Desert Hedgehog mediates stem Leydig cell differentiation through Ptch2/Gli1/Sf1 signaling axis.

Abstract

Desert Hedgehog (Dhh) mutations cause Leydig cell dysfunction, yet the mechanisms governing Leydig lineage commitment through Dhh-mediated receptor selectivity, transcriptional effector specificity, and steroidogenic coupling remain elusive. In this study, using CRISPR/Cas9-mediated gene knockout and stem Leydig cells (SLCs) transplantation, we identified a critical Dhh/Patched 2 (Ptch2)/Glioma-associated oncogene homolog 1 (Gli1)/steroidogenic factor 1 (Sf1) signaling axis essential for SLC differentiation in Nile tilapia (<i>Oreochromis niloticus</i>). Dhh deficiency resulted in defective adult Leydig cells and androgen insufficiency. Rescue experiments involving 11-ketotestosterone administration and a Dhh agonist treatment, combined with SLCs transplantation, demonstrated that Dhh regulates SLC differentiation, not survival. In vitro knockout of <i>ptch1</i> and <i>ptch2</i> in SLCs revealed that Ptch2 likely acts as the functional receptor for Dhh. This was further supported by in vivo genetic rescue experiments, where <i>ptch2</i> mutation did not impair testicular development, yet completely rescued the testicular defects in <i>dhh</i> mutants-consistent with Ptch2 acting as an inhibitory receptor whose loss alleviates Dhh pathway suppression. Luciferase assays in Gli-knockout SLCs demonstrated that Gli1 acts as the primary transcriptional effector and transactivates <i>sf1</i> expression. Additionally, functional transplantation assays confirmed that Sf1 is indispensable for SLC differentiation, as Sf1-overexpressing SLCs rescued differentiation, whereas <i>sf1</i>-mutant SLCs failed. Overall, our work delineates the Dhh-Ptch2-Gli1-Sf1 axis and provides fundamental insights into the endocrine regulation of Leydig cell lineage development.