NOVEL HOMEOBOX GENES IN DEVELOPING GENITOURINARY TRACT

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The causes of many developmental disorders affecting the kidney and genitourinary tract (e.g., renal agenesis, hypoplasia, and cystic dysplasia) remain unknown, largely because little is known about the molecular mechanisms underlying development of the normal kidney. Homeobox-containing genes are responsible for organ differentiation in many species, and disruption of one such gene (Hox-1.5) produces congenital abnormalities in the mouse. Recently, we cloned a novel class of homeobox-containing genes (named cux) from mouse. Cux genes are related to the cut gene of Drosophila melanogaster which is required for normal development of Malpighian tubules, the insect excretory organ that serves as a primitive kidney. The inferred structures of the cux gene products indicate that they also are likely to encode nuclear transcription factors involved in gene regulation. Moreover, we found that at least one of the genes (cux-1) is expressed in kidney, testis, and renal epithelial cells suggesting that it may have a role in the normal development of the mammalian kidney and genitourinary tract. Abnormalities of cux-1 expression, then, might be involved in the pathogenesis of urogenital abnormalities or neoplasms. To further test these hypotheses, full-length cDNAs encoding cux-1, cux-2, and related murine cut homologs will be cloned by library screening and PCR. The temporal and spatial patterns of expression of cux-1 and cux-2 in the developing genitourinary tract of mouse will be further investigated using in situ hybridization and semi-quantitative PCR. Specific antisera raised to cux fusion proteins will be used to identify and immunolocalize the cux gene products in the mature and developing kidney and testis. The promoter/regulatory regions of the cux-1 and cux-2 genes will be cloned, sequenced, and expressed. Whether the cux-1 gene product binds to its own promoter/regulatory region will be evaluated as a first step towards identifying its DNA-binding specificity. The functional roles of the cux-1 gene will be assessed by specific inhibition in renal epithelial cells using antisense oligonucleotides. From the effects on cell proliferation, morphology, polarity, epithelial organization, and gene expression, functional roles may be ascribed to the cux-1 gene. Whether the cux-1 gene is required for development of the kidney and genitourinary tract in mouse will be tested by disruption using homologous recombination and expression in transgenic mice. Our long-term aims will be to address the role of cux genes in a cascade of genetic regulatory mechanisms responsible for nephrogenesis. In these future studies, steps in pathways that are more proximal to cux (regulation of cux genes and gene products) and steps that are distal (target genes) will be examined in greater detail. Such studies should contribute to our fundamental understanding of genitourinary tract development. Moreover, further characterization of this novel class of homeobox-containing genes would be relevant to the overall study of eukaryotic gene regulation.