NIH Public AccessAuthor ManuscriptDev Biol. Author manuscript; available in PMC 2012 September 12.NIH-PA Author ManuscriptPublished in final edited form as:Dev Biol. 2011 July 15; 355(2): 175–182. doi:10.1016/j.ydbio.2011.02.033.The FaceBase Consortium: A comprehensive program tofacilitate craniofacial researchHarry Hochheisera,*, Bruce J. Aronowb,c, Kristin Artingerd, Terri H. Beatye, James F.Brinkleyf, Yang Chaig, David Clouthierd, Michael L. Cunninghamh, Michael Dixoni,j, LeahRae Donahuek, Scott E. Fraserl, Benedikt Hallgrimssonm, Junichi Iwatag, Ophir Kleinn, MaryL. Marazitao,p,q,r, Jeffrey C. Murrays, Stephen Murrayk, Fernando Pardo-Manuel de Villenat,John Postlethwaitu, Steven Potterv, Linda Shapirof, Richard Spritzw, Axel Viselx,y, Seth M.Weinbergz, and Paul A. Trainoraa,ab,**aDepartment of Biomedical Informatics, University of Pittsburgh, Pittsburgh PA 15232, USAbDivisionNIH-PA Author Manuscriptof Biomedical Informatics, Center for Computational Medicine, Cincinnati Children’sHospital Medical Center, University of Cincinnati College of Medicine, CHRF 8504, 3333 BurnetAve Cincinnati, OH 45229, USAcDivisionof Developmental Biology, Center for Computational Medicine, Cincinnati Children’sHospital Medical Center, University of Cincinnati College of Medicine, CHRF 8504, 3333 BurnetAve Cincinnati, OH 45229, USAdDepartmentof Craniofacial Biology, University of Colorado Denver Anschutz Medical Campus,Aurora, CO 80045, USAeDepartmentof Epidemiology, Johns Hopkins University, 615 N. Wolfe Street, Baltimore, MD21205, USAfDepartmentof Computer Science and Engineering, University of Washington, Box 352350Seattle, WA 98195-2350, USAgCenterfor Craniofacial Molecular Biology, Ostrow School of Dentistry, University of SouthernCalifornia, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033, USAhSeattleChildren’s Hospital, 4800 Sand Point Way NE, Seattle, WA 98105, USAiFacultyNIH-PA Author Manuscriptof Medical and Human Sciences, Manchester Academic Health Sciences Center,University of Manchester, Oxford Road, Manchester, M13 9PT, England, UKjFacultyof Life Sciences, Michael Smith Building, University of Manchester, Oxford Road,Manchester, M13 9PT, England, UKkJacksonLaboratory, 600 Main St., Bar Harbor, ME 04609, USAlBiologicalImaging Center Beckman Institute 133, M/C 139-74 California Institute of TechnologyPasadena, CA 91125, USAmDept.of Cell Biology and Anatomy and the Alberta Children’s Hospital Research Institute,University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, CanadanDepartmentof Orofacial Sciences and Program in Craniofacial and Mesenchymal Biology,University of California San Francisco, San Francisco, USA 2011 Elsevier Inc. All rights reserved.*Correspondence to: H. Hochheiser, Department of Biomedical Informatics, University of Pittsburgh, Pittsburgh, PA 15232, USA.**Correspondence to: P.A. Trainor, Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, [email protected] (P.A. Trainor).
