Ethanol Induces Craniofacial Defects in Bmp Mutants Independent of nkx2.3 by Elevating Cranial Neural Crest Cell Apoptosis

Background: Craniofacial malformations lie at the heart of fetal alcohol spectrum disorders (FASDs). While there is growing evidence for a genetic component in FASDs, little is known of the cellular mechanisms underlying these ethanol-sensitive loci in facial development. The bone morphogenetic protein (Bmp) signaling pathway-dependent endoderm pouch formation is a key mechanism in facial development. We have previously shown that multiple Bmp mutants are sensitized to ethanol-induced facial defects. However, ethanol does not directly impact Bmp signaling. This suggests that downstream effectors, like nkx2.3, may mediate the impact of ethanol on Bmp mutants. Methods: We use an ethanol exposure paradigm with nkx2.3 knockdown approaches to test if nkx2.3 loss sensitizes Bmp mutants to ethanol-induced facial defects. We combine morphometric approaches with immunofluorescence and a hybridization chain reaction to examine the cellular mechanisms underlying Bmp–ethanol interactions. Results: We show that Bmp–ethanol interactions alter the morphology of the endodermal pouches, independent of nkx2.3 gene expression. Knockdown of nkx2.3 does not sensitize wild-type or Bmp mutants to ethanol-induced facial defects. However, we did observe a significant increase in CNCC apoptosis in ethanol-treated Bmp mutants, suggesting an ethanol sensitive, Bmp-dependent signaling pathway driving tissue interactions at the heart of FASDs. Conclusions: Collectively, our work builds on the mechanistic understanding of ethanol-sensitive genes and lays the groundwork for complex multi-tissue signaling events that have yet to be explored. Ultimately, our work provides a mechanistic paradigm of ethanol-induced facial defects and connects ethanol exposure with complex tissue signaling events that drive development.