PROJECT SUMMARY/ABSTRACT: The long-term objective of this proposal is to understand formation of the midface skeleton, both during normal development, and in human genetic disease conditions. The midface consists of the structures around the nose, the eye, and the upper jaw. Human genetic disorders, like frontonasal dysplasia, affect these midface structures. Specifically, the ALX transcription factor encoding genes have been implicated in multiple types of frontonasal dysplasia. Uniquely, we model these diseases in zebrafish by using genetic mutants. We propose that the ALX genes function to specify an identity code which patterns the vertebrate midface. Our model draws from examples like the DLX and HOX codes, which specify identity in the dorsoventral and anteroposterior axes of the craniofacial skeleton, respectively. We propose three specific aims to test our innovative “alx-bullseye code” hypothesis, that nested alx gene expression directly regulates frontonasal skeletal identity. In Aim 1 we define the skeletal structures arising from the alx-bullseye code in wild types using in situ hybridization, live cell tracking, and lineage tracing. Aim 2 will determine whether alx gene combinations function to specify frontonasal identity. To identify these functions, we will compare wild types to alx mutants examining gene expression, skeletal cell differentiation, and misexpression phenotypes. Aim 3 will uncover how alx genes molecularly control cellular identity. We will examine an in vivo Alx direct transcriptional reporter, ChIP-seq to identify Alx protein occupancy across the genome, and Hi-C to reveal enhancer-promoter contacts mediated by Alx. Significance of this proposal is high, the genes that we propose function in the alx bullseye code have direct human orthologs that are associated with midface dysmorphologies. This proposal is innovative, as there has not yet been a patterning code proposed for the vertebrate midface. Success of this proposal will enhance our understanding of midface craniofacial development and human disease. By using the strengths of the zebrafish system to elucidate the molecular, cellular, and genetic mechanisms underlying frontonasal dysplasia, we are fulfilling the mission of the NIDCR to improve human health through research.