Hochheiser et al.Page 2oCenterfor Craniofacial and Dental Genetics, Department of Oral Biology, School of DentalMedicine, University of Pittsburgh, Pittsburgh, PA 15219, USANIH-PA Author ManuscriptpDepartmentof Human Genetics, Graduate School of Public Health, University of Pittsburgh,Pittsburgh, PA 15219, USAqDepartmentof Psychiatry, University of Pittsburgh, Pittsburgh, PA 15219, USArClinicaland Translational Science Institute, School of Medicine, University of Pittsburgh,Pittsburgh, PA 15219, USAsDepartmentof Pediatrics, University of Iowa, Iowa City, IA 52242, USAtDepartmentof Genetics The University of North Carolina at Chapel Hill120 Mason Farm RoadRoom 5046 Genetic Medicine Building Campus Box 7264, Chapel Hill, NC 27599-7264, USAuInstituteof Neuroscience, University of Oregon, Eugene, OR 97403, USAvDivisionof Developmental Biology, Cincinnati Children’s Hospital Medical Center, 3333 BurnetAvenue, Cincinnati, OH 45229, USAwHumanMedical Genetics Program, University of Colorado School of Medicine, Aurora, CO80045, USAxGenomicsNIH-PA Author ManuscriptyU.S.Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USADepartment of Energy Joint Genome Institute, Walnut Creek, CA 94598, USAzCenterfor Craniofacial and Dental Genetics Department of Oral Biology, School of DentalMedicine, University of Pittsburgh, Bridgeside Point Building 100 Technology Drive, Suite 500Pittsburgh, PA 15219, USAaaStowersInstitute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USAabDepartmentof Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City,KS 66160, USAAbstractNIH-PA Author ManuscriptThe FaceBase Consortium consists of ten interlinked research and technology projects whose goalis to generate craniofacial research data and technology for use by the research communitythrough a central data management and integrated bioinformatics hub. Funded by the NationalInstitute of Dental and Craniofacial Research (NIDCR) and currently focused on studying thedevelopment of the middle region of the face, the Consortium will produce comprehensivedatasets of global gene expression patterns, regulatory elements and sequencing; will generateanatomical and molecular atlases; will provide human normative facial data and other phenotypes;conduct follow up studies of a completed genome-wide association study; generate independentdata on the genetics of craniofacial development, build repositories of animal models and ofhuman samples and data for community access and analysis; and will develop software tools andanimal models for analyzing and functionally testing and integrating these data. The FaceBasewebsite (http://www.facebase.org) will serve as a web home for these efforts, providing interactivetools for exploring these datasets, together with discussion forums and other services to supportand foster collaboration within the craniofacial research community.KeywordsCraniofacial development; Cleft lip and palate; Human genetics; Animal models; Database;MorphometricsDev Biol. Author manuscript; available in PMC 2012 September 12.
Hochheiser et al.Page 3Motivation to create the FaceBase ConsortiumNIH-PA Author ManuscriptAmong the lessons learned from the growth of “omics” research is the demonstration thatcollections of large publicly available, thoroughly annotated and integrated gene- andbioinformatics datasets can dramatically facilitate focused, hypothesis-driven research andopen new areas of systems-level research. The availability of genome sequences in readilyaccessible user interfaces (e.g.; http://genome.ucsc.edu/; (Kent et al., 2002)), hasfundamentally changed approaches to molecular biology and genetic research. Analyses thatused to require months of benchwork in the pre-genomic era can now be performed in silicoin a fraction of that time. Likewise, access to carefully annotated repositories of publishedphenotype, developmental and expression data Bult et al., 2008);http://emouseatlas.org/emage/; (Richardson et al., 2010)) can enable systems-wideintegrative studies to identify functional gene networks that underlie the etiology andpathogenesis of human disease. The NIDCR has sought to accelerate research in craniofacialdevelopmental biology and further understanding of craniofacial and dental disorders byestablishing the FaceBase Consortium.NIH-PA Author ManuscriptTen research and technology projects currently focused on the mid-face plus a datamanagement and coordination hub were selected from a pool of peer-reviewed applicationssubmitted in response to a Request for Applications (RFA) issued in 2008. Projects wereselected based on merit as well as their potential for synergism with other projects and theintegrative goals of the central hub. For example, studies of craniofacial development inmodel systems may identify candidate genes for human disorders, and findings from humangenome-wide association studies (GWAS) may lead to the development of important newanimal models (Dixon et al., 2011).NIH-PA Author ManuscriptOther constraints were also considered in establishing this Consortium. With a goal ofincorporating many different types of bioinformatics data about specific developmentalprocesses and disorders, the FaceBase projects could not cover the entire panoply ofstructures subsumed under “dental and craniofacial” and still have enough in common forthe desired data integration with one another. The decision was therefore made to restrict thefirst iteration of FaceBase to studies investigating the “mid-facial” region (e.g., nose, upperlip, palate, etc.), because it is the anatomic site of several common craniofacial birth defectssuch as cleft lip/palate and frontonasal dysplasia (Naidich et al., 2003). Clefts involving themidface, in particular those of the lip and the palate, are among the most common birthdefects and create life-long challenges for affected individuals, including repeated surgeries,as well as speech, and dental problems and long-term health issues such as increased risksfor cancer and delayed cognitive development (Bille et al., 2005; Conrad et al., 2009). Thus,research into the development of the mid-face has relevance to the mission of the NIDCRand addresses significant health burdens.As summarized in the next section, the FaceBase Consortium researchers studying mid-facedevelopment utilize diverse organisms including fish, mice and humans. Investigatorsemploy a wide variety of experimental approaches such as GWAS, detailed phenotyping,transcriptional profiling, and conditional knockout mice; and/or are working ontechnological advances to assist in the FaceBase goals.FaceBase Consortium projectsConsortium efforts can be broadly classified into three categories: (1) five animal modelprojects (primarily mouse and zebrafish) examining the role of various developmental andfunctional elements in the midfacial region; (2) three human projects, two investigating thegenetic determinants of facial morphometrics and structural birth defects; (one of which alsoDev Biol. Author manuscript; available in PMC 2012 September 12.
