Kurly's Role in Left-Right Patterning and Congenital Heart Disease

描述(申请人提供)：脊椎动物具有沿左右轴的内部不对称性，包括器官的不对称放置。器官的正确放置，如心脏，对胚胎的正常发育和存活至关重要。由于基因表达不当，每5000名儿童中就有一名出生时患有左右花纹缺陷，通常表现为先天性心脏病。了解在胚胎中建立适当的左右模式所涉及的遗传路径，对于了解这些缺陷是如何产生的至关重要。一种新的斑马鱼突变体kurly会影响左右图案和随后的心脏发育。有趣的是，新的人类kurly同源基因映射到位于唐氏综合症临界区内的人类21号染色体上的一个基因。位于该区域的基因可能是唐氏综合症患者先天性心脏病发生率较高的原因。总人口中有0.8%患有先天性心脏病，而唐氏综合症患者中有40%-50%患有先天性心脏病。通过了解kurly在斑马鱼中的功能，我们将获得宝贵的洞察力，了解左向右模式缺陷和随后的先天性心脏缺陷可能是如何发生的。斑马鱼是一种很好的模式生物，因为它的外部发育、透明度和可以进行的遗传操作。此外，由于参与左右模式的许多基因在人类中具有保守的作用，在斑马鱼身上进行的实验和从这些实验中获得的知识显然适用于更大的社会利益。为了研究kurly对左-右模式和心脏发育的影响，我将采取多种方法。我将首先使用免疫组织化学和荧光蛋白标记来检测kurly的位置和功能部位。然后，我将通过注射分析和免疫荧光来探索kurly在这些早期模式事件中如何影响初级纤毛和细胞极性。我将最终确定在这些发育阶段通过串联亲和纯化与kurly相互作用的其他蛋白质，以便深入了解可能需要kurly功能的重要途径。相关性：这项研究通过对一种基因的深入分析极大地造福于人类，这种基因如果调控不当，会导致左向右内部器官模式缺陷以及先天性心脏缺陷。

DESCRIPTION (provided by applicant): Vertebrates have internal asymmetries along the left-right axis, including asymmetric placement of the organs. Correct placement of organs, such as the heart, is critical for proper embryonic development and survival. Due to improper gene expression as many as one in 5,000 children are born with left-right patterning defects which often manifest as congenital heart disease. Understanding the genetic pathways involved in establishing proper left-right patterning in the embryo is vital to understanding how these defects may arise. A novel zebrafish mutant, Kurly, affects left-right patterning and subsequent heart development. Interestingly, the novel human ortholog of Kurly maps to a gene located on human chromosome 21 within the Down Syndrome Critical Region. Genes located in this area are perhaps responsible for the high incidence of congenital heart defects in Down Syndrome patients. While 0.8% of the general population is affected by congenital heart defects, 40-50% of those affected by Down Syndrome have congenital heart defects. By understanding how Kurly functions in zebrafish, valuable insight will be gained into how left-right patterning defects and subsequent congenital heart defects may arise. The zebrafish is an excellent model organism due to its external development, transparency, and genetic manipulations that can be made. Also, as many genes involved in left-right patterning have conserved roles in humans, experiments performed in zebrafish and knowledge gained from these experiments are clearly applicable to the greater good of society. To investigate Kurly's effects on left- right patterning and heart development, I will take multiple approaches. I will first use immunohistochemistry and fluorescent protein tagging to examine the location and sites of function for Kurly. I will then explore how Kurly affects primary cilia and cell polarity during these early patterning events by injection assays and immunofluorescence. I will finally identify other proteins that interact with Kurly, through tandem affinity purification, during these stages of development in order to gain insight into significant pathways which may require Kurly function. Relevance: This research greatly benefits mankind through an in-depth analysis of a gene that, when misregulated, leads to left-right internal organ patterning defects as well as congenital heart defects.