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

项目摘要：所有脊椎动物在左-右(LR)轴上都有内部不对称，例如 器官围绕中线的不对称放置。正确的器官不对称放置对 它们的功能，因为它允许在发育中的血管系统内适当的连接。虽然很多基因 与LR图案有关，我们还不知道LR轴是如何建立的或 图案化，LR轴如何与背腹或前后轴对齐，或者器官如何获得 他们最终的不对称立场。这项建议集中在两个斑马鱼突变体，开关击球手(Swt)和 无党派(NPT)，具有独特的LR图案表型。SWT突变导致野生型或 完全逆转了器官模式。与这种表型相一致，swt突变体通常表现为右侧。 节点成分的表达。我们假设swt的作用是在节点的上游偏置 LR轴的方向性，NPT突变体正确地建立了不对称的节点表达，但不对称 器官形态发生受到影响。我们假设NPT作用于结节下游以影响器官。 不对称性。我们将探索SWT和NPT在LR所涉及的两个机制中的潜在作用 花纹：纤毛的形成和运动，以及侧板中胚层细胞的极性。我们将想象一颗心 野生型、npt和swt突变型活体胚胎的形态发生为理解奠定基础 不对称器官定位以及《不扩散核武器条约》在这一过程中的作用。我们将确定亚细胞 对SWT和NPT进行定位，并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确定与SWT和NPT相互作用的蛋白质。 相关性：据估计，多达五千分之一的婴儿出生时就有LR图案缺陷，这些缺陷是 通常表现为先天性心脏病(Casey 2000)。在这项提案中，我们计划将 两个斑马鱼突变在LR模式中的作用。由于许多参与LR图案形成的基因 在人类中保守的角色，拟议的研究将加强我们对LR模式和 可导致先天性心脏病的偏侧缺陷的遗传基础

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