Evidence of Sertoli cell lineage contribution to the rete testis population during embryonic development.

The rete testis (RT) develops at the gonad-mesonephros interface and links seminiferous tubules to efferent ductules, but the cellular origins of RT cells and the mechanisms driving RT expansion during embryonic development remain incompletely resolved. Here, to test whether gonadal Sertoli cell (SC) lineage cells contribute to the developing RT, we applied a chimeric organotypic culture approach combining gonadal and mesonephric compartments of GFP+ and GFP-mouse embryonic urogenital complexes of embryonic day 12.5 (E12.5). The gonadal compartments were dissected to be devoid of PAX8+ RT cells, whereas the mesonephric compartments contained distal RT structures adjacent to mesonephric tubules. After 3 days of culture, newly formed PAX8+ RT cells and continuous RT-like structures were detected in the gonadal compartments. A subset of these PAX8+ cells was localized among SCs within testis cords and co-expressed the SC marker AMH, suggesting that the SC lineage may contribute to the forming RT. When larger portions of the distal RT were retained in the mesonephric compartments, the numbers of newly formed PAX8+ cells in the gonadal compartments increased, indicating that local signaling plays a role in RT formation. In parallel, we reanalyzed a published single-cell RNA sequencing dataset of embryonic mouse testes by focused reclustering of the male supporting lineage (pre-Sertoli, SC, and RT populations), followed by RNA velocity and ligand-receptor-based inference of intercellular communication. Transcriptomic reanalysis identified C5 and SC-Lhx9 clusters with mixed pre-Sertoli, SC, and RT-associated marker profiles. RNA velocity predicted transitions from these clusters toward the RT lineage, and ligand-receptor inference indicated active signaling interactions between these clusters and the RT. In addition, quantitative analysis of RT expansion on E12.5-E16.5 showed that proliferation of resident RT cells alone cannot account for the observed increase in RT cell numbers, which supports the hypothesis that additional cells were recruited. Together, these data support a model in which a subset of proximal RT cells adjacent to testis cords may arise from the SC lineage, and proximal RT formation is promoted by signals from pre-existing distal RT structures. This refines current models of RT morphogenesis and clarifies cellular plasticity at the SC-RT interface during testis development.