Projects

Human
3D Facial Norms Database

This is a completed FaceBase project

Although ample evidence exists that facial appearance and structure are highly heritable, there is a dearth of information regarding how variation in specific genes relates to the diversity of facial forms evident in our species. With the advent of affordable, non-invasive 3D surface imaging technology, it is now possible to capture detailed quantitative information about the face in a large number of individuals. By coupling state- of-the-art 3D imaging with advances in high-throughput genotyping, an unparalleled opportunity exists to map the genetic determinants of normal facial variation. An improved understanding of the relationship between genotype and facial phenotype may help illuminate the factors influencing liability to common craniofacial anomalies, particularly orofacial clefts, which are among the most prevalent birth defects in humans. This proposal has two major goals: (1) to construct a nonnative repository of 3D facial and genetic data and (2) to utilize this data repository to identify genes that influence normal midfacial variation. The first goal will focus on data generation and resource development and will involve the collection of 3D facial surface images and DNA samples on 3500 healthy Caucasian individuals (age 5-40) drawn from the general population. Quantitative facial measures will be extracted from the 3D images and all DNA samples will be genotyped for genome-wide SNP markers. Working in conjunction with the FaceBase hub, our intent is to create a scalable. Interactive and minable data resource available to outside investigators, which will contain facial measures, 3D images and genotypes. Ultimately, it is hoped that such a database will facilitate novel research initiatives. To illustrate this potential, the second goal of this proposal will focus on identifying SNPs associated with variation in midfacial morphology, including those facial features relevant to orofacial cleft predisposition. Salient measures of midfacial morphology will be derived from 3D facial surface images, and a genome-wide association approach will then be employed to identify polymorphisms that influence quantitative variation in the facial features of interest.

 

3D Facial Norms Database

Data is now available.  Please contact help@facebase.org with your request to receive further information.

Oral Clefts: Moving from Genome-Wide Studies Toward Functional Genomics

This is a completed FaceBase project

We will follow up on signals from a genome wide association study (GWAS) of oral clefts now being conducted with support from U01-DE-004425; "International Consortium to Identify Genes & Interactions Controlling Oral Clefts", 2007-2009; TH Beaty, PI). Oral clefts are among the most common human birth defects and have a complex and heterogeneous etiology. Genotyping for this project should be completed in early 2009, and our analysis will identify genes influencing risk directly, those acting only in the presence of an environmental risk factor, and/or genes showing measurable parent-of-origin effects which may represent imprinting. In this response to the FaceBase initiative (RFA-DE-09-003), we will build upon our GWAS results by using high throughput sequencing (HTS) techniques on genes/regions yielding statistical evidence of linkage and disequilibrium in the GWAS. We will first focus on genes identified through analysis of single nucleotide polymorphic (SNP) markers from our GWAS and will use HTS to identify all variants (rare mutations and novel markers) that may be causal, directly or indirectly. We will then undertake a systematic analysis of intensity data and the 60,000 markers in regions of known copy number variants (CNV) available on this platform to test genes that may influence risk through structural variation.

 

Data is now available consisting of imputed markers generated with BEAGLE in CL/P case-parent trios from an international consortium. Imputation was performed taking family structure into account. For each population (Asian and European) ped- and map-formatted files (http://pngu.mgh.harvard.edu/~purcell/plink/data.shtml) are provided. 

 

Please submit a Data Access Request form if you are interested.  View Data Dictionary description.

Investigators 
Human Genomics Analysis Interface for Facebase 2

There are now several large human genomics databases relevant to craniofacial research, including multiple databases funded in part by the first FaceBase consortium. Direct access to the individual level data from such databases can be cumbersome, therefore the current project seeks to make analysis of pertinent genomics data available to FaceBase users without releasing the individual level data. The current project has three major goals in order to develop a genomics analysis interface for FaceBase 2.

GOAL 1: To develop a software interface that will enable FaceBase users to apply human genetics analysis software (e.g. PLINK) to human genomics data from craniofacial research, with access to these tools through the FaceBase 2 Hub. NOTE: no individual level data will be available to FaceBase users; the data for analysis will be located on a secure location of the FaceBase 2 Hub. The purpose of these tools is to allow FaceBase users to explore genomics data (e.g. GWAS, sequencing) in order to ask research questions or to decide whether to download the underlying data from its home data repository (dbGaP in most cases).

GOAL 2: To identify pertinent genomics data on dbGaP, and/or within the craniofacial research community that would then be incorporated into the above system (i.e. no access to individual level data through FaceBase, only the ability to query/analyze the data). Some of the data to be made available was created as part of FaceBase I spoke projects (Beaty, Spritz, and Weinberg/Marazita spoke projects).

GOAL 3: To create analysis results datasets to make available on FaceBase, and also data tracks for the UCSC genome browser (or other repositories designated by the FaceBase 2 Hub) from the human genomics results. This will make such results immediately available to FaceBase users, and also available to a wider audience through the genome browser or other centralized analysis results databases.

