Development of the Metanephric Mesenchyme

Cincinnati Childrens Hosp Med Ctr, Cincinnati OH

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Abstract

The goal of this proposal is to define the overlapping functions of Hox genes in kidney development. The Hox genes encode transcription factors that often serve important roles in the genetic hierarchy of development. During the previous funding period we showed that 26 Hox genes are expressed during kidney development, with Hox genes flanking each other on a single cluster often showing strongly overlapping expression patterns. In addition, through a series of coding sequence exchange gene targeting experiments we showed that flanking Hox proteins are closely related functionally. These observations lead us to the hypothesis that flanking Hox genes, as well as paralogs, have a high level of redundancy during kidney development. To test this hypothesis we propose the creation of a series of mutant mice carrying strings of frameshift mutations in flanking Hox genes. We have devised a novel variant of recombineering that allows the rapid construction of BAG targeting vectors with multiple Hox mutations. A total of eleven targeting experiments will produce mice with overlapping sets of mutations in 17 Hox genes on three clusters. Interbreedings will make mice with compound arrays of flanking and paralogous Hox frameshift mutations. The resulting phenotypes will be analyzed by a combination of histology, immunohistochemistry, in situ hybridizations, and laser capture microdissection-microarray. This study would represent the first genetic dissection of the functional redundancy of both flanking and paralogous Hox genes in kidney development. Our hypothesis predicts synergistic severity of phenotype as additional flanking Hox genes are removed. In addition, the fine expression analysis of the resulting phenotypes, using laser capture and microarrays, will be capable of detecting even subtle shifts in gene expression patterns, defining overlapping sets of Hox target genes and effector pathways.