Function and Regulation of Sema3 Genes in Palate Development and Innervation

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Abstract Cleft palate is a common birth defect in humans and causes significant problems in feeding, speech, and increased mortality. While most cleft palate patients receive surgical repair to restore an anatomically intact palate, about 30% of children with a repaired palate still experience significant functional palate deficit and/or velopharyngeal dysfunction (VPD), which impairs speech and significantly affects quality of life. Orofacial malformations, including cleft or high-arched palate and flattened nasal bridge, and nasal dysarthria are major features of congenital facial palsy conditions such as the Moebius syndrome (OMIM 157900), suggesting that facial/palatal innervation is intimately linked to palate morphogenesis and velopharyngeal function. However, nothing is currently known about the molecular mechanisms regulating palate innervation and their integration with the overall mechanisms regulating palate morphogenesis. We have discovered that mice lacking the zinc finger transcription factor Osr2 exhibit complete cleft palate and dramatic disruption of palatal innervation. We previously demonstrated that Osr2 is a key intrinsic regulator of palatal shelf growth and integrates the functions of several major molecular pathways, including SHH signaling and the transcription factor Pax9- mediated regulation of palate morphogenesis. Through RNA-seq transcriptome profiling we found that expression of three physically linked members of the Sema3 gene family, Sema3a, Sema3d, and Sema3e, was significantly upregulated in the developing palatal mesenchyme in Osr2 mutant embryos. The Sema3 proteins were originally identified as negative mediators of axonal guidance in the central nervous system and have since been shown to play crucial roles in many other developmental processes, including neural crest migration, endothelial migration and vasculogenesis, and osteoblast differentiation. Our central hypothesis is that proper regulation of expression of the Sema3 genes during palate development is crucial for not only palate innervation but also palate morphogenesis. Recently mutations in PLXND1, which encodes a receptor for SEMA3 proteins, and a complex chromosomal rearrangement disrupting both the SEMA3A and SEMA3D genes, have been associated with Moebius syndrome, suggesting that the SEMA3-PLXND1 pathway plays crucial roles in cranial nerve development and palate morphogenesis in humans as well. To better understand the function and regulation of the Sema3 genes in palate development and innervation, we will use a combination of multi-omics approaches, CRISPR/Cas9-mediated genome editing, and compound mutant mouse studies to unravel the molecular network involving Osr2 and the Sema3 genes in the regulation of palate innervation and morphogenesis. Data from these studies will fill a long standing major gap in the understanding of pathogenic mechanisms of cleft palate and VPD, which will help to significantly improve strategies for diagnosis, counseling, treatment and care of patients with these devastating craniofacial conditions.