Molecular and Developmental Analysis of Holoprosencephaly

无前脑畸形(HPE)是一种常见的前脑和面中部畸形，其原因是未能明确 早期的喙腹侧中线。HPE具有复杂的病因，并与遗传和 环境风险因素。导致高血压性肺泡灌洗风险升高的环境因素之一是产前 酒精暴露。Nodal和Hedgehog(HH)信号通路中的杂合突变是 与HPE相关联。然而，HPE的临床表现高度多变，许多突变携带者是 基本上没有受到影响。这些和其他观察结果导致了一个多因素模型，在该模型中，结果 与突变相关的基因会受到更常见的遗传变异和/或环境暴露的影响。 因此，HPE是一个很好的模型系统，用于理解基因-环境相互作用和 许多常见出生缺陷的多因素病因。我们已经在老鼠身上模拟了这种情况。CDON编码 一种多功能的细胞表面辅助受体，通过几条途径促进信号传递，包括HH 路径。带有Cdon突变的小鼠在HH信号方面只有轻微的缺陷，但与野生型小鼠不同的是， 对一过性接触乙醇(Etoh)在早期诱发各种HPE缺陷敏感 胚胎发生。尽管许多研究使用了这一模型和相关模型，但胚胎细胞类型和 作为乙醇在HPE中致畸作用的直接靶点的发育事件仍未确定。《节点》 通路位于HH的发育上游，呈轮状腹中线模式。我们最近的发现是 与以下假设一致：1)密码子自主发挥细胞功能，调节结节通路信号 在前原始条纹(APS)细胞中；2)抑制APS细胞中的Nodal信号是靶点 胎儿乙醇和密码子突变的协同作用为了验证这些假设，我们提出了以下目标：1) 为了确定CDON在乙醇诱导的HPE的APS细胞中是否以细胞自主的方式发挥作用，我们将使用条件 突变去除小鼠APS或替代结构中的密码子并评估小鼠的敏感性 至乙醇诱导的HPE。胚胎将在多个阶段进行HPE表型的形态分析 基因表达变化的分析、原位杂交和qRT-PCR，以及细胞增殖的检测 2)为了确定乙醇在HPE中的作用机制，我们将研究小鼠上皮细胞干细胞 细胞作为APS细胞的体外模型。我们将通过转录和转录技术研究这些细胞对乙醇的反应。 信号转导分析。这些实验的结果将被应用于乙醇治疗的研究 胚胎。这两个目标是协同的，因为它们融合了发育遗传学和机械性分子 探讨胎儿酒精暴露对HPE共同特征的影响 以及相关的脑和头面部缺陷。

Holoprosencephaly (HPE), a common malformation of the forebrain and midface, is caused by failure to define the early rostroventral midline. HPE has a complex etiology, and is associated with both genetic and environmental risk factors. Among the environmental factors implicated in elevated risk of HPE is prenatal alcohol exposure. Heterozygous mutations in the Nodal and Hedgehog (HH) signaling pathways are associated with HPE. However, clinical presentation of HPE is highly variable, and many mutation carriers are largely unaffected. These and additional observations have led to a multifactorial model, in which the outcome associated with a mutation is influenced by more common genetic variants and/or environmental exposures. HPE is therefore an excellent model system for understanding gene-environment interactions and the multifactorial etiology of many common birth defects. We have modeled this scenario in mice. CDON encodes a multifunctional, cell surface coreceptor that promotes signaling by several pathways, including the HH pathway. Mice with a mutation in Cdon have only a minor deficit in HH signaling but, unlike wild type mice, are sensitive to induction of a full range of HPE defects by transient exposure to ethanol (EtOH) during early embryogenesis. Despite many studies employing this and related models, the embryonic cell types and developmental events that are direct targets of EtOH’s teratogenicity in HPE remain unidentified. The Nodal pathway lies developmentally upstream from HH in rostroventral midline patterning. Our recent findings are consistent with the hypotheses that: 1) Cdon functions cell autonomously to regulate Nodal pathway signaling in cells of the anterior primitive streak (APS); and 2) inhibition of Nodal signaling in APS cells is the target of synergy between fetal EtOH and Cdon mutation. To test these hypotheses, the following aims are proposed: 1) to determine if CDON functions cell-autonomously in APS cells in EtOH-induced HPE, we will use conditional mutagenesis to remove Cdon in the mouse APS or alternative structures and assess the sensitivity of the mice to EtOH-induced HPE. Embryos will be analyzed at multiple stages for HPE phenotypes by morphological analyses, in situ hybridization and qRT-PCR for alterations in gene expression, and assays for cell proliferation and apoptosis; and 2) to determine mechanisms of EtOH action in HPE, we will study mouse epiblast stem cells as an in vitro model for APS cells. We will study responses of these cells to EtOH by transcriptomic and signal transduction analyses. Findings from these experiments will then be applied to studies of EtOH-treated embryos. The two aims are synergistic in that they merge developmental genetics and mechanistic molecular analyses in approaching the problem of how fetal alcohol exposure contributes to common features of HPE and related brain and craniofacial defects.