Gonadal gene expression in the leopard gecko
Temperature-dependent sex determination (TSD) is a prevalent process 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 pure TSD system. Through temperature-shift experiments, we precisely defined the end of the temperature-sensitive period at embryonic stage 36 (St. 36). Critically, our transcriptomic data reveal that the male and female developmental pathways diverge at St. 34, significantly preceding any histological differentiation. We found that the upregulation of key testicular genes (e.g., AMH, DMRT1, and SOX9) precedes that of canonical ovarian markers (e.g., FOXL2 and CYP19A1), although an early ovarian response is evident through the activation of Wnt signaling components. We also uncovered a unique regulatory pattern for the histone demethylase KDM6B, a key male-determining factor in turtles. Unlike in turtles, KDM6B in the leopard gecko is activated by warm (male-producing) temperatures, and its expression is transient and decouples from the later upregulation of DMRT1. Furthermore, we identified genes related to RNA splicing among the earliest 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 throughout evolution of TSD reptiles. This work addresses a critical phylogenetic gap in TSD research and establishes the leopard gecko as a powerful model for understanding the complex interplay between environment and gonadal developmental fate.