Gonadal development and gene expression in the leopard gecko during temperature-dependent sex determination

Temperature-dependent sex determination (TSD) is prevalent in reptiles, yet its molecular mechanisms, particularly in squamates, remain poorly understood. This study presents the first comprehensive histological and transcriptomic analysis of gonadal development in the leopard gecko (Eublepharis macularius), a squamate with a TSD system. Based on temperature-shift experiments, we determined the end of the temperature-sensitive period at embryonic stage 36 (St. 36). Our transcriptomic analysis revealed that the male and female developmental pathways diverge at St. 34, preceding any histological differentiation. We found that the upregulation of important testicular genes (e.g., AMH, DMRT1, and SOX9) precedes that of canonical ovarian markers (e.g., FOXL2 and CYP19A1), although activation of Wnt signaling components was observed from early stages at female-producing temperatures. We also uncovered a regulatory pattern unique to the leopard gecko. Unlike in turtles, the histone demethylase KDM6B, a key male-determining factor in turtles, was activated by warm (i.e., male-producing) temperatures in the leopard gecko, but its expression was diminished with the later upregulation of DMRT1. Furthermore, we identified genes related to RNA splicing among the early temperature-responsive factors, suggesting a potential role for post-transcriptional regulation in the TSD cascade. Our findings demonstrate that while the core gene network for gonadal differentiation is conserved, the upstream thermosensitive regulation has diversified across TSD reptiles. This work addresses a critical phylogenetic gap in TSD studies and establishes the leopard gecko as a useful model for understanding the complex interplay between environment and gonadal developmental fate.