Mutation in the Bone Morphogenetic Protein signaling pathway sensitize zebrafish and human to ethanol-induced jaw malformations

Background Fetal Alcohol Spectrum Disorders (FASD) describe a continuum of ethanol-induced developmental defects including commonly observed craniofacial malformations. While ethanol-sensitive genetic mutations are a major contributor to facial malformations, the impacted cellular mechanisms underlying these facial anomalies remain unknown. The Bone Morphogenetic Protein (Bmp) signaling pathway is a key regulator of epithelial morphogenesis driving facial development, providing a possible ethanol-sensitive mechanism to malformations to the facial skeleton. Methods Using zebrafish, we tested several mutants for Bmp pathway components for ethanol-induced facial malformations. Mutant embryos were exposed to ethanol in the media from 10-18 hours post-fertilization (hpf). Exposed zebrafish were fixed at 36 hpf to analyze anterior pharyngeal endoderm size and shape by immunofluorescence or at 5 days post-fertilization (dpf) to quantitatively examine shape of the facial skeleton stained with Alcian Blue/Alizarin Red staining. Integrating human genetic data, we screened for Bmp-ethanol associations in jaw volume of ethanol-exposed children. Results We found that mutations in the Bmp pathway sensitize zebrafish embryos to ethanol-induced malformations to anterior pharyngeal endoderm shape, leading to altered expression of fgf8a in the oral ectoderm. These changes correlate with shape changes in the viscerocranium, suggesting that ethanol-induced malformations of the anterior pharyngeal endoderm lead to facial malformations. Variants in the Bmp receptor gene, BMPR1B were associated with ethanol-related differences in jaw volume in humans. Conclusions For the first time, we show that ethanol exposure disrupts proper morphogenesis of, and tissue interactions between, the facial epithelia. These shape changes in the anterior pharyngeal endoderm-oral ectoderm-signaling axis during early zebrafish development mirror the overall shape changes observed in the viscerocranium and were predictive for Bmp-ethanol associations in jaw development in human. Collectively, our work provides a mechanistic paradigm linking the impact of ethanol to the epithelial cell behaviors that underlie facial defects in FASD.

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