The Hox genes are among the key genetic regulators of development. Mutations of these genes in simple organisms can cause dramatic transformations of body parts, with legs for example protruding from the head in place of antennae. In mammals, however, mutations generally give considerably milder effects. This is likely the result of the much greater number of Hox genes in mammals and their extensive overlapping functions. In this proposal we describe a novel recombineering strategy that allows us to simultaneously introduce frameshift mutations into sets of contiguous Hox genes, while leaving interspersed shared enhancers intact. We propose to complete the creation of a rich resource of Hox mutant mice that will allow the dissection of both flanking and paralogous Hox gene functional relationships. While 31 Hox genes show robust expression during kidney development, developmental functions have only been identified for six. We propose to unveil the roles of the other Hox genes by creating mice with stepwise reductions in Hox flanking and paralogous function. Preliminary studies of mice already made with mutations in HoxD, 3, 4, 8 and HoxA9, 10, 11, HoxC9, 10, 11 and HoxD9, 10, 11, have identified interesting phenotypes, including dramatic reductions in nephron number, glomerulomegaly, hydronephrosis, and failure to make renal vesicles, discovering the importance of additional Hox genes in kidney development. We also propose an extensive analysis of resulting kidney phenotypes, leveraging our GUDMAP consortium expertise and database. The moleular mechanisms of Hox function will be defined through characterization of overlapping sets of downstream targets and Gene Ontology analysis defining their molecular functions and biological processes. The study of disturbed kidney gene expression patterns in Hox mutants will extend to the single cell level, using novel microfluidics/RNA-Seq approaches. In addition Chip-Seq will be used to define the overlapping sets of Hox DNA binding sites and to distinguish direct targets. The results will improve our understanding of the genetic circuitry of kidney development and will thereby augment future DNA diagnosis and iPS mediated regeneration and/or replacement of diseased or malformed kidneys.
It is important to understand the genetic regulatory network that drives kidney development. This will lead to an improved future ability to diagnose, regenerate and replace defective kidneys. The proposed research will define novel roles of Hox genes in kidney development, test patterning versus proliferation models, and identify overlapping and distinct downstream targets and DNA binding sites. The end result will be a much deeper understanding of the genetic circuitry of nephrogenesis.