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 <i>Trachemys scripta</i>. 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 <i>T. scripta</i>, where the supporting lineage expresses genes required for androgen synthesis. Evolutionary reconstructions show that this lineage derives from a <i>Pax2</i>-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 <i>Twist1 or Runx1</i>, 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.