The Roles of Definitive Endoderm in Establishment of Left-Right Asymmetry

定形内胚层在左右不对称建立中的作用

• 批准号: 7948929
• 负责人: Yukio Saijoh
• 金额: $ 31.23万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7948929
• 关键词: Address; Affect; Biological Assay; Biological Models; Cells; Characteristics; Chemicals; Cilia; Collaborations; Congenital Abnormality; Connexin 43; Connexins; Defect; Development; Dominant-Negative Mutation; Dyes; Ectopic Expression; Electron Microscopy; Embryo; Embryology; Embryonic Development; Embryonic Fluid; Endoderm; Endoderm Cell; Functional disorder; Gap Junctions; Gastrointestinal tract structure; Genes; Genetic; Goals; Handedness; Health; Healthcare Systems; Heart; Human; Intracellular Transport; Iontophoresis; Knowledge; Lateral; Left; Life; Lung; Mammals; Mesoderm; Molecular; Morphology; Mus; Nature; Nodal; Organ; Output; Pattern; Phenotype; Population; Positioning Attribute; Primitive Streaks; Process; Proteins; Reporting; Research Personnel; Role; Rotation; Series; Side; Signal Transduction; Signaling Molecule; Speed; Structure; System; Tamoxifen; Techniques; Testing; Time; Transducers; Transgenic Mice; Transgenic Organisms; Work; body system; design; experience; improved; inhibitor/antagonist; insight; mouse model; mutant; novel; prevent; public health relevance; research study; smoothened signaling pathway; tool

DESCRIPTION (provided by applicant): Defects in human left right (LR) axis formation cause birth defects of the heart, vasculature, lungs, and gastrointestinal tract that are life-threatening and costly to the healthcare system. To understand how laterality defects develop, it is essential to understand the molecular mechanisms by which LR asymmetry is established. My long-term goal is to understand the mechanism of how LR asymmetry is established during early embryogenesis. My previous work including collaboration with my colleagues in this field has revealed important steps in this process in the mouse model. However, there are still gaps in our understanding of the entire process, such as the mechanisms by which the node structure and rotating node cilia develop, and how LR polarity in the node is transferred to the left LPM. Here I will address these questions by investigating roles of definitive endoderm in LR determination. To date, no report has been focused on roles of endoderm in LR asymmetry. But several series of evidence suggest importance of endoderm in this process. Our hypothesis is that endoderm cells regulate LR determination in two ways: regulating node formation and/or function, and transferring LR signal from the node to the LPM. This hypothesis will be addressed by analyzing Sox17 mutants because Sox17 is the only gene that specifically expressed in the endoderm and primary defects of the mutants are specific to the definitive endoderm. Aim 1 will study the nature of the initial LR determination defects occurring in Sox17 mutants by characterizing the node and cilia formation, in addition to the development of nodal flow. Aim 2 will examine the possible defects occurring in the process of transferring LR signals from the node to the LPM regarding the gap junctional transport and intracellular Ca2+ signaling. In addition to chemical blockers, the roles of gap junctions will be tested by a sophisticated transgenic approach using a dominant negative form of connexin that can be temporally and/or spatially controlled. Conditional mutants of Sox17 will be studied to identify the importance of endoderm cells in the signal transfer from the node to the LPM, being separated from the initial abnormalities in the node. We have found that the Nodal gene continues to be expressed in migrating endoderm in the Sox17 mutants, which could be the cause of endoderm defects in Sox17 mutants that affect LR determination. In Aim 3, I will test this possibility by transgenic techniques. I will rescue Sox17 mutants by expressing Nodal antagonists in endoderm and the Sox17 mutant phenotype will be reproduced by ectopically expressing the Nodal gene in the endoderm of wild type embryos. The 20 yrs of experience that the PI possesses in this model system has enabled us to design and optimize novel tools and techniques that are ideal for addressing the questions I have put forth in this proposal. The proposed experiments will reveal novel roles of endoderm cells in establishment of LR asymmetry in mammals. This work may ultimately provide insight into the mechanisms underlying human birth defects due to problems in laterality. PUBLIC HEALTH RELEVANCE: The proposed experiments will reveal novel roles of endoderm cells in establishment of LR asymmetry in mammals. This work may ultimately provide insight into the mechanisms underlying human birth defects due to problems in laterality.

描述（由申请人提供）：人类左右（LR）轴形成的缺陷会导致心脏、脉管系统、肺部和胃肠道的出生缺陷，这些缺陷危及生命并且对医疗保健系统造成高昂的成本。为了了解偏侧性缺陷如何发展，有必要了解 LR 不对称性形成的分子机制。我的长期目标是了解早期胚胎发生过程中 LR 不对称性的形成机制。我之前的工作，包括与该领域同事的合作，揭示了小鼠模型中这一过程的重要步骤。然而，我们对整个过程的理解仍然存在差距，例如节点结构和旋转节点纤毛发育的机制，以及节点中的LR极性如何转移到左LPM。在这里，我将通过研究定形内胚层在 LR 测定中的作用来解决这些问题。迄今为止，还没有报道关注内胚层在 LR 不对称中的作用。但一系列证据表明内胚层在此过程中的重要性。我们的假设是内胚层细胞通过两种方式调节 LR 测定：调节节点形成和/或功能，以及将 LR 信号从节点转移到 LPM。这一假设将通过分析 Sox17 突变体得到解决，因为 Sox17 是唯一在内胚层中特异性表达的基因，并且突变体的主要缺陷是定形内胚层特有的。目标 1 将通过表征节点和纤毛形成以及节点流的发展来研究 Sox17 突变体中发生的初始 LR 测定缺陷的性质。目标 2 将检查将 LR 信号从节点传输到 LPM 的过程中可能出现的关于间隙连接运输和细胞内 Ca2+ 信号传导的缺陷。除了化学阻断剂之外，间隙连接的作用还将通过复杂的转基因方法进行测试，该方法使用可以在时间和/或空间上控制的连接蛋白的显性失活形式。将研究 Sox17 的条件突变体，以确定内胚层细胞在从节点到 LPM 的信号传递中的重要性，与节点中的初始异常分开。我们发现Nodal基因在Sox17突变体的迁移内胚层中继续表达，这可能是Sox17突变体中内胚层缺陷影响LR测定的原因。在目标 3 中，我将通过转基因技术测试这种可能性。我将通过在内胚层中表达Nodal拮抗剂来拯救Sox17突变体，并且通过在野生型胚胎的内胚层中异位表达Nodal基因来复制Sox17突变体表型。 PI 在该模型系统中拥有 20 年的经验，使我们能够设计和优化新颖的工具和技术，这些工具和技术非常适合解决我在本提案中提出的问题。拟议的实验将揭示内胚层细胞在哺乳动物 LR 不对称性建立中的新作用。这项工作最终可能会深入了解由于偏侧性问题导致的人类出生缺陷的机制。 公共健康相关性：拟议的实验将揭示内胚层细胞在哺乳动物 LR 不对称性建立中的新作用。这项工作最终可能会深入了解由于偏侧性问题导致的人类出生缺陷的机制。