Liao, Eric CMacias Trevino, Claudio2022-11-2420222022-11-232022-11Macias Trevino, Claudio. 2022. Transcriptional Regulation of Esrp1 and its Role in Craniofacial Morphogenesis. Doctoral dissertation, Harvard University Graduate School of Arts and Sciences.29393975https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37373719Alternative splicing creates different messenger RNAs from the same genetic locus, an essential regulatory mechanism that increases protein diversity and regulates key developmental processes. Recent studies have identified epithelial-specific alternative splicing regulatory factors ESRP1 and ESRP2 as crucial proteins that regulate craniofacial morphogenesis during embryonic development. Animal models carrying Esrp1 and Esrp2 loss-of-function mutations lead to a cleft palate phenotype or anterior neurocranium cleft phenotype in mice and zebrafish. However, no mechanism has been proposed to describe how disruption of epithelial-specific alternative splicing events leads to morphogenic defects during development. Additionally, there is a need for experimental models to functionally annotate gene variants in ESRP1 and ESRP2 found in human cohorts of cleft lip and/or palate (CL/P) congenital malformations. Using RNA-sequencing of irf6-/- embryos, RNAscope in-situ hybridization, and phenotypic analysis of optogenetic irf6-/- and esrp1/2 DKO zebrafish embryos, we established a regulatory axis between the key transcriptional regulator of epithelial maturation and craniofacial development, Irf6, and the Esrp1/2 proteins. In order to elucidate the mechanism for the ANC cleft phenotype, we employed the Tg(sox10:kaede) photoconvertible zebrafish line to lineage trace frontonasal neural crest cells that give rise to the medial ANC in developing zebrafish. We found that cranial neural crest cells in esrp1/2 DKO fish migrate to the medial ANC but fail to differentiate into chondrocytes. Meanwhile, we developed an esrp2 morphant assay that phenocopies the esrp1/2 cleft ANC phenotype to test mRNAs encoding for 18 esrp1 or esrp2 gene variants in a phenotypic rescue assay. We found that only 4 variants found in the RNA-Recognition Motifs 1 and 3 of esrp2 are pathogenic and fail to rescue the cleft ANC phenotype in our morphant assay. We further validated our data by performing a molecular splicing assay for putative Esrp1/2 target genes Arhgef11 and Ctnnd1 in Esrp1/2 DKO mouse PY2T cells that confirmed our zebrafish morphant assay results. Lastly, we defined alternatively spliced patterns for Ctnnd1 in WT and esrp1/2 DKO zebrafish and show that Ctnnd1 transcripts partially rescue the zebrafish cleft ANC phenotype. This is a critical finding that confirms Ctnnd1 as a target gene of esrp1 and esrp2 and implicates the importance of intercellular junctions in craniofacial morphogenesis and development of the cleft ANC phenotype.application/pdfenAlternative SplicingCleft Palateesrpirf6zebrafishBiologyGeneticsDevelopmental biologyThesis or Dissertation2022-11-240000-0002-3276-2064