An ethanol-sensitive, Bmp-PCP dependent pathway regulating endoderm cell behaviors - Summary Fetal Alcohol Spectrum Disorders (FASD) describe a continuum of ethanol-induced developmental defects with jaw defects being common. Genetic risk factors lie at the heart of FASD and are major drivers of FASD etiology, providing insight into the cellular and molecular processes potentially disrupted in FASD. However, we know little of these processes. From our studies in zebrafish, we have established an in vivo model for FASD where mutations in the Bone Morphogenetic Protein (Bmp) and Wnt/Planar Cell Polarity (PCP) signaling pathways sensitize embryos to ethanol-induced jaw defects by disrupting the cell behaviors driving tissue morphogenesis during jaw development. Jaw formation involves complex signaling interactions between the neural crest and the anterior pharyngeal endoderm (anterior PE). Critical to these interactions, anterior PE morphogenesis requires Bmp- and PCP-dependent actin-based cell movements, polarity and adhesion. Bmp signaling is required for endodermal E-cadherin localization and PCP receptor expression while PCP regulates cell polarity and adhesion. Mutations in the Bmp and PCP pathways sensitize embryos to ethanol-induced malformations of the anterior PE and jaw. Work in mouse liver indicates that ethanol reduces Bmp signaling at the level of the receptors. Altogether, this suggests that an ethanol-sensitive, Bmp-PCP signaling cascade contributes to anterior PE morphogenesis. Yet, how ethanol modulates the Bmp and PCP pathways and their crosstalk, disrupting anterior PE morphogenesis remain unknown. We hypothesize that ethanol attenuates Bmp signaling there by decreasing PCP component expression levels and disrupting actin dynamics, cell polarity, adhesion and migration during anterior PE morphogenesis. Using a unique combination of zebrafish-based approaches, we will test our hypothesis in the following aims. In Specific Aim 1, we will a) analyze an ethanol dose response on total and phosphorylated Bmp receptor and Smad proteins, b) use scRNA-seq to examine endoderm-specific gene expression changes of Bmp and, downstream of Bmp, PCP genes, and c) test the epistasis of the Bmp- PCP signaling axis on jaw development. In Specific Aim 2, we will use 4D-confocal analysis in control and ethanol-treated Bmp and PCP mutants to quantify ethanol-induced disruptions to a) cell velocity, coherence, and persistence of migration and resulting changes to size and shape of the anterior PE and b) actin polarization and cellular protrusion dynamics. In Specific Aim 3, we will a) quantify shape and polarity and b) analyze E-cadherin gene expression as well as E-cadherin and PCP protein localization in anterior PE cells of control and ethanol- treated Bmp, PCP and Bmp-PCP compound mutants. We will also c) quantify ethanol-induced jaw defects in Bmp- and PCP-E-cadherin compound mutants. Overall, this work will greatly advance our understanding of the cell behaviors and molecular mechanisms driving ethanol-induced structural malformations and provide concrete signatures to pursue predictive, diagnostic, and novel therapeutic approaches to FASD.
