HOXA 13 REGULATION OF GENITOURINARY DEVELOPMENT

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DESCRIPTION (adapted from the application) Nonsense mutations in the transcription factor Hoxa 13 result in severe malformations of the genitourinary (GU) region. In humans, loss of Hoxa 13 function causes familial hand-foot-genital syndrome (HFGS), resulting in hypospadia. prostate, uterine, Mullerian duct, vaginal bladder, ureter. and rectal defects. In mice, these developmental pathways are conserved, as targeted disruption of' Hoxa 13 results in malformation or agenesis of the prostate, uterus, urethra, ureter, MuIlerian ducts, vagina, rectum, and bladder. What is intriguing about the development of these structures is that many of them form from condensations of undifferentiated mesenchyme. Recently, I demonstrated that Hoxa 13 regulates mesenchymal condensation in the limb and umbilical vasculature (UV) by controlling genes that-direct cell-cell adhesion, apoptosis, and identity. Because many of the structures affected in HFGS are derived from mesenchymal condensations. I hypothesize that the genetic pathways regulated by Hoxal13, to mediate limb and UV development also function to direct normal development of the GU region. To test this hypothesis, a Hoxa 13 mutant mouse with an in-frame GFP reporter will be used. The Hoxa 13 GFP allele produces a strong, Hoxa 13-specific signal in the affected GU regions. This signal facilitates the enrichment of homogeneous populations of cells by fluorescence-activated cell sorting (FACS) from dissected tissues expressing Hoxa 13. Mesenchyme cells purified by FACS from the affected regions of mutant and control embryos will be characterized in vitro for changes in gene expression, cell adhesion, apoptosis, and proliferation that could account for specific malformations. These FACS enriched cells will also be used as a resource for RNA-and cDNA-based gene discovery methodologies to identify additional genes regulated by Hoxa 13 that play a role in GU development. Once identified, the chromosomal localization of Hoxa 13 specific target genes will be determined using mouse/hamster radiation hybrid DNA panels. The map positions and embryonic expression patterns of any newly identified genes will be made available to the research community, providing a resource to identify unknown genetic components involved in benign prostate hyperplasias, malignancies, and GU malformations. The epistatic relationship between Hoxa 13, and dhydroxytestosterone will be determined using a Hoxa 13 GFP reporter cell line in conjunction with in vitro screen, to identify genes that activate Hoxa 13. This unique experimental design allows study of GU development from the, perspective of the cells and tissues most affected by loss of Hoxa 13 function. From this perspective, insight genetic and cellular mechanisms required for normal GU development will be gained.