Early female germline development in Xenopus laevis: Stem cells, nurse cells and germline cysts

In Drosophila, germline cysts arise through synchronous mitotic divisions and acquire a polarized architecture organized by the fusome, which guides oocyte specification and supports meiotic progression. Similar cyst structures exist in non-mammalian vertebrate ovaries, but their polarity and function have remained uncertain. Using single-cell RNA sequencing and high-resolution imaging, we reconstructed the germ cell differentiation trajectory in Xenopus laevis and uncovered striking parallels with invertebrate and mouse cyst development. We identified a distinct germline stem cell (GSC) population marked by piwil4, low translational activity, and expression of neuronal-specific and transposon-silencing genes. Downstream from GSCs, during cyst development, an asymmetric fusome-like structure (FLS) composed of stable microtubules forms a rosette-like connection between cystocytes and co-localizes with Golgi vesicles and ER, suggesting polarized trafficking. In contrast to previous claims, ~80% of EdU-prelabeled cyst cells turned over rather than forming oocytes, consistent with a nurse cell fate. The striking parallels described here between cyst and fusome formation, polarization, cyst breakdown, and nurse cell turn over to produce relatively few oocytes, argue that amphibian cysts have important functions in female gametogenesis. Overall design: We performed single-cell RNA sequencing on juvenile Xenopus laevis ovaries collected at developmental stages NF62–66 to characterize germline populations during early ovarian development. Dissected ovaries were enzymatically dissociated, and approximately 10,000 viable cells per sample were loaded onto the 10X Genomics Chromium platform. Libraries were generated from three biological replicates and sequenced on an Illumina NextSeq 500. Raw reads were processed using Cell Ranger v8.0.1, and the resulting count matrices were used for downstream analysis.