DLGH1 AND UROGENITAL DEVELOPMENT

描述（由申请人提供）：本研究的长期目标包括确定小鼠大盘同源基因Dlgh1调节泌尿生殖发育的机制。DLGH1是一种具有多个蛋白相互作用结构域的支架蛋白，包括3个PDZ结构域和1个SH3结构域。Dlgh1是果蝇圆盘大基因dlg的同源物，dlg是PDZ蛋白家族和MAGUK（膜相关鸟苷酸激酶）家族的创始成员。对果蝇的研究表明，dlg是一种肿瘤抑制因子，因为dlg的突变会导致象盘过度生长。对果蝇和哺乳动物细胞的研究表明，dlg及其同源物也参与上皮细胞极性的建立和/或维持。我们研究了缺乏DLGH1的小鼠，发现了几种泌尿生殖缺陷，包括散发性肾发育不全、先天性肾积水和生殖道异常。我们已经证明，肾积水与严重的平滑肌排列缺陷有关；正常圆形的肌肉在纵向上异常排列。这极大地损害了蠕动的挤压运动，从而使尿液保留在肾脏中。此外，通常位于尿路上皮和平滑肌层之间并表达Raldh2的输尿管基质细胞在Dlgh1突变的输尿管中缺失。这些细胞的缺失也可能直接或间接地影响输尿管功能。免疫荧光研究表明，DLGH1在尿路上皮中表达强烈，在输尿管其他细胞中表达较弱。我们将使用一个新的固定的Dlgh1等位基因和适当的Cre转基因小鼠，确定哪个细胞室必须表达Dlgh1才能使正常的平滑肌细胞对齐，并使输尿管基质细胞分化和/或迁移。此外，我们将产生嵌合小鼠来研究DLGH1是否以非细胞自主方式起作用。形态学和功能评估将进行，以确定Dlgh1表达的细胞需求。接下来，我们将使用遗传学方法来研究其他DLGH家族成员与已知或可疑的DLGH1结合蛋白的相互作用部分补偿DLGH1缺失的假设。最后，我们将使用体外方法来确定DLGH1在细胞迁移、极化和信号传导中的作用。公共卫生相关性：先天性尿路异常是人群中相对常见的健康问题，是导致大量进行性肾脏疾病的原因。我们建立了一种新的小鼠肾积水模型（Dlgh1-/-小鼠），涉及输尿管平滑肌错位，这在人类尿路梗阻病例中很常见。更好地了解DLGH1的生物学可能对理解和治疗或预防人类尿路异常具有重要意义。

DESCRIPTION (provided by applicant): The long term objective of this research involves determining the mechanism whereby the mouse discs- large homolog 1 gene, Dlgh1, regulates urogenital development. DLGH1 is a scaffolding protein with multiple protein-protein interaction domains, including three PDZ domains and one SH3 domain. Dlgh1 is a homolog of the Drosophila discs-large gene, dlg, which is a founding member of the eponymous PDZ family of proteins and also of the MAGUK (membrane-associated guanylate kinase) family. Studies in Drosophila suggest that dlg is a tumor suppressor, because mutation of dlg causes overgrowth of imaginal discs. Studies in both fly and mammalian cells suggest that dlg and its homologs are also involved in establishing and/or maintaining epithelial cell polarity. We have studied mice lacking DLGH1 and found several urogenital defects, including sporadic renal agenesis, congenital hydronephrosis, and reproductive tract abnormalities. We have shown that hydronephrosis is associated with dramatic smooth muscle alignment defects; the normally circular muscle aberrantly aligns in the longitudinal direction. This greatly impairs the squeezing motion of peristalsis, such that urine is retained in the kidney. In addition, ureteric stromal cells, which normally lie between the urothelium and the smooth muscle layers and express Raldh2, are absent from Dlgh1 mutant ureters. The absence of these cells could also affect ureteric function, either directly or indirectly. Immunofluorescence studies show that DLGH1 is expressed strongly in the urothelium and weakly in other cells of the ureter. We will determine which cellular compartment must express Dlgh1 for normal smooth muscle cell alignment and for differentiation and/or migration of ureteric stromal cells using a new floxed Dlgh1 allele and appropriate Cre transgenic mice. In addition, we will generate chimeric mice to investigate whether DLGH1 acts in a non-cell autonomous fashion. Morphological and functional assessments will be performed to determine the cellular requirements for Dlgh1 expression. Next we will use genetic methods to investigate the hypothesis that the interaction of other DLGH family members with known or suspected DLGH1 binding proteins are compensating in part for the absence of DLGH1. Finally, we will use in vitro methods to define the role of DLGH1 in cell migration, polarization, and signaling. PUBLIC HEALTH RELEVANCE: Congenital urinary tract abnormalities are a relatively common health problem in the human population and are responsible for a significant number of cases of progressive renal disease. We have generated a novel mouse model of hydronephrosis (Dlgh1-/- mice) involving the misalignment of ureteric smooth muscle, which is commonly found in human cases of urinary tract obstruction. A better understanding of the biology of DLGH1 could have important implications for understanding and perhaps for treatment or prevention of urinary tract abnormalities in humans.