EXPRESSION OF THE RENAL H+ ATPASE IN METANEPHROGENESIS

在后肾发育过程中， 肾单位和集合管开始表达蛋白标记物 成熟的、分化的细胞的特征。 我们建立 在先前工作中， 包含B亚基的B1同种型，是插入型的标记物。 细胞分化，并且首先通过抗体染色检测到， 妊娠第17天。 在使用转基因小鼠和培养的 细胞，我们已经建立了一个区域的B1亚基基因，赋予 插入细胞特异性表达。 我们最近发现， 该区域中的GAGA（“GAGA”）盒对于表达 B1亚型。该应用程序的长期目标是了解 控制肾上皮细胞分化的机制。的 这项建议的具体目标是：1。要定义时间进程， 液泡H ~+ ATP酶亚基mRNA在胚胎发育过程中的表达 原位杂交技术在体内肾脏发育中的应用 2.以隔离 并表征B1亚基所需的GA β结合蛋白 同种型表达。一株肾脏细菌的寡核苷酸筛选 表达文库，或用于筛选酵母GAL 4的单杂交方法 激活结构域质粒文库，将用于分离cDNA克隆 GA β-结合蛋白 DNA结合特性和 然后检查蛋白质在肾脏中的表达。 3.到 确定肾同种型上的启动子元件 液泡H+ ATP酶的扩增表达过程中所需的 发展 启动子-报告基因构建体将在培养的细胞中测定。 细胞，肾组织和完整的小鼠，使用几种方法， 鉴定B1亚基表达所必需顺式调节元件。 这些研究应该促进我们对发展的理解。 集合管的异常，并可能提供信息 与控制肾上皮细胞的机制有关 缺血性肾损伤后发生的分化。

During metanephric the epithelial cells in different segments of the nephron and collecting duct begin to express protein markers characteristic of the mature, differentiated cell. We have established in prior work that amplified expression of the vacuolar H+ATPase, containing the B1 isoform of the B subunit, is a marker for intercalated cell differentiation, and is first detectable by antibody staining on gestational day 17. In studies using transgenic mice and cultured cells, we have established a region of the B1 subunit gene that confers intercalated cell-specific expression. We have recently discovered that a GA eta ("GAGA") box in this region is essential for expression of the B1 isoform. The long term goals of this application are to understand the mechanisms controlling renal epithelial cell differentiation. The specific aims of this proposal are: 1. To define the time course for expression of mRNA for vacuolar H+ATPase subunits during embryonic kidney development in vivo using in situ hybridization. 2. To isolate and characterize the GA eta-binding protein(s) required for B1 subunit isoform expression. Oligonucleotide screening of a kidney bacterial expression library, or the one-hybrid method for screening a yeast GAL4 activation domain plasmid library, will be used to isolate cDNA clones for the GA eta-binding proteins. The DNA binding properties and expression of the proteins in kidney will then be examined. 3. To define the promoter elements on the kidney isoform of the kidney vacuolar H+ATPase required for amplification of expression during development. Promoter-reporter constructs will be assayed in cultured cells, kidney tissue, and intact mice using several approaches, to identify cis-regulatory elements essential for B1 subunit expression. These studies should advance our understanding of developmental abnormalities of the collecting duct, and may provide information pertinent to mechanisms controlling renal epithelial cell differentiation that occurs following ischemic renal injury.