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

描述(申请人提供)：项目总结：所有脊椎动物都有沿左右(LR)轴的内部不对称，例如器官围绕中线的不对称放置。器官的正确不对称放置对它们的功能至关重要，因为它允许发育中的血管系统内适当的连接。虽然许多与LR模式有关的基因已经被发现，但我们还不知道LR轴是如何建立或模式的，LR轴是如何与背腹轴或前后轴对齐的，或者器官是如何获得最终的不对称位置的。这项建议集中在两个斑马鱼突变体，开关打击手(Swt)和无党派(NPT)，具有独特的LR模式表型。SWT突变要么导致野生型，要么导致器官图案完全逆转。与这种表型相一致的是，swt突变体经常表现出节部成分的右侧表达。我们假设swt的功能是在结节上游偏向LR轴的方向，NPT突变体正确地建立了不对称的结节表达，但不对称器官的形态发生受到了影响。我们假设NPT在结节下游发挥作用，从而影响器官的不对称。我们将探索SWT和NPT在LR图案化中的两个机制中的潜在作用：纤毛的形成和运动，以及侧板中胚层细胞的极性。我们将在活体野生型和NPT和swt突变胚胎中成像心脏形态发生，为了解不对称器官定位和NPT在这一过程中的作用奠定基础。我们将确定SWT和NPT的亚细胞定位，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来鉴定与SWT和NPT相互作用的蛋白质。相关：据估计，每5000名婴儿中就有1人出生时患有左侧型缺陷，通常表现为先天性心脏病(Casey 2000)。在这项提案中，我们计划表征两个斑马鱼突变在LR模式中的作用。由于许多参与LR模式的基因在人类中具有保守的作用，拟议的研究将增强我们对LR模式以及可导致先天性心脏病的偏侧缺陷的遗传基础的理解。

DESCRIPTION (provided by applicant): 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.