Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, Institute for Neuroscience, Waggoner Center for Alcohol and Addiction Research, University of Texas at Austin, Austin, Texas, 78712, USA.
Department of Integrative Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, 78712, USA.
BMC Biol. 2021 Jul 1;19(1):134. doi: 10.1186/s12915-021-01062-9.
Gene-environment interactions are likely to underlie most human birth defects. The most common known environmental contributor to birth defects is prenatal alcohol exposure. Fetal alcohol spectrum disorders (FASD) describe the full range of defects that result from prenatal alcohol exposure. Gene-ethanol interactions underlie susceptibility to FASD, but we lack a mechanistic understanding of these interactions. Here, we leverage the genetic tractability of zebrafish to address this problem.
We first show that vangl2, a member of the Wnt/planar cell polarity (Wnt/PCP) pathway that mediates convergent extension movements, strongly interacts with ethanol during late blastula and early gastrula stages. Embryos mutant or heterozygous for vangl2 are sensitized to ethanol-induced midfacial hypoplasia. We performed single-embryo RNA-seq during early embryonic stages to assess individual variation in the transcriptional response to ethanol and determine the mechanism of the vangl2-ethanol interaction. To identify the pathway(s) that are disrupted by ethanol, we used these global changes in gene expression to identify small molecules that mimic the effects of ethanol via the Library of Integrated Network-based Cellular Signatures (LINCS L1000) dataset. Surprisingly, this dataset predicted that the Sonic Hedgehog (Shh) pathway inhibitor, cyclopamine, would mimic the effects of ethanol, despite ethanol not altering the expression levels of direct targets of Shh signaling. Indeed, we found that ethanol and cyclopamine strongly, but indirectly, interact to disrupt midfacial development. Ethanol also interacts with another Wnt/PCP pathway member, gpc4, and a chemical inhibitor of the Wnt/PCP pathway, blebbistatin, phenocopies the effect of ethanol. By characterizing membrane protrusions, we demonstrate that ethanol synergistically interacts with the loss of vangl2 to disrupt cell polarity required for convergent extension movements.
Our results show that the midfacial defects in ethanol-exposed vangl2 mutants are likely due to an indirect interaction between ethanol and the Shh pathway. Vangl2 functions as part of a signaling pathway that regulates coordinated cell movements during midfacial development. Ethanol exposure alters the position of a critical source of Shh signaling that separates the developing eye field into bilateral eyes, allowing the expansion of the midface. Collectively, our results shed light on the mechanism by which the most common teratogen can disrupt development.
基因-环境相互作用可能是大多数人类出生缺陷的基础。已知最常见的环境致畸物是产前酒精暴露。胎儿酒精谱系障碍（FASD）描述了由产前酒精暴露引起的所有缺陷。基因-乙醇相互作用是导致 FASD 的易感性基础，但我们缺乏对这些相互作用的机制理解。在这里，我们利用斑马鱼的遗传可操作性来解决这个问题。
我们首先表明，Wnt/平面细胞极性（Wnt/PCP）途径的成员vangl2在晚期囊胚和早期原肠胚阶段强烈与乙醇相互作用。vangl2 突变或杂合的胚胎对乙醇诱导的面中部发育不良敏感。我们在早期胚胎阶段进行了单个胚胎 RNA-seq，以评估胚胎对乙醇的转录反应的个体差异，并确定 vangl2-乙醇相互作用的机制。为了确定被乙醇破坏的途径，我们使用这些全局基因表达变化来通过集成网络细胞信号特征（LINCS L1000）数据集识别模拟乙醇效应的小分子。令人惊讶的是，该数据集预测 Sonic Hedgehog (Shh) 途径抑制剂环巴胺会模拟乙醇的作用，尽管乙醇不会改变 Shh 信号的直接靶标表达水平。事实上，我们发现乙醇和环巴胺强烈但间接相互作用，破坏面中部发育。乙醇还与另一个 Wnt/PCP 途径成员 gpc4 相互作用，Wnt/PCP 途径的化学抑制剂 blebbistatin 模拟了乙醇的作用。通过表征膜突，我们证明乙醇与 vangl2 的缺失协同作用，破坏了会聚延伸运动所需的细胞极性。
我们的结果表明，暴露于乙醇的 vangl2 突变体中的面中部缺陷可能是由于乙醇与 Shh 途径之间的间接相互作用。Vangl2 作为调节面中部发育过程中协调细胞运动的信号通路的一部分发挥作用。乙醇暴露改变了 Shh 信号的一个关键来源的位置，该来源将眼区分离成双侧眼睛，从而使面中部扩张。总的来说，我们的结果揭示了最常见的致畸物如何破坏发育的机制。
