Toddler and Apelin Signaling During Zebrafish Gastrulation

描述(申请人提供)：脊椎动物的发育依赖于细胞的精确增殖、迁移和规格，以产生有机体的不同和复杂的部分。这些过程中的任何一个失败都可能导致器官衰竭、神经综合征或胚胎死亡等缺陷。尽管这些过程很重要，但人们并不完全了解它们的途径和监管机构。这个项目将通过描述toddler的特征来解决这种差异，toddler是一种参与胚胎发育的新型、高度保守的分泌肽，在斑马鱼的原肠发育过程中，它是内胚层和中胚层正确迁移所必需的。实验室以前的工作表明，蹒跚学步的孩子调节细胞迁移，尽管幼儿刺激的细胞行为和分子机制的变化尚不清楚。这项工作将检验这一假设，即幼儿在原肠形成过程中需要细胞内化(目标1)，因为它调节E-钙粘蛋白(目标2)，并在功能上与已知的分泌肽Apelin部分重叠(目标3)。首先，将使用基因工程和显微镜分析来标记和跟踪发育过程中幼儿反应细胞，以确定幼儿信号影响的特定细胞行为。接下来，这项工作将使用Western blotting、免疫组织化学和上位性分析来分析蹒跚学步的孩子如何调节E-钙粘蛋白，这是一种已知在发育早期活跃的黏附分子。最后，这项研究将考察幼儿与Apelin的关系，Apelin是一种在发育过程中活跃的保守肽，像幼儿一样，通过Apelin受体A和B发出信号。幼儿和Apelin的通路可能有部分重叠，但这一点的程度和要求尚不清楚。这种关系将通过产生apelin突变体，然后进行单突变和双突变分析，包括细胞跟踪和形态比较和报告基因表达来评估。因此，这项研究将增加我们对细胞在发育过程中迁移的知识，从而增强我们对生物如何从单细胞过渡到复杂系统的理解。这些知识将有助于我们了解和解决与发展有关的疾病。此外，已知Apelin多肽参与心血管发育，目前正在人类身上进行治疗测试，该项目中进行的工作可能会揭示幼儿也是一个同样有希望的药物靶点。

DESCRIPTION (provided by applicant): Vertebrate development depends on precise proliferation, migration and specification of cells to generate distinct and complex parts of an organism. Failure in any of these processes can cause defects such as organ failure, neurological syndromes or death of the embryo. Despite the importance of these processes, their pathways and regulators are not completely understood. This project will address this disparity by characterizing Toddler, a novel, highly conserved secreted peptide involved in embryogenesis and required for proper migration of the endodermal and mesodermal germ layers during gastrulation in zebrafish. Previous work in the laboratory demonstrated that Toddler regulates cell migration, though the change in cellular behavior and molecular mechanism stimulated by Toddler is not known. This work will test the hypothesis that Toddler is required for cell internalization during gastrulation (Aim 1) due to its regulation of E-cadherin (Aim 2) and partially overlaps in function with the known secreted peptide Apelin (Aim 3). First, genetic engineering and microscopy analysis will be used to label and track Toddler-responsive cells during development to identify the specific cellular behavior affected by Toddler signaling. Next, this work will use Western blotting, immunohistochemistry and epistatic analysis to analyze how Toddler regulates E-cadherin, an adhesion molecule known to be active during early development. Finally, this work will examine Toddler's relationship with Apelin, a conserved peptide active in development that, like Toddler, signals through the Apelin Receptors A and B. It is likely that the Toddler and Apelin pathways are partially overlapping, but the degree and requirement for this is unclear. This relationship will be assessed by generation of an apelin mutant followed by single and double mutant analysis including cell tracking and comparisons of morphology and reporter gene expression. As a result, this study will increase our knowledge of cell migration during development thereby enhancing our understanding of how organisms transition from single cells to complex systems. Such knowledge will aid in our ability to understand and address development related diseases. Additionally, the peptide Apelin is known to be involved in cardiovascular development and is currently being tested as a therapeutic in humans and the work carried out in this project may reveal Toddler to be a similarly promising drug target.