Mypt1在斑马鱼前肾发育中功能与机制的研究

Many kidney diseases are caused by mutations in kidney development regulatory genes; studies of kidney development will promote clinical treatment of kidney diseases. All of vertebrate kidneys are derived from mesoderm, the zebrafish pronephros, the functional counterpart of metanephros in mammalians, is an ideal model to study kidney development due to conserved molecular mechanisms across vertebrates. We found that the loss-of-function mutation in zebrafish mypt1 (myosin phosphatase target subunit 1)did not disrupt specification, segmental differentiation and positioning of pronephros at 24hpf, while at 30hpf the proximal convoluted segment (PCT) in the mutant was properly differentiated, with a loss of anterior part, and the neck of mutant pronephros was located more posteriorly than the wild type. Since Mypt1 regulates mesoderm cell migration, we speculated that this mutant phenotype was caused by the defective cell migration of renal mesoderm. To address the mechanism underlying this phenotype,we propose following experiments: Firstly, detailed characterization of pronephric phenotype in mypt1 mutant. Secondly,to examine autonomy of phenotype caused by the mutation. Thirdly, to generate pronephric PCT and neck tissue specific promoters driven KEADE transgenic lines to study migration and positioning of marked cells in mutant and wild type. Finally, to test known mechanisms controlling mesoderm cell migration in the mutant and try to figure out which is our case.

许多肾脏疾病是肾脏发育调控基因突变所导致的，研究肾脏发育对于治疗肾脏疾病意义重大。肾脏发育自中胚层，斑马鱼前肾是哺乳动物后肾的对应物，其发育的分子机制是保守的，是研究肾脏发育的理想模型。我们发现斑马鱼mypt1突变体早期前肾的特化、分化及各节段定位正常，30hpf时突变体前肾进曲小管PCT前段部分缺失,颈部结构也未处于预定位置。Mypt1调控中胚层细胞的迁移，突变体的表型很可能是前肾细胞迁移异常导致的。本课题运用细胞实时追踪这一斑马鱼模式生物的优势技术，设计以下实验研究Mypt1在斑马鱼前肾发育中的功能与机制：1）mypt1突变体前肾发育缺陷分析；2）mypt1突变体表型自主性的研究；3）建立PCT细胞和前肾颈部特异性启动子驱动KEADE的转基因鱼，研究mypt1突变体中PCT细胞和前肾颈部的迁移与定位；4）检测与中胚层细胞迁移有关的因素，发掘突变体表型的内在分子机制研究。

肾脏发育自中胚层，斑马鱼前肾是哺乳动物后肾的对应物，其发育的分子机制是保守的，是研究肾脏发育的理想模型。斑马鱼mypt1 突变体中前肾发育缺陷。我们的研究结果显示突变体IM的特化和早期发育是正常的，从30hpf开始突变体前肾管的前部包括颈部和PCT前端异常，表现为颈部的定位异常及在头尾轴向长度延长，同时，PCT缺失第三至六体节对应的部分。在trpm7:eGFP背景上整体观察可初步确定突变体前肾整体后移。为研究这一表型，我们尝试建立前肾特异性基因启动子驱动KAEDE的转基因品系来研究前肾细胞的迁移行为，我们获得了pax2a、cdh17和podocin的启动子并建立了驱动eGFP的稳定遗传品系，然而，在建立pax2a和wt1a/b的KEADE转基因品系时未获成功，原因是这两个基因的完整的启动子难以获得，现正在运用genomic knock-in的方法进行。.与此同时，我们还研究了斑马鱼mypt1 突变体中胰腺的发育缺陷。我们的前期研究表明突变体中肝脏的发育是缺陷的，与肝脏同样来源于腹侧前肠的胰腺则未有仔细讨论。我们的研究结果表明突变体中外分泌胰腺的起始发育未受影响，在36 hpf突变体的外胰管缺失且外分泌胰腺较野生型偏小，至48 hpf这两种表型变得更为明显，直至72dpf外分泌胰腺几乎消失。突变体的这一表型可被注射mypt1 mRNA挽救，这从遗传上证明了mypt1的突变是导致表型的原因。TUNEL检测显示突变导致了外分泌胰腺发生细胞凋亡。Mypt1既表达于外分泌胰腺，又表达于外分泌胰腺周围的LPM组织。而突变导致在LPM中表达的bmp2a的时空表达模式发生了改变。过表达bmp2a可以挽救突变体的胰腺发育缺陷，这说明mypt1 突变通过改变bmp2a表达模式来影响外分泌胰腺的发育。.