Investigators 
Genomic and Transgenic Resources for Craniofacial Enhancer Studies

Genetic studies have shown that distant-acting regulatory sequences (enhancers) embedded in the vast non-coding portion of the human genome play important roles in craniofacial development and susceptibility to craniofacial birth defects. The mechanistic exploration of these distant-acting enhancers continues to be difficult because the genomic location and in vivo function of most craniofacial enhancers remains unknown.

 

As members of FaceBase 1, we generated first sets of annotation and functional data for distal enhancers controlling craniofacial development. These resources proved to be of significant value to the craniofacial research community. However, these efforts captured only a small proportion of the enhancers that are active during craniofacial development in vivo. Here we propose to characterize the gene regulatory landscape of craniofacial development more comprehensively using new and complementary approaches. The specific aims are: 

 

1) We will map predicted enhancers by ChIP-seq from embryonic mouse and human facial tissues. In preliminary studies, we used ChIP-seq with the enhancer-associated protein p300 to identify several thousand enhancers predicted to be active in the mouse face at e11.5 and in the secondary palate at later stages of development. Using ChIP-seq for a panel of histone modifications (H3K4me1, H3K27ac, H3K27me3), which will require less tissue and increase the sensitivity of enhancer discovery by an order of magnitude, we will obtain higher-resolution data from all subregions of the developing mouse face at three stages of development (e11.5, e13.5, e15.5). We will complement this mouse-based effort with ChIP-seq on human embryonic face tissue to identify human-specific craniofacial enhancers not functionally conserved in mice.

 

2) In initial studies we characterized ~200 craniofacial enhancers in vivo in transgenic reporter assays. Taking advantage of protocols and collaborations established during FaceBase 1, we will continue to generate critically needed in vivo transgenic assays accompanied by optical projection tomography to characterize enhancers residing in new craniofacial loci identified by FaceBase 2 investigators and outside groups. This will include testing of enhancer variants associated with craniofacial malformations.

 

The datasets, vectors and transgenic embryos produced through our efforts will be made available as resources to the craniofacial research community. We are deeply committed to our ongoing collaborative interactions with the Hub and other Spoke projects, contributing to and taking advantage of the unique research opportunities enabled through the FaceBase program.

Investigators 
Epigenetic Landscapes and Regulatory Divergence of Human Craniofacial Traits

During development, cranial Neural Crest Cells (cNCCs) play major roles in establishing craniofacial morphology and determining its species-specific variation. To understand human distinctive features it is imperative to study human cNCCs and their derivatives in addition to cNCCs from model organisms. Since human NC formation occurs at 3 to 6 weeks of gestation and is largely inaccessible to genetic studies, we have established a human pluripotent stem cell-based cNCC differentiation model in the dish with high relevance to craniofacial development. Moreover, we have extended our model to chimpanzee cNCCs, allowing us to identify molecular features that distinguish human cNCCs from those of our closest evolutionary cousins.

 

First, we propose to characterize epigenetic landscapes and transcriptomes of human and chimpanzee cNCCs and to identify conserved and species-specific cis-regulatory elements utilized by this unique cell type. Since chromatin modification maps from NCCs of any organism are not yet publicly available beyond our report, we will generate chromatin marking profiles from a cohort of human and chimp post- migratory cNCCs, complemented with transcriptome analyses. Thus, we will create ""reference epigenomes"" that will be annotated for active and poised enhancers and promoters. We have already identified over 2000 regulatory elements that show strong species-specific bias in their chromatin marks in human versus chimpanzee, arguing that human-specific cNCC molecular features do exist and may underlie human-specific craniofacial divergence. These datasets will provide a rich resource for future investigations of the transcriptional and epigenetic basis of human craniofacial evolution, development, and disease.

 

Second, we will analyze candidate human-specific craniofacial enhancer activity in vivo. To this end, transgenic reporter assays in mouse embryos will be used to analyze the activity of 50 human regulatory elements that either gained or lost active enhancer signature in human cNCCs, as compared to the 50 orthologous chimpanzee regions. Thus, we will generate a validated set of human-specific craniofacial enhancers that can be further explored in mechanistic studies. For 10 selected human-specific enhancers exhibiting gain or loss of activity, interesting activity patterns, or relevant associatin with craniofacial development or disease in humans, we will utilize BAC recombineering to further develop founder transgenic lines that will be distributed to the craniofacial community.   

Investigators 
Developing 3D Craniofacial Morphometry Data and Tools to Transform Dysmorphology

Dysmorphology is the branch of pediatrics and clinical genetics concerned with structural birth defects and delineation of syndromes. More than 1500 syndromes that include orofacial dysmorphia have been described. Today, dysmorphology remains largely descriptive, with diagnoses based on subjective or semi-quantitative clinical impressions of facial and other anatomic features.

 

Over the past decade, dramatic technological advances in imaging, quantification, and analysis of variation in complex three-dimensional (3D) shape have revolutionized the assessment of morphologic variation, permitting robust definition of quantitative morphometric phenotypes that can distinguish patients from controls in a variety of syndromes.

 

The goal of this application is to develop systems that will enable diagnostic application of craniofacial 3D morphometrics in clinical practice. We aim to define specific quantitative measures that characterize the aberrant facial shapes in a large number of human dysmorphic syndromes.

