Deciphering neural crest-specific TFAP2 pathways in midface development and dysplasia

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PROJECT SUMMARY: Craniofacial anomalies are the 2nd leading structural malformation to appear at birth, with the most common forms occurring in the mid- and upper-face regions. However, our limited understanding in the molecular mechanisms controlling their development has hindered strategies for effective treatment and prevention. Several studies have identified the pathways underlying lower face formation during embryonic development (e.g., jaw and teeth). Yet, less emphasis has been placed on the midface, and the unique mechanisms orchestrating the development of this region are poorly understood. The long-term goal of this study is to dissect the molecular underpinnings governing midface development and how their dysregulation causes dysplasia. We and others have independently uncovered key midface genes that function within the neural crest, an embryonic stem cell population that gives rise to facial bone and cartilage. For example, deletion of Alx1/3/4 (Alx), whose loss causes syndromic forms of Frontonasal Dysplasia, lead to a severe midfacial cleft in mice. Our lab has found that loss of Tfap2 in the neural crest compromises expression of Alx genes and presents the same midface cleft as the Alx knockout embryos. Thus, our overall objective in this research proposal is to leverage this new animal model for Frontonasal Dysplasia to uncover how TFAP2 transcription factors operate within the midface-unique gene regulatory networks (GRN’s). To approach this objective, we test the overall hypothesis that TFAP2 transcription factors directly regulate Alx gene expression for appropriate neural crest survival, midface fusion, and skeletal formation. In AIM 1, we will employ genome- and epigenome-wide assays (CUT&RUN, ATAC-seq) to decipher how TFAP2 targets Alx and shapes the genome landscape to regulate their expression levels. We will leverage zebrafish reporter strategies to test TFAP2-bound Alx noncoding enhancers for midface specificity. In AIM 2, we will couple sophisticated mouse genetics with immunofluorescence and skeletal analyses to characterize how Tfap2 and Alx genetically interact to control neural crest viability, behavior, and formation of the skeletal elements. Completion of these aims will fill a critical knowledge gap in our understanding for how these genes and pathways are linked to shape the developing midface. Such knowledge will contribute to the development of strategies in treating midfacial disorders or even preventing them. The principal investigator of this study, Mr. Nguyen, has extensive training in mouse genetics, embryology, and gene expression profiling techniques highlighted in this proposal. With combined mentorship from Drs. Van Otterloo and Amendt, Mr. Nguyen will develop skills in genome-wide molecular assays, next-generation sequencing approaches, bioinformatic techniques, and immunofluorescent and confocal microscopy methods; collectively, greatly advancing his doctoral training and expanding his research toolkit. The training plan, established team, and institutional environment outlined in this research proposal will not only pave the way to improving craniofacial treatment and health, but also catalyze his career towards becoming an independent researcher.