Developmental Biology

Mouse germline cysts contain a fusome that mediates oocyte development

Madhulika Pathak
Allan C Spradling author has email address

Howard Hughes Medical Institute Research Laboratories Carnegie Institution for Science, Baltimore, United States

https://doi.org/10.7554/eLife.109358.1

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Figures and data

Mouse pre-meiotic PGCs contain a fusome-like structure, “Visham.”
A. E9.5-E12.5 ovaries: EMA, DAPI. Box: EMA granule (triangle) in germ cells. B. E11.5 ovary: lineage-labeled clones (YFP), DDX4. B′. 2-cell cyst. EMA granules (boxed). Graph: EMA volume asymmetry N=16. B″. 4-cell cyst: EMA granules (triangles). Graph: EMA volume asymmetry N=18; Student’s t-test, ***p<0.001. C. E13.5 ovary and testis (C’): GCNA, EMA. Visham: (dotted line); Graph: % cysts with rosette Visham (N=26; ***p<0.001). D. E13.5 YFP-marked cyst: EMA, TEX14. D’ (boxed region). Graph: Ring canal number vs. Visham enrichment (≥10 μm³). (N=54; ***p<0.001). E-E″. EM images of rosette Visham spanning intercellular bridges: E14.5 (E, E’’); EMA granule at E11.5 (E’). F. E11.5 ovary: GM130, EMA. F’. Rab11a1, EMA. G. E11.5 ovary: WGA, EMA; G’. E13.5 ovary: GCNA, WGA labels rosette Visham. (H) Schematic: Visham transformation from granular to rosette structure. (I) % germ cells with EMA structure during E9.5-E14.5 (N=15 per stage; ***p<0.001). Scale bars: 5μm (A, F′), 10μm (B-B″, C-C′, D′, F, G-G′), 20μm (D), 2μm (E).

Stabilized spindle microtubules mediate Visham asymmetry and cyst breakage.
A. Visham interphase pericentric localization. E11.5 germ cells: EMA, Pericentrin. A′. Summary. B. Cell cycle behavior of EMA during initial 2-cell cyst formation. B’. Summary diagrams. C-D. Asymmetric Visham segregation (E10.5). C. Symmetric telophase separation. D. Asymmetry at arrested cytokinesis. E-E’’. Persistent spindle remnants presage cyst breakage. Three lineage-marked (YFP) E12.5 8-cell cysts (E-E’’) in early interphase stained to reveal Visham and AcTub. MT channel alone shown in middle column. Diagrams at right suggest sites of breakage (dashed line). The E’’ cyst has already broken into 2-cell and 6-cell cyst derivatives. F. Cell number distribution of cyst breakage products predicted from 15 lineage-labeled cysts analyzed as in E (7-cell: 3; 8-cell: 8; 9-cell:1; 10-cell:3). Binomial test: 6-cell dominance in 13/15 cysts (****p<0.0001). G. Model of PGC cyst production and breakage into four 6-cell cysts and 4 2-cell cysts. Scale bars: 5μm (B), 10μm (A, C-E).

Visham associates with Pard3 and apical polarity.
A-B. Pard3 overlaps Visham at E11.5-E12.5 (A, A’). and after rosette formation at E13.5 (B, B’). C-C’. Ring canals (RACGAP) localize within the Pard3+ apical domain. D. A lineage-labeled E13.5 cyst (YFP); channels below show overlap of Pard3 and Visham. (N=13; ***p<0.001). E. Xbp1 enrichment in EMA granule of E11.5 PGC. F-H. scRNAseq of E10.5-P5 germ cells. UMAP (F), UMI (G) NC =nurse cells. Summary (H): pre-meiotic (Pre-M), leptotene (Lp), zygotene (Zy), pachytene (Pa), diplotene (Dp), dictyate (Dc). (I-I′) Bar plots: Xbp1and target expression plots. I.’ Targets orthologous to fusome components. Scale bars: 10μm (A-C), 20μm (D).

UPR genes are active during cyst formation and controlled by Dazl.
(A) Dnmt3a and EMA levels at E12.5 are reduced in WT compared to Dazl^-/- germ cells. (B) Ring canals ar smaller and defective in E13.5 Dazl^-/-cysts compared to WT. (N=44; **p<0.05). (C) Diverging expression o WT and Dazl^-/- germ cells in UMAP plots. (C’) Xbp1, Xbp1 targets, and fusome orthologs in WT vs Dazl^-/- germ cells. (D-D″) IRE1-Xbp1 assay comparing SSEA1+ germ vs SSEA1− somatic cells at E11.5 and WT vs Dazl^-/- germ cells at E12.5. (E-E″) Proteasome activity comparing SSEA1+ germ vs SSEA1− somatic cells at E11.5 and WT vs Dazl^-/- germ cells at E12.5. (F) Golgi fragmentation in E12.5 Dazl^-/- germ cells. (F’) Failure of E13.5 Dazl^-/- germ cells to form EMA rosettes or enrich Pard3. G. Dazl^-/- effects. H. ERAD-UPR proteostasis. Scale bar: 10μm (except zoomed in 2μm).

