EphA7在肾小管发生中的功能与作用机制研究

The kidney is an essential organ that functions in filtering wastes, absorbing nutrients and water. The pronephros of Xenopus, which contains a single nephron, offers a simplified in vivo system to study the events of tubulogenesis, including the establishment of cell polarity and lumen formation. The Eph receptors are the largest sub-family of receptor tyrosine kinases (RTKs) and are widely involved in animal growth and development, including segmentation, axon guidance, cell migration, etc. However, their roles in kidney development remain largely unknown. We found that EphA7 is specifically distributed at the subapical region in Xenopus embryonic pronephric tubule epithelial cells and knockdown of XEphA7 disturbed the epithelial cell polarity and blocked pronephric tubulogenesis, suggesting that polarized Eph signaling might be essential in the control of cell polarity and lumen formation in Xenopus pronephric tubulogenesis. In this project, we propose to study systematically the dynamic distribution of EphA7 protein during Xenopus pronephros development and the signaling events downstream of EphA7 leading to the polarized accumulation of actin. The relationship of EphA7 signaling to Wnt4-PCP signaling will also be studied, which is reported to be involved in similar processes. With this project, we hope to reveal new mechanisms involved in vertebrate kidney development.

肾脏是身体中最主要的过滤和排泄器官，肾脏发育中上皮细胞的极性化和肾小管的形成是研究细胞极性的建立与管腔形成机制的重要模型。Eph受体家族广泛参与多种组织器官的发育过程，但目前关于其在胚胎肾脏早期发育中的功能仍未有报道。我们发现EphA7在非洲爪蛙胚胎原肾小管上皮细胞中呈极性分布，在肾小管形成中具有重要作用，提示这一信号通路在肾上皮细胞的极性调控与管腔形成中具有重要意义。本项目拟从EphA7蛋白在爪蛙肾小管发育中表达的动态变化及其极性分布的调节、EphA7在肾小管发育中的功能及其信号传递途径、与Wnt4信号的关系等方面，深入研究XEphA7及Wnt4在非洲爪蛙肾小管形成过程中的作用机制。本项目有望揭示肾小管发育调控的新机制。

Eph/ephrin信号通路在胚胎发育中具有广泛而重要的功能。我们发现，非洲爪蛙的EphA7在胚胎原肾小管中有特异性表达，利用反义Morpholino阻断EphA7的表达，会引起原肾细胞分化与形态发生的异常。我们还鉴定出了一种分泌型的EphA7。利用剪切特异性Morpholino可选择性抑制全长型EphA7的表达。阻断全部EphA7的表达，会显著降低细胞紧密连接相关蛋白Claudin6在原肾小管细胞的表达水平，而只抑制全长EphA7的表达则没有这种现象。在培养细胞中，EphA7可结合并磷酸化Claudin6，并降低其在细胞膜上的分布水平。有意思的是，分泌型EphA7可同样结合Claudin6并逆转EphA7的效应。我们还发现，EphA7是通过Eph/JNK1通路调节肾小管上皮细胞分化的，而分泌型EphA7同样可抑制Eph7/JNK1 通路活性。我们推测，在肾小管发育过程中，分泌型EphA7一方面可调节 EphA7/JNK1 通路活性水平，调节上皮细胞分化，另一方面解除EphA7对Claudin6稳定性的抑制作用，维持正常的细胞粘附水平，推动原肾小管的形态发生。上述结果建立了一个全新的调节原肾发育的分子通路，首次阐明了分泌型EphA7在体内发育过程中的功能。