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

<b>Background:</b> 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 <i>nkx2.3</i>, may mediate the impact of ethanol on Bmp mutants. <b>Methods:</b> We use an ethanol exposure paradigm with <i>nkx2.3</i> knockdown approaches to test if <i>nkx2.3</i> 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. <b>Results:</b> We show that Bmp-ethanol interactions alter the morphology of the endodermal pouches, independent of <i>nkx2.3</i> gene expression. Knockdown of <i>nkx2.3</i> 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. <b>Conclusions:</b> 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.