 

Specifically, we aim to build a broad and deep 3D morphometric facial scan ""library"" of defined craniofacial dysmorphic syndromes, a resource that can be shared with approved investigators for research purposes via the NIDCR FaceBase Hub; to develop 3D geometric morphometric (GM) and dense surface modeling (DSM) analytical tools to systematically analyze and distinguish dysmorphic syndromes from unaffected individuals and from each other; and finally to develop a functional, automated, prototype clinical tool that is capable of simultaneously distinguishing a large number of syndromes, and that thereby can assist real-time diagnosis of syndromes in the clinical setting.

 

We anticipate that 3D photomorphometric ""deep-phenotyping"", in conjunction with the rapid advent of exome and genome sequencing in clinical medicine, will transform dysmorphology from a clinical art into a medical science.  

Investigators 
Rapid Identification and Validation of Human Craniofacial Development Genes

The advent of new genomic sequencing technologies has made the task of gene discovery in human developmental disorders highly efficient. Simultaneously, advances in gene targeting in model organisms, specifically in zebrafish, have made semi-high throughput validation and analysis of human candidate genes feasible, including those responsible for craniofacial disorders. This application for a new spoke project in FaceBase 2 will take advantage of this convergence of new technologies to identify and functionally validate approximately two dozen genes involved in novel aspects of human craniofacial development.

 

Specifically, we will take advantage of already ascertained collections of craniofacial dysmorphoses from Boston Children's Hospital (BCH) and from King Faisal Specialist Hospital and Research Center (KFSHRC) in Saudi Arabia, where the high incidence of consanguinity makes autozygosity mapping and the identification of recessive causal loci highly feasible. We will extend the work of FaceBase beyond its current focus on disorders of palatal development by including a relatively wide range of craniofacial disorders that involve other components of the craniofacial complex. In addition, use of resources already compiled by FaceBase, including detailed gene expression data in mouse and zebrafish, enhancer analyses, and genome wide association studies, in combination with the present data and publicly available datasets, will further facilitate the functional annotation of these newly validated gene. To provide valuable deliverable resources to other FaceBase investigators and to the community at large, we will pursue three Specific Aims.

 

In Aim 1, we will ascertain and recruit patients with a wide range of craniofacial dysmorphoses of likely monogenic etiology. These patients will not only be identified at the BCH and KFSHRC referral centers, but also solicited from other clinical investigators and potentially even the FaceBase Biorepository.

 

In Aim 2, patients will be prioritized for further study based on the genetic likelihood of identifying a caual variant. We will then perform whole exome and in some cases whole genome sequence (WES/WGS) analysis, on the proband and potentially other family members, using aCGH to ensure genomic integrity and autozygosity mapping where applicable. An existing state-of-the-art computational pipeline will be used to derive a limited set of potentially causal DNA sequence variants and candidate genes.

 

Lastly, in Aim 3, in cases where causation cannot be readily established from known function and expression data, we will seek additional independent confirmatory cases and, in parallel, employ a rapid analysis strategy consisting of high-throughput gene expression analysis, morpholino knockdown, and mutagenesis and transgenesis to prepare GOF and LOF alleles. The results will be forwarded to the FaceBase 2 Coordinating Center, with the key deliverables to the community being a validated gene list of human craniofacial developmental regulatory genes and a set of corresponding zebrafish mutants that can be widely shared for further detailed study.          

Investigators 
Animal
Functional Analysis of Neural Crest and Palate: Imaging Craniofacial Development

This is a completed FaceBase project.

The research plan will refine and deploy a set of advanced tools for the imaging of tissue structure, gene expression domains and cellular dynamics during craniofacial development. Volumetric imaging tools will be used to create accurate 3D atlases that can be digitally dissected to permit the tissue interactions and cellular events to be better understood in the forming faces of normal, mutant and perturbed mouse and avian embryos. Molecular imaging agents, optimized for imaging intact tissues, will be employed to create atlases of the molecular correlates of these embryos. Finally, intravital imaging tools will be used to study cell and tissue interactions as they take place, offering a view into the dynamic events that execute craniofacial development. The data will be assembled into atlases that will offer unprecedented tools for exploring the cellular, tissue and molecular correlates of craniofacial development. These atlases will be made widely available for the use of others in the Face Base consortium and the broader community of craniofacial researchers through an online resource created locally. In addition, they will be linked to other Face Base resources through established hubs, so that the data in our atlases can be used in concert with the molecular and structural data provided by other members of the consortium. The data acquisition pipeline established here in close collaboration with the laboratories of Dr. Yang Chai (University of Southern California) and Dr. Marianne Bronner-Fraser (Caltech) will create a model that can be expanded to phenotype and analyze other experimental systems.

Genetic Determinants of Orofacial Shape and Relationship to Cleft Lip/Palate

This is a completed FaceBase project.

