Transcriptional Profiling of the Megabladder Mouse - A Unique Model of Bladder Dysmorphogenesis

Recent studies in our lab have identified a mutant mouse model of obstructive nephropathy designated mgb for megabladder. Homozygotic mgb mice (mgb-/-) develop lower urinary tract obstruction in utero due to a lack of bladder smooth muscle differentiation. This defect is the result of a random transgene insertion into chromosome 16 followed by a translocation of this fragment into chromosome 11. In an effort to identify potential gene targets affected in mgb mice, we performed transcriptional profiling on embryonic day 15 (E15) mgb-/- bladders using both a Chromosome 11/16 Custom GeneChip Array and the Affymetrix Mouse Genome 430 2.0 GeneChip. This analysis identified no definitive mis-expressed gene targets on chromosome 11. In contrast, mgb-/- mice significantly over-expressed a cluster of gene products located on the translocated fragment of chromosome 16 including urotensin II-related peptide (Urp), which was shown to be preferentially over-expressed in developing mgb-/- bladders. Immunohistochemical studies indicated that the spatial distribution of Urp was altered in mgb-/- bladders, while biochemical studies suggested a potential role for Urp in modifying smooth muscle cell phenotype in vitro. Pathway analysis of mgb microarray data showed dysregulation of at least 60 gene products associated with the differentiation of smooth muscle. In conclusion, the results of this study indicate that the molecular pathways controlling normal smooth muscle development are severely altered in mgb-/- bladders, and provide the first evidence that Urp may play a critical role in bladder smooth muscle development. Keywords: mgb mutant bladders Gene expression profiling was performed on two separate study groups. First, total cellular RNA was isolated from wildtype (n=2; 1 female/1 male), mgb+/- (n=2; 1 female/1 male), and mgb-/- (n=2; 1 female/1 male) E15 whole embryos. Second, total cellular RNA was isolated from the bladders of E15 wildtype (n=7; 3 female/4 male), mgb+/- mice (n=7; 3 female/4 male), and mgb-/- mice (n=11; 7 females/4 males).本实验室近期的研究中，我们鉴定出一种梗阻性肾病的突变小鼠模型，命名为mgb（megabladder，巨膀胱）。纯合型mgb小鼠（mgb-/-）在子宫内即出现下尿路梗阻，其病因是膀胱平滑肌分化缺失。该缺陷源于随机的转基因插入16号染色体，随后该染色体片段易位至11号染色体。为识别mgb小鼠中受影响的潜在基因靶点，我们分别采用11/16号染色体定制基因芯片阵列（Chromosome 11/16 Custom GeneChip Array）以及Affymetrix小鼠基因组430 2.0基因芯片（Affymetrix Mouse Genome 430 2.0 GeneChip），对胚胎期15天（E15）的mgb-/-膀胱组织开展转录表达谱分析。此次分析未在11号染色体上发现明确的差异表达基因靶点。与之相反，mgb-/-小鼠的16号染色体易位片段上的一组基因产物出现显著过表达，其中包括尾加压素II相关肽（urotensin II-related peptide，Urp），该肽已被证实于发育中的mgb-/-膀胱组织中优先过表达。免疫组织化学研究显示，Urp在mgb-/-膀胱中的空间分布发生改变；而生化实验则表明Urp在体外可能具有调节平滑肌细胞表型的潜在功能。对mgb小鼠芯片数据的通路分析显示，至少60种与平滑肌分化相关的基因产物存在表达失调。综上，本研究结果表明，调控正常平滑肌发育的分子通路在mgb-/-膀胱中发生严重紊乱，同时首次证实Urp可能在膀胱平滑肌发育过程中发挥关键作用。关键词：mgb突变体膀胱。本研究设置两个独立实验组开展基因表达谱分析：第一组，从野生型（n=2，1雌/1雄）、mgb+/-（n=2，1雌/1雄）及mgb-/-（n=2，1雌/1雄）的E15全胚胎中提取总细胞RNA；第二组，从E15野生型（n=7，3雌/4雄）、mgb+/-小鼠（n=7，3雌/4雄）及mgb-/-小鼠（n=11，7雌/4雄）的膀胱组织中提取总细胞RNA。