Davidian A and Spradling AC (2025), Early female germline development in Xenopus la...

XB-ART-61577

Proc Natl Acad Sci U S A 2025 Nov 18;12246:e2522343122. doi: 10.1073/pnas.2522343122.

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

Davidian A, Spradling AC.

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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 nonmammalian 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 composed of stable microtubules forms a rosette-like connection between cystocytes and contains Golgi vesicles and endoplasmic reticulum, suggesting polarized trafficking. In contrast to previous claims, ~80% of 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-like cell turnover to produce relatively few oocytes argue that amphibian cysts have important functions in female gametogenesis.

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	Fig. 1. Development of cysts based on scRNAseq and telomere FISH. (A) UMAP visualization of germline scRNA-seq clusters from three juvenile Xenopus ovary replicates. Development progresses clockwise along an outer ring, from GSCs, to proliferating oogonia, premeiotic S-phase, leptotene, zygotene, pachytene, early diplotene primordial follicles, and stage I follicles. An inner ring comprises nurse-like populations (NC1) branching from meiotic cyst stages and NC2 and NC3 from follicles. (B) Dot plot comparing marker gene expression in indicated clusters. (C) Marker gene feature plots on UMAP coordinates. (D) Germline cyst development visualized by EdU labeling (1.5 h, magenta), telomere FISH (yellow), and DAPI (cyan). Premeiotic S-phase: telomeres cluster at the nuclear envelope, while “leading” cystocytes (red arrowhead) start bouquet formation. Leptotene–zygotene: telomere bouquet formation and rDNA amplification onset (magenta arrowheads). Pachytene: nuclear size increases, rDNA amplification forms an “rDNA cap.” Early diplotene: cysts fragment to form primordial follicles, telomere dispersal. Stage I follicles: large GV, LBC, telomere detachment.
	Fig. 2. GSC identification. (A–A”) Whole-mount juvenile Xenopus ovary. HCR RNA-FISH for piwil4.S (magenta), DAPI (cyan). (B) GSC electron microscopy reveals polymorphic nucleus (blue), mitochondrial cloud (white arrowhead), ER-like structures (yellow arrowhead). (C and C’) GSC: Vasa (red), DAPI (cyan). (D) GSC stained with PDI (red), phalloidin (green), and DAPI (gray). (E) GSC stained for mitochondria (citrate synthase, red), Golgi (GM130, yellow), and DAPI (gray). (F) Dot plot comparing expression patterns of selected marker genes for GSCs across different germ cell clusters. (G and G’) Whole-mount juvenile Xenopus ovary: two-color HCR RNA FISH for piwil4.S (magenta) and nefm.S (yellow), DAPI (cyan). (H and H’) Whole-mount juvenile Xenopus ovary: OPP (2 h, red), DAPI (cyan). (H’) Magnified rectangle in H: quiescent GSCs: white dashed outlines.
	Fig. 3. FLS within germline cysts. (A) Germline cyst: EdU (~2 h, magenta), Kif23 (ring canals, green) showing fragmentation into a 6-cell group and 2-cell group. (B) Juvenile ovary imaged live (~2 h) showing motile cystocytes (yellow dashes), Hoechst (gray). (C–E) 4-,8-16-cell cysts: acetylated microtubules (red), phalloidin (green), DAPI (cyan). (Scale bar, 10 µm.) (F) 32-cell cyst: EdU (cyan), acetylated microtubules (red), Kif23 (green). (G) 8-cell cyst topology: EdU (magenta), Kif23 (green), acetylated microtubules (red); cartoon: deduced connectivity. (H) Fragmented 16-cell cyst (14-cells, 2-cells): Kif23 (green), acetylated microtubules (red); Imaris 3D-reconstruction showing cyst nuclei, FLS (pink); cartoon: deduced interconnections.
