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

描述(由申请人提供)：人类左、右(LR)轴形成缺陷会导致心脏、血管、肺和胃肠道的出生缺陷，这些缺陷危及生命，对医疗保健系统来说代价高昂。要了解偏侧缺陷是如何发展的，就必须了解建立LR不对称性的分子机制。我的长期目标是了解LR不对称是如何在早期胚胎发育过程中建立起来的。我之前的工作，包括与我在这一领域的同事的合作，已经在老鼠模型中揭示了这一过程中的重要步骤。然而，我们对整个过程的理解仍然存在差距，如节点结构和旋转节点纤毛的发育机制，以及节点中的LR极性如何传递到左侧LPM。在这里，我将通过研究最终内胚层在LR测定中的作用来解决这些问题。到目前为止，还没有关于内胚层在下丘脑不对称中的作用的报道。但一些证据表明，内胚层在这一过程中具有重要作用。我们的假设是，内胚层细胞通过两种方式调节LR的决定：调节结节的形成和/或功能，以及将LR信号从结节传递到LPM。这一假设将通过分析Sox17突变体来解决，因为Sox17是唯一在内胚层特异表达的基因，并且突变体的主要缺陷是最终内胚层特有的。目的1将通过表征结节和纤毛的形成以及节流的发展来研究Sox17突变体中初始LR确定缺陷的性质。目标2将研究在将LR信号从结点传递到LPM的过程中可能出现的关于缝隙连接运输和细胞内钙信号的缺陷。除了化学阻滞剂，缝隙连接的作用将通过一种复杂的转基因方法进行测试，该方法使用一种可以在时间和/或空间上控制的显性负性连接蛋白。将研究Sox17的条件突变体，以确定内胚层细胞在从结节到LPM的信号传递中的重要性，并将其与结节中的初始异常分开。我们发现，在Sox17突变体中，Nodal基因继续在迁移的内胚层中表达，这可能是Sox17突变体中影响LR测定的内胚层缺陷的原因。在目标3中，我将通过转基因技术来测试这种可能性。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突变体的表型。PI在这个模型系统中拥有20年的经验，使我们能够设计和优化新的工具和技术，这些工具和技术非常适合于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。这项工作最终可能为了解偏侧性问题导致的人类出生缺陷的潜在机制提供洞察力。 公共卫生相关性：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角色。这项工作最终可能为了解偏侧性问题导致的人类出生缺陷的潜在机制提供洞察力。

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.