A New Leydig Cell-Exclusive Cre Line Allows Lineage Tracing of Fetal and Adult Leydig Cell Populations in the Mouse

Leydig cells produce hormones that are required for male development, fertility, and health. Two Leydig cell populations produce these hormones but at different times during development: fetal Leydig cells, which are active during fetal life, and adult Leydig cells, which are functional postnatally. Historically, our ability to understand the origin and function of Leydig cells has been made difficult by the lack of genetic models to exclusively target these cells. Taking advantage of the Leydig cell-exclusive expression pattern of the Insl3 gene, we used a CRISPR/Cas9 gene-editing strategy to knock-in iCre recombinase into the mouse Insl3 locus. To demonstrate the Leydig cell-exclusive nature of our iCre line, lineage-tracing experiments were performed by crossing Insl3iCre mice with a Rosa26LoxSTOPLox-TdTomato reporter. iCre activity was restricted to male offspring. TdTomato fluorescence was detected both in fetal and adult Leydig cells and colocalized with CYP17A1, a classic Leydig cell marker. Prior to birth, fluorescence was observed in fetal Leydig cells beginning at embryonic day 13.0. Fluorescence was also detected in adult Leydig cells starting at postnatal day 5 and continuing to the mature testis. Fluorescence was not detected in any other fetal or adult tissue examined, except for the unexpected finding that the adrenal cortex contains some Insl3-expressing Leydig-like cells. Our Leydig cell-exclusive iCre line therefore constitutes an invaluable new tool to study not only the origin of Leydig cells but also to target genes that have been long-proposed to be important for the development and functioning of these critical endocrine cells.