Deciphering a tissue specific role for Arhgap29 during palatogenesis - PROJECT SUMMARY Orofacial clefts (OFCs), specifically cleft lip with or without cleft palate, are among the most common class of birth defects and they contribute to a significant health and financial burden. The development of OFCs is influenced by complex interactions between genetic and environmental factors. Developing a comprehensive understanding of the cellular pathways and molecular regulators of palatogenesis will be essential to enhancing treatment options and disease intervention. My overall objective of the proposed project is to identify molecular pathways that modulate palatal development. Genetic studies in the human identified over 50 loci associated with OFC. However, mutations in very few of these genes can cause isolated OFC with high penetrance, making them well-suited for mechanistic studies and better candidates for clinical interventions. One such gene is Rho GTPase activating protein 29 (ARHGAP29). ARHGAP29 contributes to cyclic regulation of the small GTPase RhoA, inactivating it. To explore the role of ARHGAP29 during craniofacial development, Arhgap29 was previously deleted in the mouse. Although Arhgap29 knockout embryos were found to die around embryonic day e8.5, i.e., before craniofacial structures develop, heterozygous loss-of-function embryos were viable and displayed intraoral adhesions, a phenotype associated with OFCs. In the craniofacial region, ARHGAP29 is expressed in cell lineages derived from both the ectoderm (periderm and epithelial cell layers) and the neural crest (mesenchymal cells). To tease apart the contributions of ARHGAP29 to each cell lineage during palatogenesis, I initiated a tissue-specific knockout strategy using the Cre-Lox system. My preliminary results from these animals show that the loss of ARHGAP29 in either ectoderm- or neural crest-derived lineages results in a delay in palatogenesis (apparent at e14.5). However, only the loss of ARHGAP29 in ectoderm-derived cells results in a cleft palate at e18.5. These findings are consistent with ARHGAP29 playing a tissue-specific role during palatogenesis. My central hypothesis is that ARHGAP29 in ectoderm- and neural crest-derived cells of the palatal shelves is required for proper palatogenesis because it promotes remodeling of adherens junctions and force transduction across the palatal shelves as they elevate. The premise for this hypothesis is that ARHGAP29 is a modulator of RhoA, which promotes the actomyosin contractility that is required for both the remodeling of cell-cell junctions and cellular contractility that are required for palatal shelf elevation and fusion. In Aim 1 I will identify the tissue(s) in which ARHGAP29 is required to promote proper palatogenesis in vivo, using a series of tissue-specific Cre recombinase driver alleles and characterizing embryos at time points critical to palate development. In Aim 2 I will define the molecular mechanisms by which ARHGAP29 regulates the epithelial shape and adhesions required during palatogenesis, using an in vitro culture system in conjunction with biochemical and confocal microscopy-based analyses. This project will provide me with in-depth training in all these techniques, as well as in the scientific communication skills that I will need to become a successful independent investigator.
