Studies in Drosophila first demonstrated the developmental significance of the homeobox genes, with mutations often resulting in homeotic transformations or structure deletions. These genes are surprisingly well conserved during evolution in sequence, organization, expression and function. The murine Hoxa 11 gene was originally cloned by the PI, using a degenerate oligonucleotide library screen. Initial characterization of this gene has included sequencing, extensive expression studies with both whole mount and serial section in situ hybridizations, and mutational analysis including both single gene targeting, and, in collaboration with another lab, double gene targeting. The results have suggested that the Hoxa 11 gene is worthy of additional investigation. The Hoxa 11 knockout showed that this gene is required for both male and female fertility, normal segment determination, and proper limb development. The double knockout, including a paralogue of Hoxa 11, resulted in even more profound malformations, with deletions of forelimb structures and absent or rudimentary kidneys at birth. The goal of this proposal is to define the molecular genetic mechanisms of Hoxa 11 function in kidney, uterus and limb development.
The first aim i s to complete the characterization of the mutant phenotype, using histologic and molecular marker analysis at multiple developmental time points.
The second aim i s to discern the regulatory mechanisms that control Hoxa 11 expression. Of particular interest is the finding of abundant processed, polyadenylated Hoxa 11 natural antisense RNAs. In the developing limbs the expression of the antisense RNA is complementary to that of the sense RNA, with antisense transcripts appearing in abundance in domains where sense transcripts are disappearing. The hypothesis that the antisense RNAs regulate stability of Hoxa sense RNA, as previously described for the bFGF gene in Xenopus, will be directly tested in transgenic mice. The homeobox genes, by encoding transcription factors, modulate expression patterns of downstream target genes. To carry our understanding of Hoxa 11 function to the next level it is therefore necessary to identify and characterize the genes that it regulates. The final specific aim proposes to accomplish this using a variety of approaches. The Hoxa 11 target binding sequence has been identified, the expression patterns of candidate target genes will be examined in Hoxa 11 mutants, and Hoxa 11 antibody will be used to immunoprecipitate chromatin with target genes. In addition differential display will be used to compare expression patterns in certain wild type and mutant tissues and cell lines. The proposed studies will extend our comprehension of Hox function beyond targeting-phenotype analysis, and into the realm of molecular mechanisms.
