Transcriptional Regulation of Esrp1 and its Role in Craniofacial Morphogenesis

Abstract

Alternative 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.