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题名	紧密连接蛋白Claudin-6及酪氨酸激酶受体EphA7在非洲爪蛙胚胎原肾发育中的功能研究
作者	孙健
学位类别	博士
答辩日期	2015-05
授予单位	中国科学院研究生院
授予地点	北京
导师	毛炳宇
关键词	胚胎原肾 形态发生 终末分化 Claudin-6 EphA7
其他题名	The roles of Claudin-6 and EphA7 in pronephros development
中文摘要	非洲爪蛙的原肾在其蝌蚪时期发挥肾脏的功能，它只包含一个巨大的肾单位，在结构和功能上与哺乳动物肾单位非常类似，是研究肾脏发育的一个很好的模型。非洲爪蛙原肾的发育过程主要包括原肾的诱导、间充质细胞向上皮细胞转化过程、原肾的图式化和终末分化。 Claudin是一类四次跨膜的紧密连接蛋白，主要参与紧密连接的形成，调解细胞间的通透性，并参与维持上皮细胞和内皮细胞的极性，但Claudin在胚胎肾脏发育过程中的作用还不清楚。我们制备了针对Claudin-6的特异性抗体，发现Claudin-6在爪蛙胚胎原肾小管和肾导管中特异性表达。利用特异性Morpholino阻断Claudin-6的表达，对原肾诱导和早期分化没有明显影响，但是会影响原肾小管的形态发生和终末分化。敲低Claudin-6的表达，会抑制原肾小管上皮细胞顶部actin的聚集，并减弱侧面紧密连接蛋白Na/K-ATPase的表达，提示Claudin-6可能是通过维持上皮细胞的细胞粘附与细胞极性而参与原肾小管发育的。 Eph/Ephrin信号在胚胎发育中具有广泛的表达并且参与了许多重要发育过程。非洲爪蛙的EphA7在胚胎原肾小管中有特异的表达。除了全长形式的EphA7（EphA7-FL），我们还鉴定出了一种分泌型的EphA7 (EphA7-S)。体内和体外的实验分析表明，EphA7-S和EphA7-FL的功能相互拮抗，共同参与原肾小管形态发生与细胞分化的调控。利用EphA7-ATG-MO同时阻断全长和分泌型EphA7的表达，会影响原肾小管的形态发生，肾小管管腔扩大，同时抑制其终末分化。利用EphA7-sp-MO 特异性抑制EphA7-FL的表达，会抑制肾小管管腔的形成和终末分化。我们发现注射EphA7-ATG-MO显著降低紧密连接蛋白Claudin-6在原肾小管上皮细胞中的表达，而EphA7-sp-MO不会影响其蛋白表达。体外细胞实验证明EphA7与Claudin-6通过它们胞外结构域相互作用，全长EphA7可以磷酸化Claudin-6，并减少其在细胞膜上的分布。这一过程可被而分泌型EphA7所抑制。全长EphA7可能通过调节JNK1的活性来影响原肾终末分化，而这一活性也可被分泌型EphA7抑制。上述结果表明分泌型与全长EphA7相互协调，分别通过稳定Claudin-6和激活JNK通路，调控原肾小管的形态发生与细胞分化，揭示了Eph信号在肾脏发育中的新功能与调控机制。
英文摘要	The vertebrate kidney is a complex organ that performs important homeostatic tasks including reabsorption of nutrients, water balance and waste products excretion. During kidney development in high vertebrates, three successive renal structures form via inductive processes: the pro-, meso- and metanephros. Although there are profound differences in anatomical structure between these three forms, they all contain nephron as their basic functional unit, which consists of three components: the glomerulus, tubule, and duct. Xenopus pronephros has been a good model to reveal the molecular network underlying kidney development. The pronephros of the frog Xenopus laevis consists of a single giant nephron, with its structural and functional segmentation compares well with that of mammalian nephrons. The development of Xenopus pronephros involves a series of complex processes including induction, mesenchymal epithelial transition, patterning and differentiation, accompanied by complicated lumenization and morphogenesis processes. The pronephric anlagen are first induced from the intermediate mesoderm lateral to somites 3-5 at about stage 12.5 by surrounding tissues, as indicated by the expression of the transcription factors Lim1 and Pax8. Once segregated from the intermediate mesoderm, the proximal condensate elongates in the dorsal-ventral direction, shaping the future pronephric tubule. Initiated at about stage 24, the cells undergo mesenchymal epithelial transition to become epithelial tubules. Later, the pronephric tubules elongate and become folded into a highly coiled structure while the pronephric duct grows caudally and fuses with the cloaca. Th Claudins are tetratransmembrane tight junction proteins and play important roles in regulating paracellular permeability in the kidney and maintaining cell polarity in epithelial and endothelial cells. There are at least 26 members of the claudin family in mammals, which are differentially expressed in the nephron segments, conferring different permeability properties to them. Claudin-6 (Cldn6) has been reported to play an important role during mouse embryonic epithelium formation and the development of endodermal tissues. Cldn6 has also been suggested to be a surface marker for mouse pluripotent stem cells and overexpression of Cldn6 is able to trigger epithelial morphogenesis in mouse stem cells. There are 2 Cldn6 genes reported in Xenopus, Cldn6.1 (also known as Cldn4L2) and Cldn6.2 (also known as Cldn4L1), both of which are expressed in the developing pronephros with similar patterns. Here we showed that Xenopus Cldn6 is required for terminal differentiation of pronephros. Knockdown of XCldn6 also interfered with pronephros morphogenesis and the maintaining of the apical-basolateral polarity of the tubule cells. We suggest that XCldn6 regulates pronephros development likely through its roles in tight junction formation. Eph/ephrin molecules are widely expressed during embryonic development, and function in a variety of developmental processes. Here we cloned the full length as well as a secreted form (EphA7-S) of Xenopus EphA7 and studied their roles in pronephros development. XEphA7 is specifically expressed in pronephric tubules at tadpole stages. In embryos with both forms knocked-down by a morpholino blocking their translation (EphA7-ATG-MO), the pronephric anlagen formed properly, however, it failed to undergo proper morphogenesis and terminal differentiation to form functional proximal tubules, which became organized into enlarged tubules instead. In embryos with only full length EphA7 knocked-down by a morpholino blocking its splicing (EphA7-sp-MO), the radialized cell condensates formed properly, however, they failed to lumenize. We found that the protein level of claudin6.1, a tetraspan transmembrane protein involved in tight junction formation, was dramatically reduced in EphA7-ATG-MO injected embryos, but not in EphA7-sp-MO injected embryos. EphA7 interacts with claudin6.1 via their extracellular domains and phosphorylates its cytoplasmic tail. EphA7-S, on the contrary, inhibited the tyrosine phosphorylation of claudin6.1 induced by EphA7 in a cell autonomous way. Interestingly, the apical accumulation of actin was dramatically reduced in EphA7-ATG-MO or EphA7-sp-MO injected PT cells. We propose that EphA7-S regulates mesenchymal epithelial transition of pronephric cells and tubulogenesis by stabilizing claudin6.1, while full length EphA7 is involved in the regulation of lumenization of the pronephric tubules and terminal differentiation of tubular cells.
语种	中文
内容类型	学位论文
源URL	[http://159.226.149.26:8080/handle/152453/10178]
专题	昆明动物研究所_发育生物学
推荐引用方式 GB/T 7714	孙健. 紧密连接蛋白Claudin-6及酪氨酸激酶受体EphA7在非洲爪蛙胚胎原肾发育中的功能研究[D]. 北京. 中国科学院研究生院. 2015.

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