RHOX10 Drives Mouse Spermatogonial Stem Cell Establishment Through A Transcription Factor Signaling Cascade

Spermatogonial stem cells (SSCs) are essential for male fertility. Here, we report that mouse SSC generation is driven by a transcription factor (TF) cascade controlled by the homeobox protein, RHOX10, which acts by stimulating the differentiation of SSC precursors called pro-spermatogonia (ProSG). We identify genes regulated by RHOX10 in ProSG in vivo, and define direct RHOX10-target genes using several approaches, including a rapid temporal induction assay we design: iSLAMseq. Together, these approaches identify temporal waves of direct targets, as well as secondary-target genes. Many of the RHOX10-regulated genes encode proteins with known roles in SSCs. Using an in vitro ProSG differentiation assay that we develop, we find that RHOX10 promotes mouse ProSG differentiation through a conserved transcriptional cascade involving the key germ-cell TFs DMRT1 and ZBTB16. Our study gives important insights into germ cell development and provides a blueprint for how to define TF cascades. RNAseq: Mice were crossed with OCT4-eGFP mice. eGFP+ cells were sorted by fluorescence-activated cell sorting (FACS). For each sample analyzed, testes from 3 individuals were pooled. Four replicate samples were analyzed per genotype. CUT&Tag: Mouse germline stem (GS) cells were dissociated from neonatal testes. Endogenous RHOX10 was knocked-down using an shRNA lentivirus and purified on the basis of GFP fluorescence by FACS. A HA-tagged Rhox10 lentivirus (unsensitive to this shRNA; expresses mCHERRY reporter) was used to induce a control level of exogenous Rhox10 expression. 1 million GS cells were used per sample and three biological replicates were performed. The negative IgG controls were performed using Rhox10-kd;HA-tagged Rhox10 GS cells. Samples from Rhox10-kd; HA-tagged empty vector GS cells served as another control (Ctrl). iSLAMseq: Endogenous Rhox10 in mouse GS was knocked-down using an shRNA lentivirus and purified on the basis of GFP fluorescence by FACS. A lentivirus expression vector carrying the Rhox10-ERT2-mCherry fusion gene was used to make a conditional inducible Rhox10 expression in response to 4-Hydroxytamoxifen. 2 millions GS cells were prepared for each sample, six time points with two biological replicates were performed. Samples from Rhox10-kd; ERT2-mCherry empty vector served as the control.