Orofacial clefts, principally cleft lip (CL), cleft palate (CP), and cleft lip and palate (CLP), are among the most common major birth defects, occurring in ~1/700 to 1/1000 live births in various populations around the world, ~70% as a sporadic, isolated abnormality. Such "non-syndromic" orofacial clefts act as complex traits, involving multiple genes and environmental risk factors. To date traditional genetic mapping approaches have identified only a few major susceptibility genes for non-syndromic orofacial clefts with certainty. Therefore, new approaches are clearly required. There is considerable evidence that orofacial malformations can occur at the extremes of the normal ranges of phenotypic variation of midfacial size and shape. Here we propose a novel approach to identify genes that regulate midfacial shape in mouse and human. We hypothesize that genes that are major contributors to normal orofacial size and shape will also have important roles in the occurrence of orofacial clefts. To identify such genes, we will perform detailed morphometric analysis of midfacial shape differences in informative mouse strains as well as in select human populations, combining these studies with genetic analyses to identify genes that control major determinants of midfacial morphometries. Our studies have shown that specific inbred strains of mice have heritable differences in measurable parameters of facial shape. We will take advantage of a valuable new resource we have developed, the mouse "Collaborative Cross" (CC), to con-elate heritable differences in facial shape among the 8 founder strains of the CC, along with select Recombinant Inbred lines and Recombinant Intercross (RIX), with detailed genetic mapping data for these mice. This approach will enable identification of quantitative trait loci (QTLs) that underlie these morphometric differences. We will complement our mouse studies with a similar analysis of humans, studying specific populations with different susceptibilities to orofacial clefts. These comparative studies will allow us to identify genes that underlie midfacial shape in humans. Together, these studies should provide a basis for understanding the relationship between human facial morphogenesis and susceptibility to orofacial clefts, and for initiating studies of the functions of these genes in animal models relevant to human orofacial development.

 

Data is now available.  Please contact help@facebase.org with your request to receive further information.

Genome-Wide Atlas of Craniofacial Transcriptional Enhancers

This is a completed FaceBase project.

Clefts of the lip and palate are among the most common craniofacial birth defects. They can co-occur with other symptoms as part of Mendelian disorders, but the majority of cases are non-syndromic and have a complex etiology. In some cases disrupted protein-coding genes have been identified as contributors to orofacial clefting risk. However, accumulating evidence from genome-wide association studies (GWAS) indicates that sequence variation in non-coding regions also strongly contributes to a variety of clinical disorders including orofacial clefting. While it is speculated that many of these variants affect disease through impacting on functional properties of distant-acting transcriptional enhancers, only very few isolated examples of such regulatory variation have been identified. This is likely due to the fact that the genomic location and function of the vast majority of distant-acting enhancers in the human genome remains unknown. To address the pressing need to identify on a genomic scale enhancers that are involved in face and palate development and likely relevant for clefting etiology, we propose here an integrated genomic and transgenic mouse strategy to identify craniofacial enhancers and characterize their activities. Specifically, we will use a ChlP-seq approach to identify genome-wide sets of enhancers that are active in mouse face and palate tissues at embryonic stages that are relevant for orofacial clefting. We will use a transgenic mouse enhancer screen to validate and characterize 130 of these enhancer predictions in detail by determining their in vivo activity patterns. Furthermore, we will identify disease-associated GWAS variants that map to craniofacial enhancers that we will have discovered. We will then test and compare the variant and normal sequences in the transgenic enhancer assay for differences in their in vivo activities. All of the genomic and in vivo datasets, as well as molecular reagents developed through these experiments will be made available to other investigators through the Face Base program in order to maximize their availability and accelerate the progress of biomedical and clinical studies of mid-face and palate development and orofacial clefting.

Investigators 
Global Gene Expression Atlas of Craniofacial Development

This is a completed FaceBase project.

The objective is to create a global gene expression atlas of craniofacial development. The central thesis is that a combination of laser capture microdissection, and FACS, combined with microarrays and next generation sequencing can be used to efficiently achieve this goal. Microarrays, for example, with essentially complete gene representation can be used to rapidly determine the expression levels of every gene in laser capture microdissected elements of craniofacial development. A single experiment, therefore, provides a comprehensive analysis of the gene expression status of one component and a limited number of experiments examining each structure and cell type can create an atlas. A combination of structure, lectin staining, and transgenic GFP expression will be used to precisely identify specific compartments and lineages, including cells driving neural crest induction, nasal placodes and pits, lateral and medial facial eminences, neural crest and paraxial mesoderm cells, maxilla and mandibular processes, signaling centers, and the structures of palatogenesis. Next generation sequencing provides a digital readout of gene expression levels, cross-validates microarray data, gives additional valuable information concerning alternative processing, and can detect expression of genes not represented on arrays. We propose, therefore, to combine the use of microarray and nexgen sequencing in the creation of the craniofacial gene expression atlas. In addition, there are target amplification technologies that make possible the global analysis of gene expression in even single cells, which we propose to use to define molecular level heterogeneity present in the different cells of individual components. Using integrative bioinformatics analysis methodologies, gene expression modules reflective of variously differentiating components will be linked to gene networks implicated by shared structural, functional, and interactome features of the hundreds of genes already known to play individual roles in craniofacial development.

Investigators 
Identification of miRNAs Involved in Midfacial Development and Clefting

This is a completed FaceBase project.

