Wnt5a-Ror细胞信号转导通路调节泌尿系统发育的新机制研究

Development of the metanephric kidney is an intricate process. It begins with the induction of single ureteric bud on the caudal Wolffian duct (WD) in response to GDNF (glial cell line-derived neurotrophic factor) produced by the adjacent metanephric mesenchyme (MM). Mutual interaction between the UB and MM maintains expression of GDNF in the MM, thereby supporting further outgrowth and branching morphogenesis of the UB, while the MM also grows and aggregates around the branched tips of the UB. The receptor tyrosine kinase Ror2 mediates β-catenin independent Wnt signaling as a receptor or coreceptor for Wnt5a. Wnt5a-Ror2 signaling has been shown to play crucial roles in developmental morphogenesis and human diseases. Our original working indicated Ror2 is predominantly expressed in the MM during UB induction and that Ror2- and Wnt5a-deficient mice exhibit duplicated ureters and kidneys due to ectopic UB induction. During initial UB formation, these mutant embryos show dysregulated positioning of the MM, resulting in spatiotemporally aberrant interaction between them and WD, which provides the WD with inappropriate GDNF signaling. These results hypothesized that Wnt5a-Ror2 signaling balance MM cells proliferation and concentration. On the other hand, it is also important to examine Ror1 Ror2-double mutants to clarify the possible functional redundancy between Ror1 and Ror2 and to elucidate further the mechanisms by which Wnt5a-Ror signaling regulates kidney development. We will continue our study to validate these hypotheses by using gene knockout mice.

肾脏发育基于午非氏管末端的输尿管芽在与后肾基质细胞的相互作用。基质细胞产生的GDNF在调控因子的作用下诱导午非氏管末端产生单个输尿管芽，继而与基质细胞协同发育形成肾脏。受体酪氨酸激酶Ror2作为Wnt5a受体，介导β-catenin非依赖性Wnt细胞信号转导通路。我们在前期研究中发现Ror2主要表达于发育早期的肾脏基质细胞，并且Ror2或Wnt5a敲除小鼠表现出单侧双肾双输尿管、肾缺如等畸形。这些畸形已被我们证实与Wnt5a-Ror2信号缺失而导致的基质细胞空间相对位置失调有关，但其发生机制并不明确。本课题利用多种转基因小鼠生物模型，从多角度研究肾脏发育过程中Wnt5a-Ror细胞信号转导机制，进一步验证该路径在肾脏发育过程中对于后肾基质细胞的迁移所起到的关键作用。本课题还将对Ror2同族受体Ror1的作用补偿机制进行研究。这些新机制的发现将对于肾脏发育以及人工肾脏构建的研究提供新的线索。

间充质-上皮转化（MET）是肾脏发育以及肾功能完全的重要过程，然而microRNA 在肾发育MET中的作用以及调控机制尚不清楚，因此探究miRNA在肾发育MET过程中的作用有助于了解肾发育疾病的病因，扩展肾发育疾病的治疗手段。本项研究首先通过qRT-PCR以及Western Blot检测miR-1，miR-802以及Wnt-4/β-catenin信号通路中相关蛋白的表达，发现肾发育过程中miR-1表达下降miR-802表达上升，同时Wnt-4/β-catenin信号通路中蛋白表达发生变化。进一步通过荧光素酶报告基因分析鉴定miR-1，miR-802靶基因，western blot检测miRNA对靶基因表达的调控，确定miR-1,miR-802负向调控其靶基因Wnt-4，β-catenin的表达。最后通过免疫荧光检测MET相关蛋白的表达，证实Wnt-4，β-catenin参与MET的过程，miR-1，miR-802通过调控Wnt-4，β-catenin的表达影响MET的过程。总之，miR-1，miR-802通过调节Wnt-4/β-catenin信号通路调控肾发育MET过程。