SOX9 is a key transcription factor and Sertoli cell fate determinant responsible for the differentiation of the gonad into a testis during embryonic development. Human SOX9 mutations cause Disorders of Sex Development (DSD) in XX males (SOX9 duplications) and XY females (SOX9 mutations/deletions), however most DSD patients don’t receive a definitive genetic diagnosis, making clinical management challenging. We hypothesise that SOX9 target genes are candidate DSD genes.
To identify Sox9 target genes, we undertook RNAseq analysis on mouse Sox9 knock-out gonads from embryonic day E13.5, when Sox9 is ablated in an intact Sertoli cell environment. We also performed Sox9 ChIPseq on wildtype E13.5 mouse testes and E90 bovine testes. 240 genes were downregulated in the Sox9 knockout testes, thus activated by Sox9. 4293 Sox9 ChIPseq peaks were common to the mouse and bovine testis. Overlapping the RNAseq and conserved ChIPseq datasets identified 119 genes whose gonadal chromatin is bound by Sox9, and whose gene expression is upregulated by Sox9. Of these, 34 have an enriched expression in Sertoli Cells compared to the other cell types of the gonads. We are now elucidating the role of these genes in the development of the testis.
One of these genes is Nedd9, a scaffolding protein in the Cas family which is involved in many cellular processes including cell polarity and migration. By manipulating SOX9 levels in a human Sertoli Cell-like culture model, we have also shown that this regulation is likely relevant to humans too. Initial analyses of Nedd9 knockout embryos has revealed abnormal testis morphology, including a mis-located ceolomic blood vessel, a male-specific feature of gonadal vasculture.
These studies may reveal a previously unsuspected role of Sox9 in the development of the vasculature of the testis and may provide another candidate gene for screening of DSD patients.