Overview
The lab has a primary interest in Hox genes, which are among the master switch genetic regulators of development. We are working to define the functions of these genes, using several strategies. Our favorite tools include kidney organ culture, microarrays, siRNA, and gene targeting, and in particular a new type of gene targeting termed recombineering that allows the generation of sophisticated multi-Hox gene hits in a single electroporation of ES cells. We study the genetic programs initiated by Hox genes, and the functional relationships of these genes, in particular in the developing kidney. Below is a summary of the work supported by our two major NIH grants.
Studying the roles of hox genes in kidney development
We are interested in understanding 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 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 BAC 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 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.
Study the gene expression states that drive kidney development
The objective is to create a global gene expression atlas of the developing kidney. The central thesis is that a combination of laser capture microdissection and microarrays can be used to efficiently achieve this goal. Microarrays with essentially complete gene representation can be used to rapidly determine the expression levels of every gene in laser capture microdissected elements of the developing kidney. A single experiment, therefore, provides a comprehensive analysis of the gene expression status of one component, and a limited number of experiments examining each strucuture and substructure can create an atlas.
Specific aim 1 is to use this strategy to produce an atlas of the gene expression profiles of specific domains of the developing mouse kidney. The initial focus will be on the E15.5 kidney, which provides a single time point with multiple stages of nephrogenesis, but earlier time points will also be examined. A combination of structure, lectin staining, immunohistochemistry and transgenic GFP expression wll be used to precisely identify specific components and lineages.
Specific aim 2 is to make transgenic mouse tools to promote both gene expression profiling and functional studies of kidney development. We propose to make a series of transgenic mice with specific promoters driving restricted expression of a Cre-GFP cassette. These mice will serve a dual purpose, to allow identification of additional discreet kidney components for Specific Aim 1 and to aid future domain specific gene knockout studies in the developing kidney.
Specific aim 3 is to perform bioinformatics analysis of the microarray data and to make results readily available to the research community. Microarrays produce large gene lists that need to be sifted. Analysis of the complex orchestrations of gene expression defined in Specific Aim 1 will provide deeper insight into the genetic basis of the development of the distinct parts of the nephron.