Hochheiser et al.Page 4NIH-PA Author Manuscriptincludes mouse models for gene discovery) and one utilizing sequencing approaches toextend GWAS results in CL/P; and (3) two technology projects developing software andanimal models to support the FaceBase Consortium goals and craniofacial researchcommunity. Importantly, the aims of several projects overlap considerably across thesedomains. The data management and coordination hub project serves as the focal point fordeveloping Consortium policies and the centralized software and hardware infrastructureneeded to support the FaceBase data resource and web portal and for housing the humanbiorepository. An overview of the various projects and key investigators is given in Table 1,with a graphical representation in Fig. 1.Animal model projectsFunctional analysis of neural crest and palate: imaging craniofacial developmentNIH-PA Author ManuscriptAs craniofacial development involves tissue movements and cell rearrangements at scalesdifficult to capture fully in conventional histological sections, techniques combining geneticdata with detailed imaging are needed to provide more detailed understanding of geneticinfluences and processes influencing craniofacial development. This research project willrefine and deploy a set of advanced tools for the imaging of tissue structure, gene expressiondomains and cellular dynamics during craniofacial development. Volumetric imaging toolswill be used to create accurate 3D atlases that can be digitally dissected to permit tissueinteractions and cellular events to be better understood in the forming faces of normal,mutant and perturbed mouse embryos. Molecular agents, optimized for imaging intacttissues, will be employed to create atlases of the molecular correlates in these embryos.Finally, intravital imaging tools will be used to study cell and tissue interactions as they takeplace, offering a view into the dynamic events that occur during craniofacial development.These data will be assembled into atlases that will be linked to other FaceBase data toprovide unprecedented tools for exploring the cellular, tissue and molecular correlates ofcraniofacial development.Genome-wide atlas of craniofacial transcriptional enhancersNIH-PA Author ManuscriptAccumulating evidence from GWAS indicates that sequence variation in non-coding regionsstrongly contributes to a variety of clinical disorders including orofacial clefting (Birnbaumet al., 2009; Visel et al., 2009b; Beaty et al., 2010). These variants may impact disease byaffecting functional properties of distant-acting transcriptional enhancers. However, veryfew isolated examples of such regulatory variation have been identified. This is in large partdue to the fact that the genomic location and function of the vast majority of enhancers inthe human genome remains unknown. This FaceBase project will use integrated genomicand transgenic mouse strategies to discover enhancers involved in face and palatedevelopment, and to characterize their activities. Specifically, a ChlP-seq approach (Visel etal., 2009a) will be employed to identify genome-wide sets of enhancers active in mouse faceand palate tissues at embryonic stages relevant for orofacial clefting. A transgenic mouseenhancer screen will be utilized to validate and characterize subsets of these enhancerpredictors in detail by determining their in vivo activity. Furthermore, disease-associatedvariants from GWAS and other human genetic studies that map to craniofacial enhancerswill be interrogated to determine how these variants affect in vivo enhancer activity. Thegenomic and in vivo datasets, as well as molecular reagents developed through theseexperiments will be made available to other researchers through the FaceBase website.Global gene expression atlas of craniofacial developmentHigh-resolution genome-wide views of temporal, spatial, and tissue-and cell-specific geneexpression patterning can provide mechanistic insights and facilitate hypotheses concerningnormal and abnormal developmental mechanisms. This project will use both laser captureDev Biol. Author manuscript; available in PMC 2012 September 12.
