Function of the Apela-APJ signaling axis in mammalian development.

 描述(由申请人提供)：正确的胚胎形成和器官发生依赖于分子信号通路，这些信号通路在整个发育过程中反复使用，对生存至关重要。APELA-APJ信号轴是最近在斑马鱼中发现的一条新途径。斑马鱼APELA的缺失会导致胚层形成的早期缺陷，以及后来各种器官的畸形。值得注意的是，这些突变体往往无法形成心脏。目前尚不清楚器官发生的缺陷是否是早期内胚层和中胚层缺陷的结果，或者apela-APJ是否也在特定的器官中发挥作用，如发育中的心脏。由于apela作为一种信号分子是最近才被发现的，它在哺乳动物发育中的作用尚未确定，尽管有证据表明apela-APJ信号通路在脊椎动物中是保守的。这个项目的目的是揭示和确定APELA-APJ信号在小鼠胚胎发育和器官发生中的功能和需求。为了做到这一点，赞助商的实验室最近创造了缺乏APELA的小鼠。APELA缺失突变体的表型将通过分析重要器官的形态以及对心脏发育、内胚层/中胚层规范和左右格局重要的各种发育标记的表达来表征。已有研究表明，APELA通过与G蛋白偶联受体APJ结合来传递信号。另一种非同源分子Apelin也激活APJ。Apelin和APJ突变表型之间的差异表明Apela可能在小鼠的发育中发挥关键作用。因此，将使用Apelin和APJ小鼠突变体来研究Apela和Apelin介导的APJ激活之间的重叠作用。内胚层和中胚层祖细胞的缺陷将使用原始条纹外植体、活力分析和荧光报告小鼠的活体成像来评估APELA突变体。为了测试APELA-APJ信号对内胚层和心脏祖细胞的规格和行为的要求，将从APELA、APELIN和APJ突变体中提取小鼠胚胎干细胞(MESCs)进行体外实验。APELIN-APJ信号通路促进mESC来源的心血管祖细胞的分化和维持，提示APELA可能发挥类似的作用。APELA-APJ信号在mESCs定向分化为内胚层和心脏前体细胞命运中的作用将被研究，从而确定其与未来治疗应用的相关性。为了确定APJ介导的信号转导的细胞自主需求，突变的和对照的mESCs将在共培养中进行检测，并将用于制造嵌合体胚胎。总而言之，这些研究将阐明新的信号轴在哺乳动物发育中的关键功能，并将为构建在发育或疾病中发挥作用的新的遗传途径提供基础知识。

 DESCRIPTION (provided by applicant): Proper embryo formation and organogenesis relies on molecular signaling pathways that are used in a reiterated manner throughout development, and are essential for viability. The Apela-APJ signaling axis is a new pathway that was only recently discovered in zebrafish. Loss of apela in zebrafish causes early defects in germ layer formation as well as later malformations of various organs. Notably, these mutants often fail to form a heart. It is not clear whether defects in organogenesis are a consequence of earlier deficiencies in endoderm and mesoderm, or if Apela-APJ also functions in particular organs, such as the developing heart. Since the discovery of Apela as a signaling molecule occurred so recently, its role in mammalian development is yet to be determined, though evidence suggests conservation of the Apela-APJ signaling pathway among vertebrates. The aims of this project are designed to uncover and define the function and requirement for Apela-APJ signaling in mouse embryonic development and organogenesis. To accomplish this, the sponsor's lab has recently created mice lacking Apela. The phenotype of Apela null mutants will be characterized by analyzing morphology of vital organs as well as expression of various developmental markers important for heart development, endoderm/mesoderm specification, and left-right patterning. It has been shown that Apela signals via binding to the G protein-coupled receptor APJ. Another non-homologous molecule, Apelin, also activates APJ. Discrepancies between Apelin and APJ mutant phenotypes suggest that Apela may play a critical role in mouse development. Therefore overlapping roles between Apela versus Apelin mediated activation of APJ will be investigated using Apelin and APJ mouse mutants. Defects in endoderm and mesoderm progenitors will be assessed in Apela mutants using primitive streak explants, motility assays, and live imaging of fluorescent reporter mice. To test the requirements for Apela-APJ signaling with respect to the specification and behavior of endoderm and cardiac progenitors, mouse embryonic stem cells (mESCs) from Apela;Apelin, and APJ mutants will be derived for in vitro experiments. Apelin-APJ signaling promotes the differentiation and maintenance of mESC-derived cardiovascular progenitors, suggesting that Apela may exert similar effects. The role of Apela-APJ signaling in directed differentiation of mESCs into endoderm and cardiac progenitor cell fates will be investigated, thereby establishing its relevance to future therapeutic applications. To determine cell autonomous requirements for APJ-mediated signaling, mutant and control mESCs will be assayed in co-cultures, and will also be used to make chimeric embryos. Collectively, these studies will shed light on the critical functions of a novel signaling axis in mammalian development, and they will provide fundamental knowledge for building new genetic pathways that function in development or disease.