PKA对斑马鱼前肾发育的影响及其机制研究

作为一个胞内蛋白激酶,PKA在中枢神经系统、眼睛和肢体等形态发生中担任了重要角色,并参与多种细胞行为,但其在肾脏发育中的作用却不十分清楚.以斑马鱼为模式动物,采用原位杂交和冰冻切片技术检测PKA在斑马鱼中的表达情况;通过显微注射反义吗啉环寡核苷酸的方式,抑制内源性PKA的表达;通过免疫荧光技术,检测斑马鱼前肾的微观结构.结果显示:编码斑马鱼PKA催化亚基的prkaa1和编码调节亚基的prkab1b 均为母源基因,并且在肾管上皮细胞中有特异性表达;分别抑制内源性prkaa1或prkab1b 的表达,胚胎于第3天呈现高比例的心脏水肿(分别为54.4%和77.3%)和体轴弯曲(分别为48.5%和72.6%)以及极低比例的多囊肾表型(<5%);进一步检测发现,抑制PKA会导致肾管上皮细胞迁移缺陷,尤其是阻碍肾管多纤毛细胞迁移,继而引起肾管前中部不同程度肿大,并最终导致胚胎死亡.该研究表明,PKA可能通过调控斑马鱼肾管上皮细胞迁移从而在肾脏发育中发挥作用.

Effects and Possible Mechanisms of PKA on the Development of Zebrafish Pronephron

As an intracellular kinase, protein kinase A (PKA) functions in many tissues morphogenesis, including the central nervous system, the eye and the limb, and regulates diverse cellular activities. However, its role in kidney development is still unclear. The expression pattern of zebrafish PKA was obtained by in site hybridization and frozen-section technology; Morpholino was used for gene knockdown through microinjection; Microstructure of the pronephron was checked by immuno-fluorescence. Results showed that prkaa1 and prkab1b, coding catalytic and regulatory subunit of zebrafish PKA respectively, were maternal genes and were specifically expressed in the renal tubule epithelial cells; Inhibition of prkaa1 or prkab1b resulted in high proportion of heart edema (54.4% and 77.3% respectively) and body curvature (48.5% and 72.6% respectively) and very low proportion of polycystic kidney disease(less than 5%); Inhibition of PKA prevented migration of the renal tubule epithelial cells, especially multi-ciliated cells, resulting in prominent dilation of pronephric duct and then embryonic death. This indicated that PKA may function in zebrafish kidney development by regulating the migration of renal tubule epithelial cells.