TESorOVA · TEStis or OVAry: Identifying the molecular and cellular processes controlling sex determination.

Sex determination is the process that allows the bipotential gonad to develop into a testis or an ovary, thus initiating the sex fate of the individual. In mammals, the supporting cells are the first to adopt a sexual identity and then direct the differentiation of the other gonadal cell types. Despite their key role, we still do not know how these important cells acquire their identity. To address this question, we will use spatial transcriptomics and lineage tracing analysis in combination with mouse models deficient for the RSPO1/WNT/ß-catenin signaling pathway as a we found that absence of Rspo1 impairs supporting cell differentiation. Ovarian determinants are still unknown in most vertebrates, but we have recently identified that an isoform of WT1 is the ovarian determinant, in mice. We will use genetic engineering in chicken and zebrafish to test whether this conserved isoform has a similar function in other sex determination systems. Notably, WT1 also interacts with WTAP, a factor involved in m6A methylation of RNA, a mechanism that regulates sex determination in drosophila. Our preliminary results suggest that m6A methylation plays a key role in the sex differentiation of supporting cells. To address how this mechanism regulates sex determination, we will determine the kinetics of RNA methylation during gonadal development followed by a functional analysis using mouse models deficient in Wtap and Mettl3, the catalytic unit of m6A methylation. Using transcriptomic analysis of methylated RNA, we will determine how the male and female programs are affected by lack of m6A methylation. We will then use genetic engineering to prevent methylation of critical genes to demonstrate the role of this mechanism in regulating sex determination in mice. This work will reveal unexplored aspects of sexual differentiation and lay the foundations for new diagnoses and better medical management for the 50% of patients affected by idiopathic differences in sex development.