Glis1-3 are novel genes recently identified in our laboratory. The Glis1-3 genes encode Kruppel-like zinc finger proteins containing five tandem zinc finger motifs that exhibit highest homology with those of members of the Gli and Zic subfamilies of Kruppel-like proteins. In addition, the zinc finger domain of Glis1 and -3 exhibit high homology with that of Drosophila gleeful/lame duck suggesting that it may be the Drosophila homologue of Glis1 and -3. Northern blot analysis showed that expression of the Glis1 and 2 mRNA is most abundant in adult kidney while Glis3 is expressed in several tissues. Whole mount in situ hybridization on mouse embryos demonstrated that Glis1-3 are expressed in a temporal and spatial manner during development. Glis1 expression was most prominent in several defined structures of mesodermal lineage, including craniofacial regions, branchial arches, somites, vibrissal and hair follicles, limb buds, and myotomes suggesting a role at different stages of development. Glis2 was expressed in kidney and neural tube suggesting a role in neurogenesis and kidney development.Glis3 is expressed in specific regions in developing kidney and testis and in a highly dynamic pattern during neurulation. From E11.5 through E12.5 Glis3 was strongly expressed in the interdigital regions, which are fated to undergo apoptosis. The temporal and spatial pattern of Glis1-3 expression observed during embryonic development suggests that they may play a critical role in the regulation of a variety of cellular processes during development.Confocal microscopic analysis showed that Glis1-3 are localized to the nucleus. The punctated pattern suggests that they are part of a larger nuclear protein complex. The zinc finger region in Glis plays an important role in the nuclear localization of these proteins. Electrophoretic mobility shift assays demonstrated that Glis1-3 are able to bind oligonucleotides containing the Gli-binding site consensus sequence GACCACCCAC. Although monohybrid analysis showed that in several cell types Glis1-3 are unable to induce transcription of a reporter, deletion mutant analysis revealed the presence of a strong activation and repressor functions suggesting that these proteins can function as repressors and activators of transcription. Glis3 was found to interact with GLi1 suggesting interaction between the Glis and Gli signaling pathways. Our results suggest that Glis1-3 may play a critical role in the control of gene expression during specific stages of embryonic development. Our hypothesis is that these proteins may act up- and/or downstream of sonic hedgehog, Wnt, BMP, or FGF signaling pathways. ? To obtain insight into the physiological functions of Glis2, mice deficient in Glis2 expression were generated. Glis2 mutant (Glis2mut) mice appear initially healthy but exhibit a significantly shorter lifespan than littermate WT mice due to the development of progressive chronic kidney disease. Histopathological analysis revealed a number of changes in the renal cortex of adult Glis2mut mice. These included tubular atrophy and basement membrane thickening affecting the proximal convoluted tubules and glomeruli. This was accompanied by infiltration of mononuclear (lymphocytic) inflammatory cells and interstitial/glomerular fibrosis. The severity of the fibrosis, inflammatory infiltrates, and the glomerular and tubular changes progressed with age and correlated with increases in blood urea nitrogen and creatinine, the development of proteinuria and increased water consumption and urine output. Ultimately Glis2mut mice die prematurely of renal failure. Comparison of the gene expression profiles of kidneys from 25 and 60 day old WT and Glis2mut mice by microarray analysis showed that a large number of genes involved in immune responses/inflammation and fibrosis/tissue remodeling are induced in kidneys of Glis2 mutant mice, and included several cytokines, adhesion and extracellular matrix proteins. Our data demonstrate that deficiency in Glis2 expression leads to tubular atrophy and progressive fibrosis that ultimately results in renal failure. Our study indicates that Glis2 plays a critical role in the maintenance of normal kidney functions.? To study the physiological function of Glis3, mice deficient in the expression of Glis3 were generated. These mice develop diabetes and polycystic kidney disease.
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