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

斑马鱼 npt 和 swt 突变体左右模式分析

• 批准号: 7929986
• 负责人: REBECCA D. BURDINE
• 金额: $ 6.38万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7929986
• 关键词: Address; Affect; Affinity Chromatography; Anterior; Biological Models; Cell Polarity; Cilia; Cloning; Defect; Development; Dorsal; Embryo; Embryology; Foundations; Genes; Genetic; Goals; Handedness; Heart; Histology; Human; Image; Immunohistochemistry; Infant; Laboratories; Lateral; Left; Life; Mesoderm; Mesoderm Cell; Modeling; Morphogenesis; Movement; Mutation; Nodal; Organ; Pattern; Phenotype; Positioning Attribute; Process; Proteins; Role; Side; Signal Pathway; Site; Techniques; Transgenes; Vertebrates; Zebrafish; base; cell motility; congenital heart disorder; genetic analysis; heart imaging; insight; mutant; novel; yeast two hybrid system

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.

描述（由申请人提供）： 项目摘要：所有脊椎动物沿左右（LR）轴都具有内部不对称性，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其功能至关重要，因为它可以在发育中的脉管系统内实现适当的连接。虽然许多与 LR 模式有关的基因已被识别，但我们还不知道 LR 轴是如何建立或模式化的，LR 轴如何与背腹轴或前后轴对齐，或者器官如何获得其最终的不对称位置。该提案重点关注两种斑马鱼突变体：switch hitter (swt) 和 non-partisan (npt)，它们具有独特的 LR 模式表型。 swt 突变体导致野生型或完全逆转的器官模式。与这种表型一致，swt 突变体通常表现出节点成分的右侧表达。我们假设 swt 在节点上游发挥作用，以偏向 LR 轴的方向性，npt 突变体正确建立不对称节点表达，但不对称器官形态发生受到影响。我们假设 npt 在节点下游发挥作用，影响器官不对称性。我们将探讨 Swt 和 Npt 在 LR 模式相关的两种机制中的潜在作用：纤毛形成和运动，以及侧板中胚层细胞极性。我们将对活体野生型以及 npt 和 swt 突变体胚胎中的心脏形态发生进行成像，为了解不对称器官定位以及 npt 在此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 的婴儿出生时就有 LR 模式缺陷，通常表现为先天性心脏病 (Casey 2000)。在本提案中，我们计划描述两种斑马鱼突变在 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，因此拟议的研究将增强我们对 LR 模式和可导致先天性心脏病的偏侧性缺陷的遗传基础的理解。