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.调控脊椎动物性腺分化的遗传调控网络，其保守性较此前认知更低。本研究以墨西哥丽脂鲤（Astyanax mexicanus）帕琼洞穴鱼为研究对象，通过观测原始生殖细胞（primordial germ cells，PGCs）的迁移与增殖、性腺组织学特征以及基因表达模式，对其性腺发育过程展开探究。研究发现，PGCs最早可在受精后80%外包期被检测到，并于受精后1天（days post-fertilization，dpf）迁移至性腺原基处。但与此前普遍报道的"PGCs在迁移阶段无增殖"的结论不同，本研究中洞穴鱼的PGCs数量在早期神经胚期至8~9体节期期间翻倍。结合性腺组织学特征与vasa（生殖细胞标记基因）的表达模式，本研究观察到：雌性个体的卵巢分化与雄性个体的精巢分化分别发生于65 dpf与90 dpf左右，且存在显著的个体间差异。dmrt1、gsdf与amh的表达模式显示，洞穴鱼性腺发育过程中，这三个基因均呈现保守的雄性优势表达特征；但包括foxl2a、cyp19a1a与wnt4b在内的卵巢分化基因，均未表现出早期性别二态性表达，且令人意外的是，这些基因在成年精巢中均呈现优势表达。综上，本研究结果为墨西哥丽脂鲤（A. mexicanus）的性别决定与分化相关后续研究奠定了基础，同时也为"脊椎动物性腺分化的下游调控网络保守性较此前认知更低"这一新兴观点提供了实证支持，至少在硬骨鱼类中是如此。