The goal of this project is to identify microRNAs (miRNAs) involved in vertebrate mid-face development and to determine their function in this process. Our hypothesis is that a limited number of miRNAs play crucial roles in the patterning, proliferation and differentiation of the mid-face through precise spatial and temporal expression of specific protein-coding genes. Further, we hypothesize that both loss- and gain-of-function of these miRNAs leads to mid-facial and orofacial defects/clefting. In humans, defects in mid-facial development, including cleft lip/palate, account for the largest number of birth defects annually. Alone, over 400 syndromes result in cleft lip and palate, which has an occurrence rate of 1 in 800 live births. Our laboratories have a long-standing interest in determining the gene regulatory networks that control normal orofacial development, many of which are causative in animal models of mid-facial clefting. In our Preliminary Data, we show that microarray technology can generate comprehensive miRNA expression profiles and that bioinformatic analysis can define miRNAs whose expression patterns are conserved across vertebrates. In this proposal, we will investigate the identity and function of miRNAs expressed in the developing mid-face in three Specific Aims. In Aim 1, we will determine and validate the temporal and spatial expression patterns of miRNAs in the developing mouse maxillary/frontonasal prominences using miRNA microarray technology. In Aim 2, we will determine the in situ expression patterns of identified miRNAs in both mouse and zebrafish embryos to define those that show a conserved pattern of expression. In Aim 3, we will use zebrafish to determine the regulatory function and morphogenetic mode of action of the miRNAs by loss- and gain-of-function analysis. We propose using both mouse and zebrafish systems because their complementary well-established experimental and genetic methods make a detailed analysis of this process feasible. By defining miRNAs involved in mouse mid-facial development and then quickly and efficiently determining miRNA function in zebrafish, we will be able to elucidate the miRNAs involved in vertebrate mid-facial development. This is crucial to further our understanding of genetic events leading to human mid-facial birth defect syndromes.

Research on Functional Genomics, Image Analysis and Rescue of Cleft Palate

This is a completed FaceBase project.

The development of the Face Base Consortium calls for a comprehensive research collaboration to facilitate data collection, organization, and optimized utilization of new and existing data on mid-facial development and malformations. Our laboratory has a long history of investigating the molecular and cellular mechanism of cleft palate. We have developed a proposal that builds on our strength and will focus on genomic and imaging analysis of selected and highly clinically relevant cleft palate animal models. Specifically, we will use the Tgfb, Tgfbr, Smad4, Msx1 and Fgfr2 mutant animal models that represent complete and sub-mucous cleft palate defects in humans as our entry point. Taking advantage of these animal models, we will work closely with several scientists to address the regulatory mechanism of CNC cell fate determination. Specifically, working with Dr. Marianne Bronner-Fraser at California Institute of Technology (Caltech), we will investigate whether the neural crest gene regulatory network of traditional vertebrate models is conserved and may exert its regulatory function during palatogenesis. In collaboration with Dr. Joseph Hacia at USC, we will discover critical components of the Tgf-b signaling network that are specifically involved in regulating the fate of CNC cells during palatogenesis. Working with Dr. Scott Fraser at Caltech, we will generate comprehensive and dynamic three-dimensional images of palatogenesis and malformations using microMRI and microCT. Finally, we have developed a strategy to screen for specific points of intervention within the gene regulatory network that will allow us to develop therapeutic strategies to prevent and rescue cleft palate. Our collective effort will not only generate tremendous resources for the Face Base Consortium but will also offer opportunities for extensive collaborations for future translational research on craniofacial birth defects.

Investigators 
Integrated Research of Functional Genomics and Craniofacial Morphogenesis

In this application, we propose to integrate our research in functional genomics and craniofacial morphology/dysmorphology within the FaceBase Consortium. Specifically, we will focus on the development of the mandible and maxilla.

Congenital malformations involving these facial bones significantly impact quality of life because our face is our identity. For example, mandibular dysmorphogenesis ranging from agenesis of the jaw to micrognathia is a common malformation and appears in multiple syndromes. Micrognathia not only presents as a facial deformity but can also cause cleft palate and airway obstruction, such as in Pierre-­Robin sequence. The maxilla contributes to mid-­facial formation. Maxillary hypoplasia is often associated with cleft palate and has been described in more than sixty different syndromes.

Despite their importance, the mechanisms that regulate facial bone development are relatively uncharacterized. This is a significant gap in our knowledge and an important opportunity to generate invaluable resources for the research community. The proposed work is a logical progression from our current spoke project within the FaceBase Consortium on palatal development. Over the past five years, we have deposited nearly 200 hard and soft tissue scans and 125 microarray gene expression datasets in the FaceBase hub. These datasets have demonstrated their utility, as shown by other researchers’ presentations at major international conferences and publications.

Equally importantly, our team has played a significant role in the FaceBase Consortium, the hub website design, data organization and presentation. Building on our experience and in alignment with RFA-DE-14-004, we propose to investigate facial bone development and malformations.

In Specific Aim 1, we will perform global and specific gene expression profiling analysis of mandible development, and will integrate these datasets with cell lineage and 3D dynamic imaging analyses. In collaboration with the ontology group within the FaceBase consortium, we will define anatomical landmarks and morphometric parameters of the developing mandible.

