The Jackson Laboratory

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Scientific report

Dissecting the mechanism of secondary palate fusion

Cleft lip with or without cleft palate is seen at a frequency of approximately 1 in 700 newborns, making it one of the most common congential birth defects in humans. In addition to disabilities in speech, feeding and breathing, these malformations have a dramatic impact on the physical appearance of the affected individual, often with devastating psychological and social consequences. The etiology of orofacial clefting is known in only a minority of cases, emphasizing the need for better understanding of the genes responsible for the normal development of the lip and palate.

Genetic regulation of secondary palate formation can be divided into three stages: vertical palate shelf growth and patterning, palate shelf elevation, and palate shelf fusion. Defects in any of these processes can result in a cleft palate phenotype, and the mouse has proven to be a critical tool in identifying some of the genes that regulate these events. Fusion of the paired palate shelves requires a series of specific changes in the epithelium. Initially, the periderm cells of the bilayered epithelium bulge along the medial aspects of the shelves and extend lamellipodia on their surface. The events following fusion are a matter of some controversy, but current evidence suggests migration of the periderm cells to the oral and nasal aspects of the fusing shelf allows for intercalation of the underlying basal epithelial layer, forming the medial epithelial seam (MES), which subsequently undergoes apoptosis to yield a fully fused secondary palate. Defects in this process are evident in both Snai2-/- mice and Snai2-/-;Snai1+/- compound mutant mice, where the palate shelves appear to approach and contact, but fail to form an epithelial seam despite normal formation of lamellipodia on the surface of the shelves.

Our current work aims to understand how interactions between the secondary palate shelf epithelium and mesnechyme interact to regulate shelf fusion. In particular, we are currently exploring the mechanism by which Snai1 and Snai2 regulate this process. To address this, we are asking three basic questions. First, is the cleft palate phenotype due to a failure in normal migration of the periderm cell layer? Second, is the cleft palate phenotype due to Snai1/Snai2 deletion cell autonomous to the mesenchyme, basal epithelium or periderm cell layer? Finally, what are the downstream pathways affected by Snai1/Snai2 deletion?

Novel models of cleft lip and palate

To extend our understanding of the genetic mechanisms that govern craniofacial development, we employ a forward genetic approach to identify novel genes and pathways, including positional cloning of spontaneous and ENU-induced craniofacial mutants. In collaboration with Dr. Leah Rae Donahue and the Craniofacial Mutant Resource, we have been characterizing a number of semi-dominant spontaneous mutants that, when rendered homozygous, display robust developmental abnormalities. Using both regional and exome capture, combined with Next-Gen sequencing, we have dramatically increased the speed with which candidate genes can be identified and validated. Several new developmental mutants have been identified as part of this screen, including models of cleft palate and holoprosencephaly.

As part of an ENU mutagenesis screen, we have recently identified a novel mouse model of cleft palate, clfp4, which displays cleft secondary palate, omphalocele and skeletal malformations with high penetrance. Preliminary histological analysis has revealed that the cleft palate in these animals results from a failure of palate shelf elevation. The skull of mutant embryos is misshapen and displays defects in calvarial ossification. Defects in ventral body wall fusion are accompanied by a failure of the sternum to fuse. Overall, mutant embryos show some degree of axial truncation and display widespread sub-epidermal hemorrhaging.  
 
Preliminary mapping data placed the mutation at the distal end of Chromosome 10, a region where this combination of phenotypes has not been reported. Through targeted array capture of the exome within our critical region and Illumina sequencing, we identified a single non-synonymous coding mutation in a gene that has not previously been reported to play a role in craniofacial development. Due to the early lethality of mice null for this gene, its role in the development of the palate and craniofacial complex has not been investigated. However, significant evidence suggests the mutant gene function as a co-receptor for several signaling molecules known to play a role in craniofacial development. Indeed, our amino acid change is predicted to disrupt a highly conserved motif within the receptor interaction domain. We are currently exploring the impact the clfp4 mutation has on these pathways during neural crest migration, development of the facial structures and growth of the palate shelves, and will present our current findings. The identification of this novel gene function will provide important insight into the molecular networks that regulate craniofacial and palate development.

Cre Repository

The Jackson Laboratory Repository distributes a large, diverse collection of Cre driver strains to scientists around the world, including over 200 strains as live colonies. The utility of existing Cre driver strains critically depends upon careful characterization of their function. Characterization data for most Cre strains is limited to that provided by the donating investigators, which is often incomplete and focused on a specific tissue of interest to their research question. Thus, complementing the published data with a standardized, unbiased, and extensive characterization pipeline promises to maximize the value of these resources for the scientific community.

To address this issue, our group has embarked on an ambitious project to add value to these strains by comprehensively characterizing Repository Cre lines. Despite the best efforts of those developing new Cre lines, the fidelity of Cre activity is not always ideal. Many difficulties have been reported in various Cre lines, including mosaic or incomplete deletion in a target tissue/cell type, inconsistent activity, expression in non-target tissues, and/or Cre-related toxicity. In many cases, however, this data is not reported or available to the potential user. Our preliminary results indicate the vast majority of Cre driver strains exhibit unexpected recombinase activity in a number of tissue types, highlighting the need for extended analysis. We have standardized our data annotation scheme to include 11 broad organ systems, 30 individual organs/structures and 89 substructures, all of which are consistent with the mouse Anatomical Dictionary. Slide-scanned images and associated annotations are published on a dedicated website (cre.jax.org) and submitted to Creportal.org. This information will allow users to make informed judgments about the suitability of a particular line for their experiments, and enhance the power of large-scale mouse gene targeting projects.

FaceBase Consortium

The overall goal of the JAX FaceBase project is to facilitate orofacial clefting research by generating new mouse genetic tools (Cre strains) and by providing a repository of mouse strains critical for clefting research community (linky to sites). Our initial set of planned Cre drivers includes four BAC transgenic drivers, deltaNp63-CreERT2, Krt6a-Cre, Tbx22-CreERT2, Lhx7/8-Cre.  These strains are designed to target the oral basal epithelium, the oral peridem cell layer, the posterior secondary palate shelf, and the mesenchyme adjacent to the fusing facial prominences, respectively. We are also employing a novel approach to Cre driver generation, using highly conserved enhancer sequences identified by our collaborator, Dr. Axel Visel at Lawrence Berkley National Laboratory (http://enhancer.lbl.gov/). Individual enhancer elements that have been empirically validated to direct expression to the midface and palate will be used to drive Cre expression from the ROSA26 locus. Our first three elements all target different parts of the fusing facial prominences at E11.5, and future drivers will use new elements identified by Dr. Visel that are expressed during secondary palate development. All of these strains will be characterized for orofacial-specific activity and examined for specificity using our Cre characterization pipeline. Together, these activities will provide both FaceBase members and the general scientific community with new research tools, novel genetic models, and comprehensive mouse repository services to enhance research in orofacial clefting.

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