ROLES OF THE MFH-1 AND WT1 GENES IN KIDNEY DEVELOPMENT

我们培育了MIFH-1基因敲除小鼠。MIFH-1基因缺失小鼠表现出心血管系统和骨骼的表型。前者为腹主动脉弓中断和室间隔缺损，后者为脊椎骨和颅面骨的畸形和畸形。这些结果表明，MFH-1基因对主动脉弓的形成和骨骼发育是必不可少的。MFH-1基因在肾脏中也有表达。在MFH-1基因敲除小鼠中，肾脏略小，骨盆畸形。肾小球和肾小管结构未见异常。我们解释了其他叉头家族基因，如BF-1和Mf-1，可能对MFH-1基因的功能起到补偿作用。我们用抗小鼠MFH-1的单抗筛选细胞系，以研究MFH-1基因是否在细胞中表达。我们发现MFH-1蛋白在Wilms肿瘤细胞系、骨肉瘤细胞、软骨肉瘤细胞、主动脉内皮细胞、主动脉平滑肌细胞和成纤维细胞、NIH/3T3和Balb/3T3细胞中均有表达。为了探讨MFH-1基因在肾脏中转录调控的分子机制，我们将与报告基因荧光素酶相连的MFH-1基因启动子的3.Okbp和5‘-侧翼区分别导入Wilms肿瘤细胞系。该载体含有3.0kbp的侧翼区，在Wilms肿瘤细胞系中具有很强的转录活性。这一结果表明，在肾母细胞瘤细胞中，3.0kbp区域存在正向调控MFH-1基因表达的元件。目前，已尝试通过凝胶漂移实验和与WT1表达载体的共转染来鉴定3.0kbp区域内的WT1结合位点。

We generated the knockout mice of the MIFH-1 gene. The MIFH-1 null mice displayed phenotypes of cardiovascular system and skeleton. The formers were interruption of the aortic arch and ventral septal defect, and the latters were defects arid malformation of the vertebrates and craniofacial bones. These results indicate that the MFH-1 gene is essential for aortic arch formation and skeletogenesis.The MFH-1 gene is also expressed in the kidney. In the MFH-1 knockout mice, the kidneys were a little small and the pelvis was malformed. However, the structures of glomerulus and renal tubules had no abnormalities. We interpret that other forkhead family genes, for example BF-1 and MF-1, might compensate the function of the MFH-1 gene.Next we investigated the target genes of MFH-1 gene. We screened the cell lines with monoclonal anti-mouse MFH-1 antibody in order to study whether the MFH-1 gene is expressed in the cells. We found that the MFH-1 protein was expressed in the Wilms tumor cell line, osteosarcoma cells, chondrosarcoma cells, aortic endothelial cells, aortic smooth muscle cells and fibroblast cells, NIH/3T3 and Balb/3T3. In order to investigate the molecular mechanism governing the transcriptional control of the MFH-1 gene in the kidney, we transfected the 3.Okbp of promoter and 5'-flanking region of the MFH-1 gene which was connected to the reporter gene luciferase into the Wilms tumor cell line. The construct which contains 3.0kbp flanking region showed a strong transcriptional activity in the Wilms tumor cell line. This result indicates that the 3.0 kbp region has elements regulating the expression of the MFH-1 gene positively in the nephroblastoma cells. Currently, the WT1-binding sites within the 3.0kbp region have been tried to identify by gel shift assay and cotransfection with the WT1-expression vector.