Genetic regulation of urogenital system development in the chicken embryo

© 2013 Dr. Andrew David CuttingThe vertebrate urogenital system is comprised of the urinary and reproductive structures. These structures develop during embryogenesis, and remain sexually indifferent until sex differentiation initiates. It is at this point that male and female genetic pathways initiate that masculinise and feminise the reproductive structures, respectively. This includes differentiation of the gonads and sex ducts into either testes and Wölffian ducts in males, or ovaries and Müllerian ducts in females. Although many genes involved in these pathways have been identified, exactly how these genes are regulated and how they interact remains to be determined. This thesis investigates the role, regulation, and interaction of genes that promote male development, namely, DMRT1, SOX9, and components of the anti-Müllerian hormone (AMH) signalling pathway, in the chicken model. This includes characterisation of the first avian AMH type-II receptor (AMHR2) to be discovered. Discordance between AMH mRNA and protein expression in the embryonic gonad are highlighted and described. In male gonads, the AMH transcript is detected two-days prior to AMH protein. In female gonads, only the AMH mRNA is detected, never the protein. This pattern suggests AMH may be post-transcriptionally regulated, potentially by microRNAs (miRNAs). Selected gonadal miRNAs are described, revealing that some exhibit expression patterns that correlate with that of AMH. A subset of gonadal miRNAs was predicted to target AMH, which was confirmed in vitro. These data therefore support the hypothesis that miRNAs regulate gonadal AMH expression. The SOX9 transcription factor is the primary activator of AMH expression during mammalian testis differentiation. Although highly conserved, this regulatory relationship has not been validated in the chicken. Furthermore, previous avian studies show AMH is expressed in the absence of SOX9. Therefore, expression patterns of SOX9 and AMH were precisely compared. Contrary to previous studies, results here showed that SOX9 was present at all time points when AMH was detected. Furthermore, AMH protein was only detected in cells expressing SOX9. However, over-expression of SOX9 did not up-regulate AMH in females. Therefore, SOX9 may be responsible for the up regulation, but not activation, of AMH. The DMRT1 transcription factor is critical for testis development in the chicken, yet an explicit role has not been demonstrated. Here, it is shown that DMRT1 regulates AMHR2 and is required for Müllerian duct development. DMRT1 and AMHR2 are co-expressed in gonadal Sertoli cells, and in the Müllerian duct. In the gonad, up-regulation and down-regulation of DMRT1 in females results in parallel increases to AMHR2. DMRT1 may therefore regulate testis differentiation by via AMH signalling and AMHR2. In the Müllerian duct, loss DMRT1 precludes Müllerian ridge formation, and thereby the subsequent formation of Müllerian duct components. The studies in this thesis present new data and theories on the role and regulation of genes underlying the formation of the urogenital system in the chicken model, and strengthen the foundations for further research into vertebrate sex differentiation