SOX9 is a key transcription factor responsible for the differentiation of the gonad into a testis during embryonic development. SOX9 turns on hundreds of genes but few have been studied and shown to be necessary. Utilising RNA-seq dataset on mouse Sox9 knock-out gonads and SOX9 ChIP-seq datasets we identified a set of genes directly upregulated by SOX9 (Rahmoun et al. 2017, NAR). 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 analyseTyro3, as candidate SOX9 target gene with a potential role in one or more cellular processes during testis differentiation. The human EC Sertoli-like cell line, NT2/D1 expresses SOX9 together with its target gene AMH, thus validating its use as a model. We found by RT-PCR and IF that TYRO3 is expressed in NT2/D1 cells. In NT2 cells we established that SOX9 is required for cell adhesion and proliferation using xCELLigence. When NT2 cells were incubated with aTYRO3 inhibitor, increasing concentrations led to a dose-dependent reduction in cell proliferation but not cell adhesion.
To determine the role of TYRO3 in mouse gonad development, E11.5 XY gonads were cultured with TYRO3 inhibitor. While overall morphology is not impacted, the germ cell marker MVH is lost, with no apoptosis apparent.
Our study suggests that TYRO3 plays a role downstream of SOX9-mediated Sertoli cell proliferation and germ cell survival in the developing testis. Future rescue experiments are aimed at determining whether TYRO3 is the exclusive mediator of SOX9 driven Sertoli cell proliferation and germ cell survival.