Cellular Mechanisms in Fetal Alcohol Spectrum Disorders

与胎儿酒精谱系障碍（FASD）相关的许多结构和功能异常 已经被发现，但在我们对相关发病机制的理解方面仍然存在重大差距， 机制等例如，众所周知，原肠胚形成期间的乙醇暴露导致经典的 低位FAS面和中线前脑发育不全;然而，暴露稍晚，在神经胚形成期间， 导致中线脑结构扩张和距离过远。有趣的是，这些异常类似于 （表型学）许多遗传性纤毛病的那些，如Joubert综合征。中枢致病机制 纤毛病的一个重要特征是初级纤毛的结构和/或功能受到干扰， 整合细胞内外信号的细胞。拟议的研究测试了整个新的假设 神经形成阶段的乙醇暴露诱导“暂时性纤毛病变”（即，临时中断的主要 纤毛功能），这是中线脑结构扩张和端粒延长的基本细胞机制。 畸形所提出的实验旨在满足以下综合具体目标。目的 1将定义早期产前乙醇暴露对初级纤毛结构和功能的直接影响。 为此，将使用共聚焦显微镜和免疫组织化学来检查乙醇对 初级纤毛数，而基因表达测定将用于评估纤毛功能。它是假设 乙醇暴露会导致纤毛数量和/或功能异常，减少Shh信号传导的激活 通路目的2将描述神经管中的继发性细胞致病事件， 酒精引起的短暂性纤毛病变这一目的的实验将检验以下假设： 乙醇诱导的短暂性纤毛病变和随后的Shh通路下调将减少 下游细胞增殖基因在腹侧神经管和扩大形态梯度的模式， 背神经管乙醇暴露后，将评估已知在细胞增殖中起作用的基因 使用qRT-PCR，并使用原位杂交评估腹侧和背侧形态发生素的梯度。 这些数据将有助于确定乙醇改变发育的确切机制。目标3是 确定乙醇诱导的短暂性纤毛病变的主要细胞机制事件。 这最后一个目标将使用RNA-seq以公正的方式确定乙醇如何破坏正常的纤毛发生 通过在乙醇暴露后立即检查几个时间点的总转录组谱。我们 假设乙醇将改变关键的纤毛发生基因;然而，使用这种无偏见的方法将有助于 确定其他潜在变化。最后，我们测试了替代/补充假设，即乙醇改变了 微管蛋白的翻译后修饰，从而破坏正常纤毛的稳定性和功能。总之，这些小说 这些实验将为乙醇的致病机制提供基本的见解 暴露在发展过程中，并推动酒精研究进入新的初级纤毛相关研究。

Many of the structural and functional abnormalities associated with Fetal Alcohol Spectrum Disorders (FASD) have been uncovered, yet major gaps remain in our understanding of the associated pathogenesis and mechanisms. For example, it is well known that ethanol exposure during gastrulation results in the classic hypoteloric FAS face and midline forebrain dysgenesis; yet, exposure just slightly later, during neurulation, induces expanded midline brain structures and hypertelorism. Interestingly, these abnormalities resemble (phenocopy) those of many genetic ciliopathies, such as Joubert syndrome. The central pathogenic mechanism of ciliopathies is a perturbation of the structure and/or function of primary cilia, hair-like organelles found on most cells that integrate extra- and intra-cellular signals. The proposed research tests the overall novel hypothesis that neurulation-stage ethanol exposure induces a “transient ciliopathy” (i.e., a temporary disruption of primary cilia function) that is the basic cellular mechanism for the expansion of midline brain structures and hyperteloric dysmorphologies. The proposed experiments are designed to meet the following integrated specific aims. Aim 1 will define the direct effects of early prenatal ethanol exposure on primary cilia structure and function. For this, confocal microscopy and immunohistochemistry will be used to examine the effects of ethanol on primary cilia number while gene expression assays will be used to assess cilia function. It is hypothesized that ethanol exposure causes abnormal ciliary number and/or function, reducing activation of the Shh signaling pathway. Aim 2 will characterize the secondary cellular pathogenic events in the neural tube resulting from an ethanol-induced transient ciliopathy. The experiments in this aim will test the hypothesis that the ethanol-induced transient ciliopathy and subsequent down-regulation of the Shh pathway will decrease downstream cell proliferation genes in the ventral neural tube and expand morphogen gradients that pattern the dorsal neural tube. Following ethanol exposure, genes with known roles in cell proliferation will be assessed using qRT-PCR and the gradients of ventral and dorsal morphogens will be assessed using in situ hybridization. These data will help to determine the precise mechanisms by which ethanol alters development. Aim 3 is to determine the primary cellular mechanistic events underlying an ethanol-induced transient ciliopathy. This final Aim will use RNA-seq to determine in an unbiased manner how ethanol disrupts normal ciliogenesis by examining the total transcriptomic profile at several time points immediately following ethanol exposure. We hypothesize that ethanol will alter key ciliogenesis genes; however, using this non-biased approach will aid in identifying other potential changes. Finally, we test the alternative/complementary hypothesis that ethanol alters tubulin post-translational modification, thereby disrupting normal cilia stability and function. Together, these novel experiments will provide fundamental insights into the pathogenic mechanisms underlying the effects of ethanol exposure during development, and propel alcohol research into new primary ciliary-related studies.