Supplementary Material for: Primordial Germ Cell Migration and Histological and Molecular Characterization of Gonadal Differentiation in Pachón Cavefish <b><i>Astyanax mexicanus</i></b>

The genetic regulatory network governing vertebrate gonadal differentiation appears less conserved than previously thought. Here, we investigated the gonadal development of <i>Astyanax mexicanus</i> Pachón cavefish by looking at primordial germ cells (PGCs) migration and proliferation, gonad histology, and gene expression patterns. We showed that PGCs are first detected at the 80% epiboly stage and then reach the gonadal primordium at 1 day post-fertilization (dpf). However, in contrast to the generally described absence of PGCs proliferation during their migration phase, PGCs number in cavefish doubles between early neurula and 8–9 somites stages. Combining both gonadal histology and <i>vasa</i> (germ cell marker) expression patterns, we observed that ovarian and testicular differentiation occurs around 65 dpf in females and 90 dpf in males, respectively, with an important inter-individual variability. The expression patterns of <i>dmrt1</i>, <i>gsdf</i>, and <i>amh</i> revealed a conserved predominant male expression during cavefish gonadal development, but none of the ovarian differentiation genes, i. e., <i>foxl2a</i>, <i>cyp19a1a</i>, and <i>wnt4b</i> displayed an early sexually dimorphic expression, and surprisingly all these genes exhibited predominant expression in adult testes. Altogether, our results lay the foundation for further research on sex determination and differentiation in <i>A. mexicanus</i> and contribute to the emerging picture that the vertebrate sex differentiation downstream regulatory network is less conserved than previously thought, at least in teleost fishes.