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, many DSD patients do not receive a definitive genetic diagnosis. SOX9 regulates the expression of thousands of genes in the developing testis but a few of these genes have been studied and shown to be essential for the proper development of the testis. To identify SOX9 target genes, we previously undertook RNA-seq analysis on mouse Sox9 knock-out gonads from E13.5, to ablate SOX9 in an intact Sertoli cell environment and SOX9 ChIP-seq on wildtype E13.5 mouse testes. Overlapping the RNA-seq and conserved ChIP-seq datasets revealed 63 genes whose gonadal chromatin is bound by SOX9, and whose gene expression is upregulated by SOX9. Of these, 35 genes show enriched expression specifically in Sertoli cells. These include Amh, Sox9, Sox10 and Dhh - already known SOX9 targets, thereby validating the approach.
Here we analyse some of the 31 genes regulated by SOX9 in order to elucidate a potential role in testis differentiation and DSD. We developed an in vitro cellular model to evaluate candidate genes more rapidly by measuring cell proliferation, cell adhesion and/or polarity in the embryonic Sertoli cell line, NT2/D1, which are reduced by SOX9 siRNA treatment. Based on the availability of targeted inhibitors, Tyro3 and Trpc3 were chosen for further analysis. TYRO3 is a receptor tyrosine kinase and TRPC3 forms a non-selective calcium channel. TYRO3 or TRPC3 inhibitors reduced cell proliferation of NT2/D1 cells, while TYRO3 inhibitor also reduced cell adhesion. In cultured XY mouse gonads, both inhibitors showed disrupted cord development. Our study suggests that SOX9 divides its labour amongst its target genes.