Cell-cell adhesion in regulation of mammalian palatogenesis

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ABSTRACT This application is for a Ruth L. Kirschstein National Research Service Award (NRSA) Individual Predoctoral Fellowship (F31) for Kendall J. Lough to study the role that the nectin-afadin cell adhesion pathway plays in regulating mammalian palatogenesis. Mr. Lough has demonstrated that loss of afadin results in highly penetrant cleft palate in mouse models. This award will allow Mr. Lough to further develop an expertise in cell and developmental biology while expanding his understanding in craniofacial morphogenesis and oral biology. This proposal will expand the understanding of mammalian palatogenesis by describing a novel cleft palate model while developing an additional model of an uncharacterized human cleft lip and cleft palate (CL/P) syndrome. Mr. Lough will take full advantage of the opportunities presented with this award to further develop skills essential to his career including 1) scientific communication through writing and oral presentation, 2) statistics and computational biology, 3) physiology in cell and developmental biology, 4) additional techniques relevant to biomedical research, and 5) significant mentoring experience. Mr. Lough has assembled a strong environment and research mentoring team to ensure the success of this proposal and his career goals. This award will prepare Mr. Lough to continue in his pursuit of a career as an independent academic researcher studying the genetics and cell biology of the oral mucosa, gastrointestinal tract and associated pathologies. Human CL/P is the most common birth defect in America and results in thousands of deaths globally each year, particularly in developing nations. While modern sequencing studies have identified numerous candidate loci associated with human CL/P, very few of these have been functionally characterized. One such disorder is CLPED1, which is associated with homozygous nonsense mutations in the extracellular domain of the nectin-1 encoding gene, PVRL1. Nectin-1 is part of the larger nectin family of transmembrane cell adhesion molecules, which bind to the cytoplasmic adapter protein afadin. Current mouse models lacking these genes are either embryonic lethal or fail to develop CL/P. Utilizing a novel genetic technique to inactivate gene expression via lentiviral delivery to early mouse embryos, Mr. Lough has demonstrated that loss of afadin results in CL/P, providing the first functional data linking this pathway to mammalian palatogenesis. This technique allows Mr. Lough to generate additional genetic mouse models at an unprecedented rate – 6 weeks as opposed to 6 months for standard techniques. This proposal outlines experiments to further characterize the mechanism of CL/P presentation in afadin null embryos through a combination of in vivo and in vitro techniques to evaluate changes in cell-cell adhesion. This application also proposes to generate an elegant genetic model expressing the human CL/P – associated Pvrl1W185X mutation in mice. This proposal will provide the scientific community with novel genetic tools and models for studying orofacial clefting candidates in an epithelial-specific manner.