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

项目简介：所有脊椎动物都有沿左右（LR）轴的内部不对称，例如器官在中线附近的不对称位置。器官的正确不对称放置对其功能至关重要，因为它允许在发育中的脉管系统内进行适当的连接。虽然已经确定了许多与LR模式有关的基因，但我们还不知道LR轴是如何建立或模式的，LR轴如何与背腹轴或前后轴对齐，或者器官如何获得最终的不对称位置。这一建议的重点是两个斑马鱼突变体，切换击球手（swt）和非党派（npt），具有独特的LR模式表型。SWT突变导致野生型或完全相反的器官模式。与这种表型一致，swt突变体通常显示右侧表达淋巴结成分。我们假设swt在淋巴结上游的功能使LR轴的方向性偏倚，npt突变体适当地建立了不对称淋巴结表达，但不对称器官形态发生受到影响。我们假设npt在淋巴结下游起作用，影响器官不对称。我们将探讨Swt和Npt在两种涉及LR模式的机制中的潜在作用：纤毛的形成和运动，以及侧板中胚层细胞的极性。我们将对野生型、npt和swt突变胚胎的心脏形态发生进行成像，为理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。相关性：据估计，多达1/5000的婴儿出生时具有LR模式缺陷，通常表现为先天性心脏病（Casey 2000）。在这项提议中，我们计划描述斑马鱼在LR模式中的两种突变的作用。由于许多参与左脑模式的基因在人类中具有保守的作用，因此所提出的研究将增强我们对左脑模式和可导致先天性心脏病的侧性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.