Genetic dissection of fluid flow signaling in Left-Right patterning of zebrafish

项目摘要/摘要 液体流动转导通路对发育至关重要，然而，人们对细胞如何感知和 对流体流动信号进行转换。流动和流动感觉缺陷会导致发育性疾病，包括 原发纤毛运动障碍、异位和先天性心脏病。而多囊藻毒素跨膜 蛋白质参与了液体流动的感觉和转导，很少有其他调节流动的蛋白质 感觉和转导已经被确认。本研究旨在刻画新的心流感觉通路 调节器，并使用高度易处理的左右(L-R)图案构建多囊藻毒素流动感官复合体 斑马鱼胚胎系统。在这个系统中，纤毛细胞感觉到不对称的流体流动，并破坏L-R 通过抑制关键靶基因dand5来实现对称性，从而导致下游基因的不对称表达。至 在L-R图案化系统中鉴定流动感觉通路组件，我进行了反向遗传学筛选 并确定了几种新型的调节器。关注其中的四个，我的首要假设是它们 作为流动信号感觉或转导的一部分，位于dand5抑制的上游。我要测试一下这个 假设通过两个特定的目的：1)测试新的调节剂对纤毛形成，运动， 使用我正在构建的新型路径传感器的流产生和流感觉路径输出，以及2)测试 假设pkhd1l1，或纤维囊藻毒素，作为多囊藻毒素复合体的一部分，在纤毛的流觉中起作用。 总体而言，这项工作将通过机械地放置小说来扩展我们对流动感觉通路的理解 调节因子进入L-R图案化通路。此外，这项工作的目的是在目前的多囊蛋白流的基础上 通过解剖多囊藻蛋白-纤维囊藻蛋白相互作用的感觉复合体，这将对 L-R纹样与肾脏疾病。

Project Summary/Abstract Fluid flow transduction pathways are critical for development, however it is little known how cells sense and transduce fluid flow signals. Defects in flows and flow sensation result in developmental diseases including primary ciliary dyskinesia, heterotaxy, and congenital heart disease. While the Polycystin transmembrane proteins have been implicated in fluid flow sensation and transduction, few other proteins regulating flow sensation and transduction have been identified. This study aims to characterize novel flow sensory pathway regulators and build on the Polycystin flow sensory complex using the highly tractable left-right (L-R) patterning system of zebrafish embryos. In this system an asymmetric fluid flow is sensed by ciliated cells and breaks L-R symmetry by repressing a key target gene, dand5, leading to asymmetric expression of downstream genes. To identify flow sensory pathway components in the L-R patterning system, I performed a reverse genetics screen and identified several novel regulators. Focusing on four of these, my overarching hypothesis is that they function as part of flow signal sensation or transduction, upstream of dand5 repression. I will test this hypothesis through two Specific Aims: 1) Test the requirement of novel regulators for cilia formation, motility, flow generation and flow sensory pathway output using a novel pathway sensor I am building and 2) Test the hypothesis that pkhd1l1, or Fibrocystin, functions in flow sensation in cilia as part of the Polycystin complex. Overall, this work will expand our understanding of flow sensory pathways by mechanistically placing novel regulators into the L-R patterning pathway. Moreover, this work aims to build on the current Polycystin flow sensory complex by dissecting Polycystin-Fibrocystin interactions, something which will have implications for L-R patterning and kidney diseases.