In Specific Aim 2, we will expand our gene expression profile analyses in the developing maxilla. We will correlate this information with 3D imaging of the maxilla and define anatomical landmarks and parameters in collaboration with the ontology group within the FaceBase consortium. Our data will facilitate the investigation of the molecular regulatory mechanism of facial bone formation.

This study will showcase how our datasets at the hub can facilitate the generation of hypothesis-­driven research and collaborations. Because of the prevalence of facial bone defects in orofacial clefting patients and the lack of quantitative studies in this area, our proposed study will fill a void and provide a significant resource for the research community 

Investigators 
Genomic and Transgenic Resources for Craniofacial Enhancer Studies

Genetic studies have shown that distant-acting regulatory sequences (enhancers) embedded in the vast non-coding portion of the human genome play important roles in craniofacial development and susceptibility to craniofacial birth defects. The mechanistic exploration of these distant-acting enhancers continues to be difficult because the genomic location and in vivo function of most craniofacial enhancers remains unknown.

 

As members of FaceBase 1, we generated first sets of annotation and functional data for distal enhancers controlling craniofacial development. These resources proved to be of significant value to the craniofacial research community. However, these efforts captured only a small proportion of the enhancers that are active during craniofacial development in vivo. Here we propose to characterize the gene regulatory landscape of craniofacial development more comprehensively using new and complementary approaches. The specific aims are: 

 

1) We will map predicted enhancers by ChIP-seq from embryonic mouse and human facial tissues. In preliminary studies, we used ChIP-seq with the enhancer-associated protein p300 to identify several thousand enhancers predicted to be active in the mouse face at e11.5 and in the secondary palate at later stages of development. Using ChIP-seq for a panel of histone modifications (H3K4me1, H3K27ac, H3K27me3), which will require less tissue and increase the sensitivity of enhancer discovery by an order of magnitude, we will obtain higher-resolution data from all subregions of the developing mouse face at three stages of development (e11.5, e13.5, e15.5). We will complement this mouse-based effort with ChIP-seq on human embryonic face tissue to identify human-specific craniofacial enhancers not functionally conserved in mice.

 

2) In initial studies we characterized ~200 craniofacial enhancers in vivo in transgenic reporter assays. Taking advantage of protocols and collaborations established during FaceBase 1, we will continue to generate critically needed in vivo transgenic assays accompanied by optical projection tomography to characterize enhancers residing in new craniofacial loci identified by FaceBase 2 investigators and outside groups. This will include testing of enhancer variants associated with craniofacial malformations.

 

The datasets, vectors and transgenic embryos produced through our efforts will be made available as resources to the craniofacial research community. We are deeply committed to our ongoing collaborative interactions with the Hub and other Spoke projects, contributing to and taking advantage of the unique research opportunities enabled through the FaceBase program.

Investigators 
RNA Dynamics in the Developing Mouse Face

Craniofacial morphogenesis is a complex process requiring coordinated proliferation, movement and differentiation of six distinct facial prominences. The complexity of this process leaves it vulnerable to environmental and genetic perturbations, such that craniofacial malformations are one of the most common classes of birth defects.

 

Facial prominences are made up of a mono-layer of ectoderm encasing a large core of neural crest- and mesodermally-derived mesenchymal cells. Signaling from this minor population of ectodermal cells directs and coordinates the behavior of the underlying mesenchyme, and thence facial morphogenesis. Manipulations that alter these signaling processes and tissue interactions have grave consequences for facial development, resulting in various types of medically important dysmorphology including orofacial clefting. Thus a detailed knowledge of geno-dynamics of the ectoderm is an essential component of the overall description of facial development.

 

In this proposal a multi-disciplinary team has been assembled with expertise in craniofacial biology, mouse molecular genetics, bioinformatics and computer biology to gain a Systems Biology level understanding of early mammalian facial development.  The ectoderm and the mesenchyme of the wild-type facial prominences will be separated at critical timepoints encompassing facial morphogenesis, then these separated tissues will be used to generate contrasting dynamic spatio-temporal profiles of gene expression and post-translational RNA regulation at the level of splicing, turnover, and translation. These combined studies should provide a valuable resource detailing the dynamic interplay of ectoderm and mesenchyme during normal facial development.

Anatomical Atlas and Transgenic Toolkit for Late Skull Formation in Zebrafish

The final form of the adult skull is achieved through a complex series of morphogenetic events and growth, largely during post-embryonic development. Many common human congenital defects in the skull have their foundation in these developmental events. The treatment options in human patients are far from perfect, and improvements demand a more complete understanding of the biology underlying post-embryonic skull formation. However, by their complex development and relatively late occurrence, these clinically relevant stages in skull development have been less accessible in experimental organisms.

 

The zebrafish displays fundamental similarity in skeletogenesis to mammals, including in formation of the vault of the skull and the cranial sutures. Although the later events of skull and suture formation have been relatively less well studied in zebrafish, they are nonetheless accessible for manipulations and imaging, making the zebrafish an ideal system to further our understanding of these complex events. Through a set of interconnected Aims, we propose to establish and make available to the community tools that will lay the foundation for the use of zebrafish to examine skull and suture formation.

