Cellular Mechanisms in Fetal Alcohol Spectrum Disorders

与胎儿酒精谱障碍(FASD)相关的许多结构和功能异常 已经被发现，但在我们对相关的发病机制和 机械装置。例如，众所周知，在原肠培养过程中暴露于乙醇会导致经典的 低钾Fas脸和中线前脑发育不全；然而，暴露仅稍晚，在神经形成期间， 导致中线脑部结构扩张和过度调理。有趣的是，这些异常现象类似于 (表现性)许多遗传性纤毛疾病，如Joubert综合征。中枢致病机制 纤毛病变是指初级纤毛的结构和/或功能的紊乱，毛状细胞器在大多数 整合细胞内外信号的细胞。这项拟议的研究检验了整个新奇假设 神经形成阶段的酒精暴露会导致一种“一过性睫毛病变”(即，暂时性的主睫毛中断) 纤毛功能)，这是中线大脑结构扩张和端粒亢进的基本细胞机制 畸形症。拟议的实验旨在满足以下综合具体目标。目标 1将确定早期产前酒精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测乙醇对 初级纤毛数量，而基因表达分析将用于评估纤毛功能。据推测， 酒精暴露导致纤毛数量和/或功能异常，减少Shh信号的激活 路径。目标2将描述神经管中继发性细胞致病事件的特征 死于酒精引起的一过性睫毛病变。这个目标的实验将检验这样一个假设，即 乙醇诱导的一过性纤毛病变和随后Shh通路的下调将会减少 在腹侧神经管中下游的细胞增殖基因，并扩大形态梯度，使 背侧神经管。酒精暴露后，将对已知在细胞增殖中起作用的基因进行评估。 使用qRT-PCR，腹侧和背侧形态原的梯度将通过原位杂交进行评估。 这些数据将有助于确定乙醇改变发育的精确机制。目标3是 确定酒精诱导的一过性纤毛病变背后的主要细胞机制事件。 这一最终目标将使用rna-seq以公正的方式确定乙醇如何干扰正常的纤毛发生。 通过检查酒精暴露后几个时间点的总转录图谱。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 确定其他潜在的变化。最后，我们检验了乙醇改变的替代/互补假设。 微管蛋白翻译后修饰，从而扰乱正常纤毛的稳定性和功能。总而言之，这些小说 实验将提供对酒精影响的致病机制的基本见解。 在发育过程中暴露，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.