato, Momoe; Garvin, Tyler H.; Akiyama, Jennifer A.; Afzal, Veena; Lopez-Rios, Javier; Rubin, Edward M.; Dickel, Diane E.; Pennacchio, Len A.; Visel, Axel. Cell. vol. 167(3), 633–642.e11. October 2016.

    The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution. PAPERCLIP.

  5. 52 Genetic Loci Influencing Myocardial Mass

    van der Harst, Pim; van Setten, Jessica; Verweij, Niek; Vogler, Georg; Franke, Lude; Maurano, Matthew T.; Wang, Xinchen; Mateo Leach, Irene; Eijgelsheim, Mark; Sotoodehnia, Nona; Hayward, Caroline; Sorice, Rossella; Meirelles, Osorio; Lyytikäinen, Leo-Pekka; Polašek, Ozren; Tanaka, Toshiko; Arking, Dan E.; Ulivi, Sheila; Trompet, Stella; Müller-Nurasyid, Martina; Smith, Albert V.; Dörr, Marcus; Kerr, Kathleen F.; Magnani, Jared W.; Del Greco M, Fabiola; Zhang, Weihua; Nolte, Ilja M.; Silva, Claudia T.; Padmanabhan, Sandosh; Tragante, Vinicius; Esko, Tõnu; Abecasis, Gonçalo R.; Adriaens, Michiel E.; Andersen, Karl; Barnett, Phil; Bis, Joshua C.; Bodmer, Rolf; Buckley, Brendan M.; Campbell, Harry; Cannon, Megan V.; Chakravarti, Aravinda; Chen, Lin Y.; Delitala, Alessandro; Devereux, Richard B.; Doevendans, Pieter A.; Dominiczak, Anna F.; Ferrucci, Luigi; Ford, Ian; Gieger, Christian; Harris, Tamara B.; Haugen, Eric; Heinig, Matthias; Hernandez, Dena G.; Hillege, Hans L.; Hirschhorn, Joel N.; Hofman, Albert; Hubner, Norbert; Hwang, Shih-Jen; Iorio, Annamaria; Kähönen, Mika; Kellis, Manolis; Kolcic, Ivana; Kooner, Ishminder K.; Kooner, Jaspal S.; Kors, Jan A.; Lakatta, Edward G.; Lage, Kasper; Launer, Lenore J.; Levy, Daniel; Lundby, Alicia; Macfarlane, Peter W.; May, Dalit; Meitinger, Thomas; Metspalu, Andres; Nappo, Stefania; Naitza, Silvia; Neph, Shane; Nord, Alex S.; Nutile, Teresa; Okin, Peter M.; Olsen, Jesper V.; Oostra, Ben A.; Penninger, Josef M.; Pennacchio, Len A.; Pers, Tune H.; Perz, Siegfried; Peters, Annette; Pinto, Yigal M.; Pfeufer, Arne; Pilia, Maria Grazia; Pramstaller, Peter P.; Prins, Bram P.; Raitakari, Olli T.; Raychaudhuri, Soumya; Rice, Ken M.; Rossin, Elizabeth J.; Rotter, Jerome I.; Schafer, Sebastian; Schlessinger, David; Schmidt, Carsten O.; Sehmi, Jobanpreet; Silljé, Herman H. W.; Sinagra, Gianfranco; Sinner, Moritz F.; Slowikowski, Kamil; Soliman, Elsayed Z.; Spector, Timothy D.; Spiering, Wilko; Stamatoyannopoulos, John A.; Stolk, Ronald P.; Strauch, Konstantin; Tan, Sian-Tsung; Tarasov, Kirill V.; Trinh, Bosco; Uitterlinden, Andre G.; van den Boogaard, Malou; van Duijn, Cornelia M.; van Gilst, Wiek H.; Viikari, Jorma S.; Visscher, Peter M.; Vitart, Veronique; Völker, Uwe; Waldenberger, Melanie; Weichenberger, Christian X.; Westra, Harm-Jan; Wijmenga, Cisca; Wolffenbuttel, Bruce H.; Yang, Jian; Bezzina, Connie R.; Munroe, Patricia B.; Snieder, Harold; Wright, Alan F.; Rudan, Igor; Boyer, Laurie A.; Asselbergs, Folkert W.; van Veldhuisen, Dirk J.; Stricker, Bruno H.; Psaty, Bruce M.; Ciullo, Marina; Sanna, Serena; Lehtimäki, Terho; Wilson, James F.; Bandinelli, Stefania; Alonso, Alvaro; Gasparini, Paolo; Jukema, J. Wouter; Kääb, Stefan; Gudnason, Vilmundur; Felix, Stephan B.; Heckbert, Susan R.; de Boer, Rudolf A.; Newton-Cheh, Christopher; Hicks, Andrew A.; Chambers, John C.; Jamshidi, Yalda; Visel, Axel; Christoffels, Vincent M.; Isaacs, Aaron; Samani, Nilesh J.; de Bakker, Paul I. W.. Journal of the American College of Cardiology. vol. 68(13), 1435–1448. September 2016.

    BACKGROUND: Myocardial mass is a key determinant of cardiac muscle function and hypertrophy. Myocardial depolarization leading to cardiac muscle contraction is reflected by the amplitude and duration of the QRS complex on the electrocardiogram (ECG). Abnormal QRS amplitude or duration reflect changes in myocardial mass and conduction, and are associated with increased risk of heart failure and death. OBJECTIVES: This meta-analysis sought to gain insights into the genetic determinants of myocardial mass. METHODS: We carried out a genome-wide association meta-analysis of 4 QRS traits in up to 73,518 individuals of European ancestry, followed by extensive biological and functional assessment. RESULTS: We identified 52 genomic loci, of which 32 are novel, that are reliably associated with 1 or more QRS phenotypes at p \textless 1 × 10(-8). These loci are enriched in regions of open chromatin, histone modifications, and transcription factor binding, suggesting that they represent regions of the genome that are actively transcribed in the human heart. Pathway analyses provided evidence that these loci play a role in cardiac hypertrophy. We further highlighted 67 candidate genes at the identified loci that are preferentially expressed in cardiac tissue and associated with cardiac abnormalities in Drosophila melanogaster and Mus musculus. We validated the regulatory function of a novel variant in the SCN5A/SCN10A locus in vitro and in vivo. CONCLUSIONS: Taken together, our findings provide new insights into genes and biological pathways controlling myocardial mass and may help identify novel therapeutic targets.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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, http://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).

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  21. 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.

  22. 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.

  23. 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.

  24. 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.


  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.


  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  21. 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.

  22. 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.

  23. 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.

  24. 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.

  25. 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.


  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  21. 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.

  22. 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.

  23. 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.


  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.


  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

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

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

  14. 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.


  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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].

  7. 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

  8. 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.

  9. 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.


  1. 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.