Title: Tissue interactions and molecular factors involved in indifferent gonad specification in the avian embryo
Description: The indifferent gonad is a unique organ that has the potential to give rise to either an ovary or a testis. Little is known of the developmental events that lead to the specification of this organ. Delineating the mechanisms of indifferent gonad specification is crucial for understanding its basic development as well as furthering our knowledge of what occurs in somatic disorders of the gonad. The work presented in this dissertation uses the avian embryo to investigate the mechanisms of indifferent gonad specification through an analysis of the tissue-tissue interactions and molecular factors required for its formation. We designed an in ovo microsurgery technique to test the requirement of the mesonephric kidney for gonad specification and demonstrated that placing an aluminum barrier posterior to the mesonephric duct primordium of a HH Stage 10 embryo successfully blocks posterior migration of the mesonephric duct and ablates subsequent mesonephros development. Under these experimental conditions, we showed that the mesonephros is required for gonad development and that in its absence, the gonad does not form and indifferent gonad markers Lhx9 and Dmrt-1 are not expressed.

To further evaluate required tissue relationships and investigate molecular regulation, we designed an in vitro culture system for the analysis of isolated gonad precursor tissues. At HH Stage 17, explants containing gonad precursors can be isolated and cultured for three days and go on to express the gonad marker, Lhx9. We demonstrate that an explant containing only the coelomic lining, its adjacent mesenchyme, and the intermediate mesoderm is capable of initiating robust gonad specific gene expression. This finding suggests that by HH Stage 17, gonad precursor tissue is already determined and committed to a gonad fate. We examined the potential role of Fgf signaling in gonad specification. The Fgf-receptor inhibitor, SU5402, eliminated expression of Lhx9 in explant cultures. Since Fgf9 knockout mice do form gonads, this result indicates a requirement for Fgf signals in addition to Fgf9 for the initiation of Lhx9 expression. Application of Fgf9 to trunk mesenchyme which is not specified to form gonad did not induce Lhx9 expression, suggesting that the reported gonad-promoting activity of Fgf9 is likely due to promotion of gonad proliferation and/or survival rather than early induction of gonad precursors.