Investigating the Roles of IRF6 in Epithelial Maturation, Craniofacial Development, and Orofacial Cleft Pathogenesis

Abstract

Cleft lip and/or palates (CL/P) are common congenital malformations, and mutations in the transcription factor IRF6 are the most significant genetic contributors to cleft pathogenesis. IRF6 is a master regulator of epithelial maturation and is expressed in the oral epithelium hypothesized to play critical signaling functions during palatogenesis. Despite the importance of IRF6 in craniofacial development, much still remains unknown about its biological function. To complement the studies of CL/P pathogenesis in other models, we used CRISPR to produce a zebrafish irf6 null model and discovered that maternal-null irf6-/- embryos displayed embryonic lethality due to periderm rupture. The zebrafish periderm has been previously used as a model of the mammalian oral epithelium with conservation of both morphologies and molecular pathways. Due to strong cross-species sequence conservation in IRF6, either zebrafish or human IRF6 could rescue the maternal-null irf6-/- periderm rupture phenotype. This allowed us to test the protein functions of IRF6 missense gene variants of unknown significance from CL/P patients, functionally categorize them by residual protein function, and provide biological data to complement the traditional statistical/computational approaches for variant pathogenicity assignment. Next, because the early irf6-/- periderm rupture precluded studies of potential irf6 functions later during craniofacial development, we employed a novel optogenetic gene expression system with dominant-negative Irf6 to spatiotemporally inhibit Irf6 functions during palatogenesis. The results revealed a striking orofacial cleft phenotype in the zebrafish model, the molecular mechanisms of which are currently being investigated. Finally, our irf6-/- model provided an opportunity to elucidate Irf6 downstream transcriptional targets, enhance our understanding of orofacial cleft pathogenesis, and identify potential nodes for intervention to prevent CL/Ps in utero. ChIP-seq and mRNA-seq were employed to compare wild type to maternal-null irf6-/- embryos and identify Irf6 transcriptional target genes significantly downregulated in the absence of irf6 functions during zebrafish development. Analyses of the candidate target genes have begun to reveal novel aspects of Irf6 biological function and developmental pathways previously uncharacterized during palatogenesis. These pathways could represent yet unexplored mechanisms by which IRF6 in the mammalian embryonic oral epithelium regulates epithelial maturation and epithelial-mesenchymal interactions during craniofacial development.