Analysis of zebrafish npt and swt mutants in left-right patterning

项目概述：所有脊椎动物都有内部的不对称性，沿着左右（LR）轴，如 器官在中线周围的不对称放置。器官的正确不对称放置对于 它们的功能，因为它允许发育中的脉管系统内的适当连接。虽然许多基因 与LR模式有关的基因已经被确定，我们还不知道LR轴是如何建立的， 模式，LR轴如何与背腹或前后轴对齐，或器官如何获得 他们最终的不对称位置这项建议的重点是两个斑马鱼突变体，开关击球手（SWT）和 无党派（NPT），具有独特的LR模式表型。SWT突变体导致野生型或 完全逆转了器官模式与这种表型相一致的是，SWT突变体通常表现为右侧 节点成分的表达。我们假设swt在nodal的上游起作用， LR轴的方向性，npt突变体适当地建立不对称的结表达，但不对称的 器官形态发生受到影响。我们假设npt在nodal下游发挥作用， 不对称我们将探讨Swt和Npt在LR的两种机制中的潜在作用。 图案：纤毛的形成和运动，侧板中胚层细胞极性。我们将图像的心 活的野生型和npt和swt突变体胚胎的形态发生，为理解 不对称的机构定位以及《不扩散条约》在这一进程中的作用。我们将确定亚细胞 定位Swt和Npt，并使用镶嵌分析来确定它们的功能位点。最后，我们将使用 串联亲和纯化和双杂交分析以鉴定与Swt和Npt相互作用的蛋白质。 相关性：据估计，多达1/5000的婴儿出生时患有LR图案缺陷， 通常表现为先天性心脏病（凯西2000）。在本提案中，我们计划描述 两个斑马鱼突变在LR模式中的作用。由于许多参与LR模式的基因具有 在人类中保守的作用，拟议的研究将加强我们对LR模式和LR基因的理解。 偏侧性缺陷的遗传基础可导致先天性心脏病

Project Summary: All vertebrates have internal asymmetries along the left-right (LR) axis, such as the asymmetric placement of organs about the midline. Correct asymmetric placement of organs is critical for their function, as it allows for proper connectivity within the developing vasculature. While many genes implicated in LR patterning have been identified, we do not yet know how the LR axis is established or patterned, how the LR axis is aligned with the dorsal-ventral or anterior-posterior axes, or how organs obtain their final asymmetric positions. This proposal focuses on two zebrafish mutants, switch hitter (swt) and non-partisan (npt), with distinctive LR patterning phenotypes. swt mutants result in either wildtype or completely reversed organ patterning. Consistent with this phenotype, swt mutants often display right-sided expression of nodal components. We hypothesize that swt functions upstream of nodal to bias the directionality of the LR axis, npt mutants properly establish asymmetric nodal expression, but asymmetric organ morphogenesis is affected. We hypothesize that npt functions downstream of nodal to affect organ asymmetries. We will explore the potential roles for Swt and Npt in two mechanisms implicated in LR patterning: cilia formation and movement, and lateral plate mesoderm cell polarity. We will image heart morphogenesis in live wildtype and npt and swt mutant embryos to lay the foundations for understanding asymmetric organ positioning and the role of npt in this process. We will determine the subcellular localization for Swt and Npt and use mosaic analysis to determine their sites of function. Finally, we will use tandem affinity purification and two-hybrid analysis to identify proteins that interact with Swt and Npt. Relevance: It is estimated that as many as 1/5000 infants are born with LR patterning defects which are often manifested as congenital heart disease (Casey 2000). In this proposal, we plan to characterize the roles of two zebrafish mutations in LR patterning. Since many genes involved in LR patterning have conserved roles in humans, the proposed studies will enhance our understanding of LR patterning and the genetic basis of laterality defects that can result in congenital heart disease