中国科学院昆明动物研究所机构知识库

非洲爪蛙是研究神经管与耳泡发育的优良模型。神经管与耳泡的发育都经历了相似的诱导、形态发生与分化过程，也拥有类似的细胞事件，比如：聚集延伸、顶部收缩，提示二者的发育可能拥有共同的分子调控机制。本论文探讨了受体酪氨酸激酶EphA7在爪蛙神经板与耳泡发育中的功能与作用机制。 EphA7特异表达于非洲爪蛙胚胎神经板与耳泡中。用特异性的Morpholino敲低EphA7在神经板的表达，不影响神经板的诱导，但是会干扰前部神经管的闭合，导致脑部开裂。敲低EphA7会阻断后脑神经上皮细胞的顶部收缩，并抑制F-actin在细胞顶部的聚集，提示细胞表面张力减弱。进一步的实验证明，EphA7能在体内与体外调控粘着斑激酶FAK（actin聚集的关键调控因子）的磷酸化水平和活性。上述研究揭示了EphA7调控后脑神经上皮细胞的顶部收缩与后脑闭合的分子机制。 我们在非洲爪蛙发育早期鉴定了EphA7的分泌形式(EphA7-S)，它与全长(EphA7-FL)都表达于神经板和耳泡中，但是各自的功能尚不清楚。在耳泡中，我们利用EphA7-ATG-MO同时阻断EphA7-S与EphA7-FL的表达，观测到耳泡减小，球囊斑变薄，毛细胞和耳石的缺陷，并伴随着运动失衡。我们设计了EphA7-sp-MO特异地抑制EphA7-FL的表达，发现耳泡有中等程度地减小，而毛细胞和耳石的分化也受到干扰，表明EphA7-FL参与了耳泡的分化。但两种MO都不影响耳泡的诱导、模式形成及耳腔的建立。在细胞水平上，EphA7-ATG-MO显著降低了紧密连接蛋白CLDN6在耳上皮细胞中的表达，而EphA7-sp-MO不影响其表达。我们的体外实验证明，EphA7的两种形式都能结合CLDN6，EphA7-FL能增强CLDN6的酪氨酸磷酸化并减弱后者在细胞膜上的定位。而EphA7-S通过拮抗EphA7-FL的这种效应，来稳定CLDN6的膜水平。进一步研究表明，CLDN6膜水平的减弱促进了耳上皮细胞外排到耳腔中，并发生凋亡。我们的结果阐明了EphA7在耳泡发育中的一个新功能及其作用机制。 为了探究EphA7-S的功能，我们通过免疫沉淀-质谱测序技术鉴定了一个与EphA7-S相互作用的蛋白Nicalin。Nicalin是一种I型跨膜蛋白，在内质网应激条件下参与了预先的质量控制过程。实验证明，Nicalin与EphA7-S共定位于内质网，Nicalin通过调控EphA7-S的易位限制其进入ER被修饰，而抑制EphA7-S的分泌。EphA7-S会增强EphA7的多聚体水平，但是Nicalin会降低这种水平。我们推断，Nicalin可能通过调节EphA7-S进入内质网的量而影响EphA7之间的多聚体水平，其生理功能还需进一步探究。

Xenopus has been an excellent model to study the early development of neural plate (NP) and the otic vesicle (OV), which share similar morphogenetic events, including convergence extension and apical contraction. Here we studied the roles and mechanisms of EphA7, a member of the receptor tyrosine kinase family, in the development of NP and OV. EphA7 is specifically expressed in the neural plate and the otic vesicles in Xenopus embryos. Knockdown of EphA7 by EphA7-ATG-MO did not affect the induction of NP, but disrupted cranial neural tube closure and disturbed apical constriction of hindbrain neuroepithelial cells, indicating weakened cell surface tension. In neural plate explants, EphA7 knockdown inhibited apical filamentous actin (F-actin) accumulation. We further showed that EphA7 is involved in the phosphorylation and activation of focal adhesion kinase (FAK) in vivo and in vitro, a key regulator of actin assembly. Our findings reveal that EphA7 functions as a critical regulator of apical constriction of hindbrain neuroepithelial cells.During Xenopus early development, a secreted form of EphA7 (EphA7-S) was identified in NP and OV, but the functions of EphA7-S and EphA7-FL (full length form) are not clear. Knockdown of EphA7 by EphA7-ATG-MO caused reduced OV, thinner otic epithelium, defected or absent otoconia, aberrant hair cells, and companied by imbalanced swimming motion. We designed an EphA7-sp-MO to specifically inhibit the expression of EphA7-FL, which caused mildly reduced OV, defected otoconia and hair cells, indicating that EphA7 is required for the differentiation of OV. However, EphA7 did not affect the induction, pattern formation or lumen formation of OV. We found that the protein level of CLDN6, a tetraspan transmembrane protein involved in tight junction formation, was dramatically reduced in EphA7-ATG-MO injected OVs, but not in EphA7-sp-MO injected OVs. Co- immunoprecipitation experiments showed that both EphA7-S and EphA7-FL could bind CLDN6. In HEK293 cell, co-expression of EphA7-FL but not EphA7-S with CLDN6 dramatically increased the level of tyrosine phosphorylated CLDN6, but markedly reduced the membrane level of CLDN6. Interestingly, EphA7-S blocked tyrosine phosphorylation of CLDN6 induced by EphA7-FL when co-expressed, and rescued the membrane level of CLDN6. Further evidence showed that reduced CLDN6 triggered OV epithelial cells to be extruded into the OV lumen. We propose that EphA7 maintains OV size likely through stabilizing CLDN6.By immunoprecipitation-mass spectrometry, we identified a potential EphA7 interacting protein named Nicalin, which is a type 1 transmembrane protein, functions in proximity to translocon in pre-emptive quality control. Our results showed that Nicalin and EphA7 were colocalized in the ER, and Nicalin inhibited the secretion of EphA7-S by controlling the translocation of EphA7-S into the ER. EphA7-S increased the level of polymerized EphA7, while Nicalin reduced EphA7 polymerization level. We speculate that Nicalin might affect the polymerization state of EphA7 by regulating the translocation of EphA7 into the ER, but the physiological role of Nicalin in EphA7 signaling remains to be explored.