	Fig. 4. FLS during germline cyst development. (A) Metaphase germline cysts (8-cell, 2-cell): α-tubulin (red). (B) 16-cell cyst during late telophase: α-tubulin (red) reveals 8 midbodies. (C) 8-cell cyst: midbodies elongating to span IBs without gaps. The dashed box is magnified in the upper right corner to show 2 cells (DAPI, gray) interconnecting by IB (green) and MT (red). (D) During interphase, oogonial cysts form FLS interconnecting all sister cells via microtubules: EdU (blue), acetylated microtubules (red), γ-tubulin (centrioles, cyan). (E–H) Juvenile ovaries: acetylated microtubules (red), γ-tubulin (cyan), EdU (green), DAPI (gray). Maximum projections of selected optical slices. (E) Premeiotic S-phase cyst: EdU (green), acetylated MT (red), shows FLS microtubules extending from MTOCs (γ-tubulin, cyan). (F) Reduced FLS structure in leptotene–zygotene cyst. (G) Pachytene-stage cyst with further reduced FLS. (H) Diplotene primordial follicle: MTs accumulating (vegetal pole) near forming Bb. (I) Stage I follicle with multiple nucleoli (fibrillarin, green) and forming LBC: acetylated MTs are enriched around GV.
	Fig. 5. Vesicles, ER, and mRNA, but not photoconvertible KikGR, move through ring canals. (A–A’’) Golgi-derived vesicles within IBs. (A) EM of IB (black dashed line) shows multiple vesicles (red arrowheads) within its lumen. The blue dashed box shows a high-magnification image of the vesicle in the upper-right corner. (A’ and A’’) Whole-mount ovary: acetylated tubulin (magenta), phalloidin (green), GM130 (red) reveals Golgi-derived vesicles localized along microtubules (arrowheads). (A’) The dashed box highlights the region at the arrow tip showing Golgi-derived vesicles in the IB. (B–B’’) ER-like structures within IBs. (B) EM shows ER-like structures (red arrowhead) within IB lumen (black dashed line). (B’ and B’’) Whole mount ovary: acetylated tubulin (magenta), phalloidin (green), PDI (red) shows ER colocalization with microtubules crossing IBs (arrowheads). (C–C’’) HCR RNA-FISH with a rec8 probe (green), acetylated tubulin (red), ring canals (Kif23, magenta), and DAPI (cyan). (C’ and C’’) Boxed region in C: showing rec8 mRNA enriched on microtubules in a cystocyte with four ring canals. (D–D’’’) HCR RNA-FISH with rec8 probe (green), acetylated tubulin (red), ring canals (Kif23, magenta), DAPI (cyan). (D’–D’’’) Boxed region in D showing rec8 RNA molecule located within ring canal lumen. (E–E’’’) Photoconversion of KikGR (green to red) within a single cyst cell. CellMask (cell membranes, magenta). Converted cell (white dashes). (E) Before conversion. (E’) ROI showing single cell conversion. (E’’) 1 h postconversion. (E’’’) 24 h postconversion. (F) Red signal intensity in neighboring cystocytes vs. an unconnected follicle (F). Y-axis: relative red intensity; X-axis: time. Sister cells 1 and 2 correspond to those in (E–E’’’); F (follicle) used for normalization.
	Fig. 6. Microtubule interconnections between cystocytes are actively maintained. Juvenile ovaries showing microtubule networks in oogonial and early meiotic cysts. (A) Control, DMSO-treated. (B) 0 h after nocodazole treatment—microtubules are reduced to centrioles, ring canals remain intact. Two boxed regions on the left are magnified in the panels on the right. (C) MT regrowth after nocodazole washout. The bottom-right panel in (C) shows another example of MT recovery in the IB. (D) Quantitation: bridges with microtubules (%) in control and recovery. Acetylated tubulin (red), Kif23 (green), EdU (1 h before recovery, magenta), DAPI (cyan). (Scale bar, 5 µm.)
	Fig. 7. Cysts contain mostly nurse cells that turn over. (A and A’) Juvenile ovary 4 d after EdU injection. Germline cells (Vasa, magenta), EdU (green). (A’) Manual quantification (Imaris: “Spot,” “Cell” functions). Boxes indicate germline cyst from (A) reconstructed (A’). (B and B’) Analysis as in (A), 33 d after EdU injection. (C) 18 d post–EdU injection. Dying EdU-positive (green) germline cells (dashed regions) in late pachytene/early diplotene cysts, (Vasa, magenta), (DAPI, gray). (D) Declining EdU-labeled oocyte numbers 2 to 57 d after EdU pulse. (E) Distribution: nCount_RNA (UMI counts) per cell in indicated germline clusters. (F) Extensive programmed cell death of germ cells visualized by acidified nuclear remnants (Lysotracker, red), (Vasa, green).