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）轴的内部不对称，例如器官在中线上的不对称放置。正确的不对称位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通性。尽管已经确定了与LR模式相关的许多基因，但我们尚不知道LR轴是如何建立或模式化的，LR轴如何与背腹或前后轴对齐，或者Organs如何获得最终的不对称位置。该提案着重于具有独特的LR模式表型的两个斑马鱼突变体，开关击球手（SWT）和非党派（NPT）。 SWT突变体会导致野生型或完全逆转器官形成。与这种表型一致，SWT突变体通常显示出淋巴结成分的右侧表达。我们假设Nodal上游的SWT功能会偏向LR轴的方向性，NPT突变体正确地建立了不对称的淋巴结表达，但不对称器官形态发生受到影响。我们假设NODAL下游的NPT功能会影响器官不对称。我们将以涉及LR模式的两种机制来探索SWT和NPT的潜在作用：Cilia形成和运动，以及侧板中胚层细胞极性。我们将在活野生型和NPT和SWT突变胚胎中对心脏形态发生，以奠定理解不对称器官定位的基础以及NPT在此过程中的作用。我们将确定SWT和NPT的亚细胞定位，并使用马赛克分析来确定其功能部位。最后，我们将使用串联亲和力纯化和两个杂交分析来识别与SWT和NPT相互作用的蛋白质。相关性：据估计，多达1/5000个婴儿出生时患有LR模式缺陷，通常表现为先天性心脏病（Casey 2000）。在此提案中，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，因此拟议的研究将增强我们对LR模式的理解以及可能导致先天性心脏病的侧向缺陷的遗传基础。