Flexibility of cell fates and functions across sex determination systems revealed by comparative single-cell analyses.

Sex determination in vertebrates can be initiated by a wide range of genetic or environmental triggers. Yet, the degree to which gonadal cell types and genetic programs are conserved remains unresolved. Here we employed single-cell transcriptomics to characterize the temperature-dependent sex determination (TSD) program in gonads from the turtle species Trachemys scripta. Comparative analyses against species with genetic sex determination, like mouse (XY) and chicken (ZW), revealed a marked divergence in cell type repertoires and functions during vertebrate evolution. Unlike mammals, fetal Leydig cells are absent from the early gonads of T. scripta, where the supporting lineage expresses genes required for androgen synthesis. Evolutionary reconstructions show that this lineage derives from a Pax2-positive mesenchymal population, suggesting an ancestral condition in Archelosauria that differs from the primarily coelomic epithelium origin in the mammalian clade. Transcriptional dynamics and co-expression analyses revealed the recruitment of lineage-specific transcription factors, including Twist1 or Runx1, into the genetic programs of vertebrate clades. Our findings reveal extensive plasticity of the cellular and genetic mechanisms of vertebrate sex determination and suggest that this flexibility is a key feature of gonadal evolution.