斑马鱼前肾管突变体的遗传筛选及机制研究

The zebrafish pronephric duct is similar to human renal tubule in function, structure, segmental differentiation and developmental mechanism, and is an ideal model to study development of renal tubule. To systematically investigate developmental mechanism of zebrafish pronephric duct, we will take advantage of the feasibility of this animal, the only suitable vertebrate, for a large scale mutant screen.We propose to use a typical ENU mutagenesis method to create founder and a classic F3 screen for recessive pronephric duct mutants. Mutants will be grouped according to their phenotype, and allelism test will be performed among mutants in the same group. Detailed phenotype characterization of mutants will include inspection on the timing of phentype appearance,polarity of ductal epithelium cell, cell-cell junctions and cilia in pronephric duct. Some typical mutants will be selected for map-based cloning for the responsible mutations. In the previous reports, screens were carried out by morphological observation under dissection microscope,however, we utilized a substrate of alkaline phosphatase to clearly visualize pronephric duct according to the high alkaline phosphatase activity in ductal epithelium.It is expected to harvest more mutants than before because of ease of observation.By this way we have isolated 34 mutants from 106 mutangenized genomes screened, these mutants were grouped into three classes based on phenotypes:(1)diluted duct,(2)wrong positioning of pronephric duct and (3)hypoplastic pronephric duct.We hope that novel developmental mechanisms will be discovered in our long term screen and study of pronephric duct mutants.

斑马鱼前肾管与人类肾管在功能、结构、组织分化和发育分子机制上高度保守，因此被认为是研究肾管发育的理想模式。斑马鱼是唯一适合于大规模遗传筛选的脊椎模式生物，为系统地研究肾管发育的调控机制，我们拟开展斑马鱼ENU前肾管突变体筛选，主要工作包括标准方法的ENU诱变、经典的F3代胚胎筛选、突变体表型的分型及互补测试，筛选获得的突变体将进行详尽的表型分析，包括表型出现的时间、细胞极性、胞间连接和纤毛结构等，我们将克隆部分代表性突变体的致变基因并研究其分子机制。与既有报道中采用光镜下直接观察的筛选方法不同，我们利用肾管上皮细胞中高活性的碱性磷酸酶显色，可鉴定出以往筛选所发现不了的突变体。在已经完成的工作中，我们从106个突变基因组中筛选获得了三类共34个斑马鱼前肾管突变体，包括肾管膨大、位置异常和发育不全。随着筛选的进行和突变体机制的研究，我们有希望揭示以前未报到的肾管发育机制。

斑马鱼是研究肾脏发育的理想模型，且适合于大规模遗传筛选。我们运用碱性磷酸酶底物显色的方法，采取经典的F3代胚胎筛选ENU诱变的斑马鱼，并经过三次传代纯化遗传背景，总共在212个突变基因组中获得了24个独立的稳定遗传突变体。通过对突变体肾管、肾小球和纤毛的表型分析归类，我们获得了包括2个肾管囊肿突变体，多个肾小球（5个）和肾管（5个）特异性突变体，这为后续的斑马鱼前肾发育研究提供了很好的材料。我们挑选了5个代表性突变体进行突变基因的图位克隆，所有的突变体都完成了染色体定位和突变所在区域的限定。由于V3-C突变位于端粒附近，难以以图位克隆的方式继续；V47突变所在区域的基因组DNA序列排列紊乱，只能等新版序列公布后继续；V3-A和V43突变的精确定位正在进行中；唯一成功克隆的L27的突变基因是osr1，有关这个基因已有较多在斑马鱼前肾发育的研究。尽管项目的进度较原计划稍有延后，但是项目的实施获得了多个前肾特异性斑马鱼突变体，有望在后续的研究中发现出新的肾脏发育调控机制，为临床肾脏疾病的诊断和治疗提供理论参考。