Hochheiser et al.Page 5NIH-PA Author Manuscriptmicrodissection and fluorescence activated cell sorting (FACS), together with microarraysand next generation sequencing, to create a global gene expression atlas of craniofacialdevelopment. A combination of morphologic, lectin staining, and transgenic GFP expressionfeatures will be used to identify specific compartments and lineages including the neuralfolds, the epidermal ectoderm, neural crest, paraxial mesoderm, nasal placodes and pits,lateral and medial facial eminences, maxilla and mandibular processes, signaling centers,and the palatal shelves. Laser capture will allow purification of discrete structures, whiletransgenic GFP/FACS will isolate specific cell types. In addition, to better define geneexpression heterogeneity within individual cell types, we will perform extensive single cellanalyses. Whole genome microarrays will provide global, sensitive and quantitativemeasures of gene expression levels. Next-generation RNA-seq will provide a digital readoutof gene expression levels, cross-validate microarray data, provide additional valuableinformation concerning alternative processing, and detect expression of genes not wellrepresented on arrays. Using integrative bioinformatics analysis approaches, gene expressionpatterns that are reflective of variously differentiating components will be linked to genenetworks implicated by specific or shared structural, functional, and interactome features ofthe hundreds of genes already known to play individual roles in craniofacial development.This will provide an important framework for integrating other craniofacial projects.Identification of miRNAs involved in midfacial development and cleftingNIH-PA Author ManuscriptThe regulation of biological processes occurs through intricate and continuous refinement ofgene expression. MicroRNAs (miRNAs) are small non-coding RNAs implicated as amechanism for controlling gene expression in a wide variety of developmental processes(Bernstein et al., 2003). Previous results have shown that miR-140-mediated regulation ofplatelet-derived growth factor a (Pdgfa) is required for normal migration of a subset ofneural crest cells towards the oral ectoderm, where they later take part in palate development(Eberhart et al., 2008). This current project will identify other miRNAs involved invertebrate midface development, and determine their function in this process. The temporaland spatial expression patterns of miRNAs in the developing mouse maxillary/frontonasalprominences between E10.5 and E14.5 will be characterized using massively parallelmiRNA sequencing (miRNA-seq). In situ expression patterns of identified miRNAs willthen be compared in mouse and zebrafish embryos to define those that show a conservedpattern of expression. Finally, gain-and loss-of-function analysis in zebrafish will be used todetermine the function of individual miRNAs. Bioinformatic interrogation of putativemiRNA targets and comparisons to gene expression atlases will further elucidate the geneticnetworks that regulate craniofacial development.Functional genomics, image analysis and rescue of cleftNIH-PA Author ManuscriptThe analysis of mutant animal models has significantly improved our understanding of thegenetic causes of cleft palate. Human linkage studies have also shown that genetic mutationsare a major contributing factor in the etiology of cleft palate. For example, mutations intransforming growth factor-β (Tgf-β) signaling can cause cleft palate in both mice andhumans (Nawshad et al., 2004). In mice, loss of Tgf-β signaling in cranial neural crest(CNC) cells (Tgfbr2fl/fl;Wnt1-Cre) results in complete cleft palate whereas loss of Tgf-β inmidline epithelial cells (Tgfbr2fl/fl;K14-Cre) results in submucous cleft (Ito et al., 2003; Xuet al., 2006). These two animal models represent two common types of cleft palate inhumans. To improve the utility of mutant animal models for investigating genetic causes ofcleft palate in humans, this project will develop a cleft palate classification system to greatlyfacilitate the organization of data and assist the coordination between mouse and humancleft palate research. This classification system will serve to standardize vocabulary andphenotypic descriptions for all researchers to communicate effectively. Furthermore, incollaboration with other projects of the FaceBase Consortium, global and specific geneDev Biol. Author manuscript; available in PMC 2012 September 12.