 

We will first construct an online, interactive atlas of normal skull development, encompassing the stages during which the vault of the skull is forming. The foundation of the atlas will be images generated by high-resolution computed tomography (micro-CT), which will be annotated and available for download. These will be complemented by images of transgenic zebrafish expressing fluorophores in critical cell populations, such as chondrocytes and osteoblasts at different stages of development.

 

For the transgenics, we will optimize recently developed methods for fixation and clearing of large (>1 mM) tissue samples and use a versatile zoom macro-confocal scope. This approach will allow creation of lower resolution data sets from which we can generate three-dimensional reconstructions of gene expression in an entire skull, and will also allow high resolution imaging of specific structures.

 

The transgenic lines used for the imaging studies will also serve as the basis for a transgenic system, using phiC31 recombinase, to allow replacement of the transgene coding sequences in genomic context while preserving tissue-specific expression patterns; the reagents (fish lines and plasmids) will available to the community.

 

Finally, both of the laboratories in this application are engaged in ongoing genetic screens to identify mutations causing defects in the juvenile or adult skull. Using a select set of  mutants with clinically relevant phenotypes, we will apply the imaging approaches above to describe the defects in morphology and gene expression during skull development.

 

Through the combined generation of a comprehensive atlas and a set of transgenic and genetic tools, we will substantially advance the use of zebrafish in the study of skull development, and greatly facilitate comparative studies with mammals that will advance treatment options in human patients.      

Investigators 
Technology
Genetic Tools and Resources for Orofacial Clefting Research

This is a completed FaceBase project.

Orofacial clefting is one of the most common birth defects in humans, affecting approximately 1 in 700 live births. This frequency highlights the complexity of craniofacial morphogenesis, which requires precise regulation of gene expression changes, alterations in cell physiology and morphogenic movements. The mouse has played an instrumental role in advancing our understanding of the mechanisms that govern mid- face and palate development. Future progress, however, will require an increasingly sophisticated set of genetic models and tools. The overall goal of this project is to facilitate orofacial clefting research by generating new mouse genetic tools and by providing a repository of mouse strains critical for clefting research community. First, we will generate both inducible and constitutive Cre recombinase driver lines as new genetic tools. These new mouse strains will be designed specifically to support orofacial clefting research, with input from members of the Face Base consortium. These lines will be characterized in our established pipeline for evaluation and quality control of Cre-expressing mouse strains. Second, we will provide a repository for importation, cryopreservation, genetic quality control, and distribution of new and existing mouse models and tool strains important for orofacial clefting research. Such strains will include genetically engineered, spontaneously occurring, and ENU-induced models. New models for orofacial clefting will be actively solicited from members of the Face Base consortium and from the scientific community at large. We will create a public website for the Jackson Face Base Repository to promote the use of this resource and to facilitate access to mouse models and tools for orofacial research. Together, these projects will provide both Face Base members and the general scientific community with new research tools, novel genetic models, and comprehensive mouse repository services to enhance research in orofacial clefting.

 

Supplement Grant Number
3U01DE020052-04S1

 

Supplement Abstract
The objectives of this revision application are to identify and characterize craniofacial (CF) disorders in mice that are reliable genetic and physiological models for human craniofacial dysmorphologies and to share these models with the scientific public. Discovery of the genetic cause of CF disease in human populations is difficult due to extreme heterogeneity in humans and to the diversity of environmental and nutritional variables, all of which have a role in resulting CF diseases. Consequently, animal models with defined genetic backgrounds maintained in controlled environments are important for CF gene discovery. The goal of this revision application is to support CF research by providing new mouse models of CF dysmorphologies to FaceBase investigators, and the broader CF research community, via the FaceBase Mouse Repository. Utilizing our well-established program to screen for phenotypic deviants in The Jackson Laboratory’s large breeding and research colonies, our ongoing ENU mutagenesis program, and access to newly developed strains via our participation in the NIH-wide Knockout Mouse Phenotyping Program (KOMP2), we will identify, characterize phenotypically and genetically, and distribute novel mouse models of CF disorders. These will include the full range of CF phenotypes, including defects in skull morphology, dentition, vision, and hearing as well as models of orofacial clefting. For most models, we will identify the causative gene using high throughput sequencing technologies. Together, the models within this resource will provide critical tools for understanding the basis of CF disorders in humans.
 

Shape-Based Retrieval of 3D Craniofacial Data

This is a completed FaceBase project.

