Investigating the role of ESRP2 in craniofacial development using the cleft primary palate (cpp) avian model. - Project Summary/Abstract The frontonasal prominence (FNP) is the keystone of the midface, which gives rise to the forehead, bridge and tip of the nose, the philtrum, and the primary palate. The FNP is populated by cranial neural crest cells (CNCCs) that migrate from the neural tube and differentiate to give rise to structural facial features like bone and cartilage. These processes are tightly regulated by signaling from adjacent FGF8-expressing ectodermal centers that include the midbrain-hindbrain boundary (MHB), frontonasal ectodermal zone (FEZ), and olfactory epithelium (OlfE). Failure of formation and fusion of the FNP contribute to several congenital craniofacial abnormalities including clefting and hypotelorism. The cleft primary palate (cpp) avian mutant is a model for failed FNP development as the FNP never fuses and is severely hypoplastic. The cpp was first characterized in the 1960s, but the pathogenic-causing variant was only recently identified as a 1 base pair (bp) deletion in Epithelial Splicing Regulatory Protein 2 (ESRP2) that causes a frameshift. We are the first to genotype the cpp and study the molecular mechanisms that cause its severe phenotype. In preliminary studies, we have performed bulk RNA sequencing at day 4 (HH23), before phenotypic expression, and revealed several differentially expressed genes (DEGs) relating to CNCC migration (Semaphorin 3F (SEMA3F)), development (Bone morphogenetic protein 4 (BMP4)), and differentiation (Ovo Like Zinc Finger 2 (OVOL2)) in the cpp. This has led us to our central hypothesis that the cpp phenotype is due to altered ESRP2-dependent splicing of target genes essential for regulating CNCC migration to, and/or differentiation within the FNP. ESRP2 is a regulator of FGFR2 alternative splicing into epithelial (FGFR2b) and mesenchymal (FGFR2c) isoforms, which dictate FGF ligand binding specificity. In Aim 1, we will use BaseScope, RNAscope, and immunostaining to investigate how variant ESRP2 affects FGF signaling within the cpp and RCAS::FGFR2b to attempt to rescue the cpp phenotype via overexpression of FGFR2b. In Aim 2, we will investigate candidate genes identified through our bulk RNA-seq data to determine if ESRP2 has an expanded role in regulating genes important for craniofacial development. We will use enhanced crosslinking and immunoprecipitation (eCLIP) to determine if ESRP2 directly binds to regulate craniofacial development genes as well as FGF8-soaked bead implantation to test the alternative hypothesis that ESRP2 modulates the expression of candidate DEGs indirectly via FGF signaling. This proposal will increase the understanding of alternative splicing in craniofacial development, which will further contribute to advancement of diagnoses and therapeutics for midfacial abnormalities.