Hochheiser et al.Page 6NIH-PA Author Manuscriptexpression profiling analysis in Tgfbr mutant animal models will be used to continuouslygenerate candidate genes critical for CNC cell fate determination during palatogenesis. Inparallel, sophisticated imaging analysis (microMRI and microCT) will be used to build acomprehensive database for investigation of the regulatory mechanisms of palatogenesis.Dissecting distinct signaling pathways and identifying the point(s) of intersection wheremultiple signaling pathways converge will aid in developing therapeutic strategies to preventcleft palate and/or restore palate formation.Human projectsGenetic determinants of orofacial shape and relationship to cleft lip/palateNIH-PA Author ManuscriptApproximately 70% of all cleft lip and/or cleft palate occur as sporadic and isolatedabnormalities (Stanier and Moore, 2004). Such “non-syndromic” orofacial clefts act ascomplex traits, involving multiple genetic and environmental risk factors. There isconsiderable evidence that orofacial malformations can occur at the extremes of the normalranges of phenotypic variation of midfacial size and shape. Therefore, genes which controlnormal orofacial size and shape could have important roles in the occurrence of orofacialclefts. To identify such genes, this project will undertake detailed morphometric analysis ofmidfacial shape differences in informative mouse strains, as well as in select humanpopulations. Combining these studies with genetic analyses will identify genes controllingmidfacial morphometries. Specific inbred strains of mice have heritable differences inmeasurable parameters of facial shape. This project takes advantage of a valuable newresource, the mouse “Collaborative Cross” (CC; (Churchill et al., 2004)) to correlateheritable differences in facial shape among the eight founder strains of the CC, along withselect Recombinant Inbred lines and Recombinant Intercross (RIX), with detailed geneticmapping data for these mice. It will also generate the largest repository of mouse microCTscan data that can be used for multiple future genetic and morphometric studies. Thisapproach will enable identification of quantitative trait loci (QTLs) that underlie thesemorphometric differences. The mouse studies will be complemented by a similar analysis ofhumans, studying specific populations with different susceptibilities to orofacial clefts.These comparative studies will identify genes that underlie midfacial shape in humans.Together, these studies should provide a basis for understanding the relationship betweenhuman facial morphogenesis and susceptibility to orofacial clefts, and for initiating studiesof the functions of these genes in animal models relevant to human orofacial development.3D analysis of normal facial variation: data repository and geneticsNIH-PA Author ManuscriptAlthough ample evidence exists that facial appearance and structure are highly heritable,there is little information regarding how variation in specific genes relates to the diversity offacial forms evident in our species. With the advent of affordable, non-invasive 3D surfaceimaging technology, it is now possible to capture detailed quantitative information about theface in a large number of individuals (Kau et al., 2010). The combination of state-of-the-art3D imaging with advances in high-throughput genotyping provides an unparalleledopportunity to map the genetic determinants of normal facial variation. An improvedunderstanding of the relationship between genotype and facial phenotype may helpilluminate the factors influencing sensitivity to common craniofacial anomalies, particularlyorofacial clefts, which are among the most prevalent birth defects in humans. This projectwill construct a normative repository of 3D facial and genetic data and utilize this datarepository to identify genes that influence normal midfacial variation. This will involvecollecting 3D facial surface images and DNA samples on 3500 healthy Caucasianindividuals (age 3–40) drawn from the general population. Quantitative facial measures willbe extracted from the 3D images and all DNA samples will be genotyped for genome-wideSNP markers. All of the 3D images, quantitative measures, and genotype data will beDev Biol. Author manuscript; available in PMC 2012 September 12.
Hochheiser et al.Page 7NIH-PA Author Manuscriptavailable to outside investigators through the FaceBase repository. The project will focus onidentifying SNPs associated with variation in midfacial morphology, including those facialfeatures relevant to orofacial cleft predisposition. Salient measures of midfacial morphologywill be derived from 3D facial surface images, and a genome-wide association approach willthen be employed to identify polymorphisms that influence quantitative variation in thefacial features of interest.Oral clefts: moving from genome-wide studies toward functional genomicsNIH-PA Author ManuscriptThe FaceBase Consortium provides a timely opportunity for follow-on GWAS of oral clefts(Beaty et al., 2010). A systematic analysis of intensity data for single nucleotidepolymorphic (SNPs) markers and monomorphic probes in regions of known copy numbervariants (CNV) available from our genome wide association study will be performed toidentify genes that influence risk through structural variation. CNV markers will then beused in a second genome wide test for linkage and association under a case-parent triodesign, which may identify additional genes of interest. The case-parent trio design offers aunique opportunity to identify de novo CNVs, and this information will be combined withevidence from transmitted CNVs to identify influential genes. In collaboration with