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Sexual development begins with the process by which the bipotential gonads of the embryonic urogenital ridge develop into either testes or ovaries. In the mouse, sex determination occurs at around 11.5 dpc and depends on the presence or absence of the Y chromosome and the associated activity of the testis-determining gene, Sry, in supporting cell precursors. The mutually antagonistic male and female developmental pathways are regulated by many cellular and molecular processes, disruption of which can lead to disorders of sex development (DSDs). However, many of the molecular mechanisms regulating the differentiation of the two gonads are still unknown. The boygirl (byg) mutant was identified in an ENU-based forward genetic screen for embryos with gonadal abnormalities. On the C57BL/6J background, XY byg/byg homozygotes exhibited complete embryonic gonadal sex reversal. The defective gene in byg, Map3k4, is a component of the mitogen-activated protein (MAP) kinase signalling pathway and provides the first evidence for a function of this pathway in sex determination. This thesis describes experiments aimed at investigating the cellular and molecular basis of the sex reversal phenotype associated with the XY Map3k4byg/byg mutant. Cellular characterisations revealed a defect in male-specific proliferation at 11.5 dpc, which was attributed to a defect in Sry up-regulation. Elucidation of the downstream kinases activated by MAP3K4 during sex determination was attempted, with particular focus on identifying a role for p38α MAP kinase (MAPK). Using a conditional knockout approach, the function of p38α in Steroidogenic factor-1 (Sf1)-positive somatic cells was assessed. However, specific inactivation in these cells did not affect gonad development. Conditional inactivation of Map3k4 itself in these Sf1¬-positive cells also did not disrupt gonad development, suggesting that this pathway is either initiated in a different cell lineage or at an earlier stage than deletion driven by Sf1-Cre can disrupt. Conditional inactivation of p38α in the Müllerian duct mesenchyme and ovarian granulosa cells using Amhr2-Cre did reveal a function for p38α in female fertility, but did not disrupt embryonic sexual development. Gene knockdown in organ culture was attempted to determine a role for multiple p38 MAPKs in all cell types of the gonad. Therefore, this thesis details further characterisations of a novel signalling pathway important for the expression of Sry, focussing on the role of the p38 MAPKs. |