Craniofacial malformations are among the most common structural malformations in humans. Researchers studying disorders of the craniofacial anatomy have many 3D imaging tools available to them, including computed tomography, magnetic resonance imaging, and 3D surface scans. Craniofacial researchers studying particular disorders are constructing large image databases of subjects in their studies. The Face Base Consortium will provide a central HUB for collection of data from numerous sites, enabling studies that would otherwise not be possible. Since shape is the critical factor in the classification of most craniofacial disorders, tools for analyzing 3D shape are essential to these studies. Quantitative shape descriptors allow for reproducible shape description, while similarity-based shape retrieval allows comparisons to be made between individuals or populations. The goal of this project is to develop tools for shape-based retrieval of 3D craniofacial image data. The specific aims of the project are to: 1) develop software tools that produce quantitative representations of craniofacial anatomy that can assist in the study of mid-face hypoplasia and cleft lip and palate; 2) develop tools for quantifying the similarity of craniofacial data between two individuals, between an individual and an average over a selected population, or between two populations; 3) develop mechanisms for organization and retrieval of multimodality 3D craniofacial data based on their quantitative representations; and 4) design and implement a prototype system for Craniofacial Information Retrieval (CIR) that incorporates quantification, organization, and retrieval; evaluate it on 3D craniofacial data and make it available to the Face Base HUB. The design of these tools and a pilot system will lead to a general methodology that is immediately applicable to studies of mid-face hypoplasia, cleft lip and cleft palate, but is also scalable and modifiable to all craniofacial abnormalities. NIDCR has awarded a supplement to this project to develop an Ontology of Craniofacial Development and Malformation (OCDM), and to demonstrate its usefulness for both the parent project and the FaceBase consortium as a whole. More information is available

 

The CranioGUI application provides web-based support for using our tools to analyze 3D facial mesh images.

Algum

This is a completed FaceBase project.

Algum is an open-source web application developed at Carnegie Mellon University for generating Diamond filters using machine learning on marked up images.


Algum was originally developed by Shiva Kaul and was implemented using JRuby on Rails. It has been completely reimplemented by Jan Harkes using Python and the Django web framework.

Investigators 
The Ontology of Craniofacial Development and Malformation

The purpose of the national FaceBase consortium is to systemically acquire and integrate multiple forms of data in order to facilitate a systems level understanding of the  causes and possible treatments for craniofacial abnormalities. A basic component of any such data integration effort is a controlled set of terms or keywords that can be  associated with the data through data annotation, so that diverse data can be related via common terms. If in addition, the terms are related to each other in an ontology, then  integration can occur at the level of meaning rather than simply via keywords. 
As part of FaceBase1, we designed and partially implemented the Ontology of  Craniofacial Development and Malformation (OCDM), based on our Foundational Model of Anatomy ontology (FMA). The OCDM currently consists of components for representing human and mouse adult and developmental anatomy and malformations, as well as mappings between homologous structures in the two organisms.
Since the  focus of FaceBase1 was cleft lip and palate, the initial focus of the ontology was representation of structures and developmental relations relevant to these conditions in mouse and human. In our proposed work we will greatly extend the OCDM to accommodate conditions of interest to FaceBase2 researchers, such as human and mouse facial, palatal, and cranial vault development, and dysmorphology such as craniosynostosis, midface  hypoplasia, frontonasal dysplasia, craniofacial microsomia and microtia.
These malformations will require extensive structural and developmental representation of the entire musculoskeletal system of the head, as well as associated soft tissue anatomy that includes the integumentary system, deep fascial system, viscerocranial mucosa, adipose tissue, eyes, ears, tongue, vasculature and neural network.
In addition we will incorporate and add relations to the Zfin ontology to reflect the inclusion of zebrafish in FaceBase2.  Versions of the OCDM will be released to the FaceBase Hub in OWL 2. By conforming  to ontology best practices such as OWL 2 the OCDM will be interoperable with other  efforts that are contributing to the overall world wide semantic web of linked data and  knowledge.

Investigators 
Facebase 2 Coordinating Center

The FaceBase consortium is a distributed network of researchers investigating craniofacial development and dysmorphology. The consortium includes research projects directly participating via funded ""spoke"" projects as well as members of the craniofacial research community at large. The collection, sharing and integration of heterogeneous data, including genetic, imaging, and anatomical, from human and animal models are essential for advancing craniofacial research.

 

We will develop and maintain a FaceBase 2 Data Management and Integration Hub infrastructure that will properly store, represent, and serve these data to the research community, and in addition provide access to tools for visualizing, integrating, annotating, linking and analyzing the data. Our three broad aims will provide not only the needed data migration and management, but also a set of user-centered tools for data visualization, comparison and annotation. These are:

 

Aim 1: Create an infrastructure driven by principles of ease of use and user centered design to manage data throughout its lifecycle. By taking a user centric approach and supporting the use of data throughout its lifecycle, the rate of discovery and utility of the hub can be maximized.

 

Aim 2: Provide tools within the Hub that accelerate craniofacial research by enabling data annotation, integration and analysis. To maximize the utility of the Hub for both spoke projects and outside users, our proposed infrastructure will allow any user to define a personal workspace and organize datasets, visualization tools, and analysis tools into specific user-driven workflows.

 

Aim 3: Promote use and collaboration across the FaceBase research network. The Hub will provide a wide range of collaboration tools and services to the consortium as well as organizing face-to-face meetings in order to enhance collaboration. We have assembled an experienced team of experts in bioinformatics, imaging, genetics, mouse and human studies, including FaceBase participants who bring firsthand knowledge of the needs within the craniofacial research community. The FaceBase team is intentionally diverse, to provide each spoke group at least one informed contact who will play an integral role in the development and implementation of the Hub, thereby ensuring that the data of each spoke project will have maximum impact on the community of researchers

Investigators 
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