otherresearchers in FaceBase, we have the opportunity to undertake high-throughput sequencing(HTS) studies of specific genes and chromosomal regions identified in our GWAS toidentify both rare and common variants that may play a causal role in the etiology of oralclefts. The genes/ regions identified in our case-parent trio design will be the first area ofHTS studies, but we will go on to further conduct whole exome HTS studies using affectedpairs of relatives drawn from multiplex families. Finding from these sequencing studiesshould identify new candidates for functional studies in animal models through collaborationwith other FaceBase projects. We are particularly interested in genes that show someevidence of gene–environment interaction in human data, because animal models offer theopportunity to further explore the combined effects of genes and environmental risk factors.Technology projectsShape-based retrieval of 3D craniofacial dataNIH-PA Author ManuscriptAs shape is a critical factor in the classification of most craniofacial disorders,computational tools for analyzing 3D shape are essential to better describe individualconditions as well as understand their pathogenesis. Quantitative shape descriptors allow forreproducible shape description, while similarity-based shape retrieval allows comparisons tobe made between individuals or populations. Researchers studying disorders of craniofacialanatomy have access to a number of 3D imaging tools, including computed tomography,magnetic resonance imaging, and 3D surface scans (Robb, 1999). This project will (1)develop software producing quantitative representations of craniofacial anatomy to assist inthe study of mid-face hypoplasia and cleft lip and palate; (2) develop tools for quantifyingthe similarity of craniofacial data between two individuals, between an individual and anaverage over a selected population, or between two populations; (3) develop mechanisms fororganization and retrieval of multimodality 3D craniofacial data based on their quantitativerepresentations; (4) design and implement a prototype system for Craniofacial InformationRetrieval (CIR) that incorporates quantification, organization, and retrieval; and (5) utilizethe CIR System on 3D craniofacial data, such as that generated by other FaceBase projects.The design of these tools and a pilot system will lead to a general methodology that is bothimmediately applicable to studies of mid-face hypoplasia, cleft lip and cleft palate, and alsoscalable and modifiable to all craniofacial abnormalities.Dev Biol. Author manuscript; available in PMC 2012 September 12.
Hochheiser et al.Page 8Genetic tools and resources for orofacial clefting researchNIH-PA Author ManuscriptThe mouse is a powerful genetic model for understanding developmental mechanisms andthe etiology and pathogenesis of human syndromes. As future progress requires anincreasingly sophisticated set of genetic models and tools, this project will generate new Crerecombinase driver strains for orofacial clefting research and serve as a mouse repository forthese and other relevant strains for the community as a whole. Four specific BAC transgenicCre drivers, (Lhx7/8-Cre; deltaNp63-CreERT2, Krt6a-Cre, Tbx22-CreERT2) and threeconserved enhancer driven Cre strains (IRF6(MCS-9.7)-CreERT2, HCES-546-CreERT2,HCES-809-CreERT2) were chosen by a working group of experts in the field and are inprogress. These strains were chosen to fill gaps in the existing repertoire of Cre strains, andto provide tools to interrogate the mechanisms of orofacial development in detail. Eightadditional constructs will be selected based on data generated in other FaceBase projectscentered on global gene expression analyses and identification of transcriptional enhancers.NIH-PA Author ManuscriptThe Lhx7/8-Cre driver will target the mesenchymal compartment directly adjacent to midfacial fusion events, facilitating investigations of mesenchymal–epithelial interactions priorto and during mid-face fusion. Expression of Lhx7/8 is first observed at E9.5 in the firstbranchial arch and by E10.5 is strongly expressed in the maxillary and mandibularcomponents of the arch, proximal to the cleft that separates them (Grigoriou et al., 1998).Because expression in the mesenchyme of the medial nasal prominence only is noted justprior to fusion with the maxillary prominence, a standard Cre driver is the best approach inthis case.During secondary palate development, the deltaN alpha isoform of Trp63 is expressedthroughout the basal epithelial cell layer of the oral ectoderm, but is excluded from theperiderm cell layer (Thomason et al., 2008). The deltaNp63-CreERT2 driver line will allowinvestigators to specifically target the basal epithelial cell layer in the oral cavity withoutaffecting the periderm and mesenchyme. Because expression of deltaNp63 occurs prior tostratification of the periderm, an inducible strategy is required to produce a Cre line with thedesired specificity.The Krt6a-Cre driver will serve to specifically target the periderm cell layer during palateshelf fusion. Expression of mouse Krt6a begins at approximately E14.5 in the periderm celllayer and persists until the periderm disappears before birth (Mazzalupo and Coulombe,2001; Wong et al., 2000). Because expression is activated just prior to the fusion events inthe secondary palate, a constitutive Cre driver is best suited to target the periderm duringpalate fusion.
**Correspondence to: P.A. Trainor, Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA. [email protected] (P.A. Trainor). NIH Public Access Author Manuscript Dev Biol. Author manuscript; available in PMC 2012 September 12. Published in final edited form as: