Molecular and Developmental Analysis of Holoprosencephaly

前脑无裂畸形的分子和发育分析

• 批准号: 10647779
• 负责人: Robert S. Krauss
• 金额: $ 60.51万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10647779
• 关键词: Acute; Affect; Alcohols; Anterior; Apoptosis; Biological Models; Brain; CDON gene; Cell Proliferation; Cell physiology; Cell surface; Cells; Cellular Assay; Clinical; Complex; Congenital Abnormality; Craniofacial Abnormalities; Defect; Development; Embryo; Embryonic Development; Environmental Exposure; Environmental Risk Factor; Epiblast; Erinaceidae; Ethanol; Etiology; Event; Failure; Fetal Alcohol Exposure; Gene Expression; Genetic; Genetic Variation; Heterozygote; Holoprosencephaly; Human; Hydrophobicity; In Situ Hybridization; Metabolism; Minor; Modeling; Molecular; Molecular Analysis; Morphology; Mus; Mutagenesis; Mutation; Nature; Nodal; Outcome; Pathway interactions; Pattern; Penetrance; Phenotype; Phosphorylation; Population; Primitive Streaks; Prosencephalon; Quantitative Reverse Transcriptase PCR; Risk; SHH gene; Signal Pathway; Signal Transduction; Structure; Teratogens; Testing; Wild Type Mouse; alcohol exposure; cell type; developmental genetics; embryo cell; experimental study; fetal; gastrulation; gene environment interaction; genetic pedigree; genetic risk factor; genetic variant; in vitro Model; insight; malformation; morphogens; mouse model; mutation carrier; receptor; response; smoothened signaling pathway; stem cells; synergism; transcriptomics

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.

前脑无裂畸形（HPE）是前脑和面中部的一种常见畸形， 早期的吻腹中线HPE具有复杂的病因学，并且与遗传和 环境风险因素。在与HPE风险升高有关的环境因素中， 酒精暴露Nodal和Hedgehog（HH）信号通路中的杂合突变是 与HPE有关。然而，HPE的临床表现是高度可变的，并且许多突变携带者 基本上不受影响。这些和其他观察结果导致了一个多因素模型，其中结果 与突变相关的基因突变受更常见的遗传变异和/或环境暴露的影响。 因此，HPE是了解基因-环境相互作用的优秀模型系统， 许多常见出生缺陷的多因素病因。我们在小鼠中模拟了这种情况。CDON编码 一种多功能的细胞表面辅助受体，可通过多种途径促进信号传导，包括HH 通路具有Cdon突变的小鼠在HH信号传导中仅具有轻微缺陷，但与野生型小鼠不同， 在早期HPE过程中，对瞬时暴露于乙醇（EtOH）诱导的全方位HPE缺陷敏感， 胚胎发生尽管许多研究采用了这种模型和相关模型，但胚胎细胞类型和 作为HPE中EtOH致畸性的直接目标的发育事件仍未确定。节点 通路位于HH在口腹中线图案的发育上游。我们最近的发现是 这与以下假设一致：1）Cdon自主地发挥细胞功能以调节Nodal途径信号传导 在前原条（APS）细胞中; 2）抑制APS细胞中的Nodal信号传导是 胎儿EtOH和Cdon突变之间的协同作用。为了验证这些假设，提出了以下目标：1） 为了确定在EtOH诱导的HPE中，CDON是否在APS细胞中自主发挥功能，我们将使用条件 诱变以去除小鼠APS或替代结构中的Cdon，并评估小鼠的敏感性 EtOH诱导的HPE。将通过形态学方法在多个阶段分析胚胎的HPE表型。 基因表达改变的原位杂交和qRT-PCR分析，以及细胞增殖试验 和凋亡; 2）为了确定EtOH在HPE中的作用机制，我们将研究小鼠上胚层干细胞， 细胞作为APS细胞的体外模型。我们将通过转录组学和免疫组化研究这些细胞对EtOH的反应。 信号传导分析。然后将这些实验的结果应用于EtOH处理的 胚胎这两个目标是协同的，因为它们合并了发育遗传学和机制分子生物学。 分析胎儿酒精暴露如何导致HPE的共同特征 以及相关的大脑和颅面缺陷。