Development of the Metanephric Mesenchyme
The goal of this work 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. We previously 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 are creating 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 BAC-targeting vectors with multiple Hox mutations. A total of 11 targeting experiments will produce mice with overlapping sets of mutations in 17 Hox genes on three clusters. Interbreeding will make mice with compound arrays of flanking and paralogous Hox frameshift mutations.
The resulting phenotypes are being analyzed by a combination of histology, immunohistochemistry, in situ hybridizations and laser capture microdissection-microarray.
This study represents 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.