Diseases that impair kidney function, both inherited and acquired, represent a major medical problem. This proposal is designed to further our understanding at the molecular level of normal kidney development and function and to identify perturbations that occur in disease states by studying the expression of transcription factors that are highly expressed in specific subsets of kidney cells.
The specific aims and methods to be used are:
(AIM 1). To determine the biological function of the two major isoforms of WT1 by identifying targets of regulation by this transcription factor. Reverse transcription and polymerase chain reaction will be used to identify genes that are either induced or repressed in stable transfectants that overexpress WT1 protein. The putative targets identified by the approach will be further characterized by northern analysis and RNA in situ hybridization ina variety of cell lines, normal fetal kidney and Wilms tumors to determine which of these genes are most likely to be important in nephrogenesis and the process of Wilms tumorigenesis.
(AIM) 2). To determine the biological function of the two major isoforms of WT1 by identifying targets of regulation by this transcription factor. Reverse transcription and polymerase chain reaction will be used to identify genes that are either induced or repressed in stable transfectants that overexpress WT1 protein. The putative targets identified by this approach will be further characterized by northern analysis and RNA in situ hybridization in a variety of cell lines, normal fetal kidney and Wilms tumors to determine which of these genes are most likely to be important in nephrogenesis and the process of Wilms tumorigenesis., (AIM 2). To characterize Tbx2, a member of a new family of putative transcription factors that is expressed in lung and kidney by a) studying its pattern of expression in fetal and adult tissues and b) determining its cellular function through in vivo and in vitro tbx2 expression studies. Tbx2 is expressed in fetal and adult kidney an lung and exhibits striking homology within its putative DNA binding domain to the DNA binding domain of several other murine and drosophila genes. The research design entails a) the identification of the subset of kidney cells expressing Tbx2 by RNA in situ hybridization and immunocytochemistry, b) the use of gel mobility shift assays to define the specific DNA sequences that are bound by the Tbx2 protein and the use of transient transfection assays with reporter constructs to demonstrate that Tbx2 encodes a transcription factor.
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