Visham and Pard3 Associate with ER and mitochondria prior to Balbiani body formation.
A-A′. E17.5 ovary stained for WGA, GCNA, and TEX14 ; Graph: Visham volume versus ring canal numbe (N=65; ANOVA, *p<0.1, ***p<0.001). B-B′. E18.5 ovary shows Visham enrichment in large medullary oocytes vs smaller nurse cells; graph compares Visham volume versus Germ cell nucleus diameter (N=54; **p<0.01). line: medulla/cortex boundary; dotted circle: large medullary oocytes; white dotted area: small nurse cells. C. Single-cell lineage-labeled E18.5 ovary shows Visham volume difference according to germ cell nucleus size (N=10; ****p<0.0001). D. Pard3 enrichment along Visham proportional to number of ring canals. E-E’. Pard3 enrichment within newly arising medullary Oocytes (Big cells, GCNA) along with Visham (WGA). F. Lineage-labeled cells (YFP): Pard3 enrichment versus germ cell (GCNA) nucleus size. G-G′. Dazl+/-E18.5 ovary-Visham (WGA) and Pard3 enrichment failure in medullary Oocytes (GCNA). H-H″. Organelle enrichment analysis: E18.5 (WT, H and Dazl+/-ovary, H”) stained for WGA, mitochondrial marker ATP5a and GCNA. H’ -EM image of Golgi-rich Visham (arrow) surrounded by mitochondria. I-I′. ER-mitochondria association in E18.5 ovary: I-EM image of ER tubules (arrow) wrapping mitochondria and I’-GCNA, ER and Mitochondria tracker staining. Scale bars: 20μm (A-F, G-G′ H, I′), 5μm (B′, E′), 0.5μm (EM images H′, I).

Primer details.

EMA/Lectin-stained aggregate (Visham) distribution in pre-meiotic PGCs
(A) Immunostaining of E9.5-E12.5 ovary for germ cell specific marker EMA (Red) and DAPI (Blue) and for E13.5 ovary (EMA in Red, GCNA in green, Z-stack video see Video S1A). (B) Z-stack video of E12.5 ovary stained for EMA (Red), DAPI (Blue) and lineage labelling shown via YFP (Green) (See Video S1B) (C) Volume rendering of EMA aggregate (White arrows) within lineage labelled cyst (Green, YFP) using Imaris software. (D-D’) Videos of at E13.5 ovary stained for EMA (Red), GCNA (Green) and DAPI (Blue) (see Video S1D1 and S1D2) (E) Random 3-D sampling using Imaris for E13.5 ovary showing branched Visham structure within GCNA labelled female germ cells. (F) Images of different E13.5 ovary stained for EMA (Red), GCNA (Green), Tex14 (Yellow) and DAPI (Blue). Central region with enriched Visham is often associated with higher number of ring canals. (G) E11.5 ovary stained for WGA (Red) and YFP (Green) as lineage labelling marker. Dotted line marks the WGA aggregate within germ cells. (G’) E13.5 ovary stained for WGA (Red), GCNA (Green) and Tex14 (yellow) forming branched WGA-stained Visham structure (dotted lines). (H and H’) E11.5 gonad stained for general fucosylation specific lectins, AAL/LCA (Green), DAPI (Blue) and Visham marker EMA (Red). (I and I’) Localization of ER markers in vicinity to Visham is validated by staining ER-specific Sec63 or Calnexin (Green), DAPI (Blue) and EMA (Red). (K) Electron microscopy of E11.5 gonad depicting Golgi clusters (Dotted red lines, labelled as Visham) near intercellular bridges (IB marked by red solid arrows). Scale bar. 100 μm (A), 20 μm (B), 10 μm (in D-H).

EMA/Lectin-stained aggregate (Visham) distribution in pre-meiotic PGCs
(A) Immunostaining of E9.5-E12.5 ovary for germ cell specific marker EMA (Red) and DAPI (Blue) and for E13.5 ovary (EMA in Red, GCNA in green, Z-stack video see Video S1A). (B) Z-stack video of E12.5 ovary stained for EMA (Red), DAPI (Blue) and lineage labelling shown via YFP (Green) (See Video S1B) (C) Volume rendering of EMA aggregate (White arrows) within lineage labelled cyst (Green, YFP) using Imaris software. (D-D’) Videos of at E13.5 ovary stained for EMA (Red), GCNA (Green) and DAPI (Blue) (see Video S1D1 and S1D2) (E) Random 3-D sampling using Imaris for E13.5 ovary showing branched Visham structure within GCNA labelled female germ cells. (F) Images of different E13.5 ovary stained for EMA (Red), GCNA (Green), Tex14 (Yellow) and DAPI (Blue). Central region with enriched Visham is often associated with higher number of ring canals. (G) E11.5 ovary stained for WGA (Red) and YFP (Green) as lineage labelling marker. Dotted line marks the WGA aggregate within germ cells. (G’) E13.5 ovary stained for WGA (Red), GCNA (Green) and Tex14 (yellow) forming branched WGA-stained Visham structure (dotted lines). (H and H’) E11.5 gonad stained for general fucosylation specific lectins, AAL/LCA (Green), DAPI (Blue) and Visham marker EMA (Red). (I and I’) Localization of ER markers in vicinity to Visham is validated by staining ER-specific Sec63 or Calnexin (Green), DAPI (Blue) and EMA (Red). (K) Electron microscopy of E11.5 gonad depicting Golgi clusters (Dotted red lines, labelled as Visham) near intercellular bridges (IB marked by red solid arrows). Scale bar. 100 μm (A), 20 μm (B), 10 μm (in D-H).

Validating microtubule dependent Visham formation and its distribution during cyst fragmentation.
(A) Z-stack videos for images used in figure 3A (See Videos from S2A1 to S2A5). (B) E11.5-E12.5 ovary stained for EMA (Red), Pericentrin (PCNT, Green), Ac. Tub (Grey) and DAPI (Blue). Arrows mark duplicated centrosome during Interphase. Dotted circles mark centrosomes. Smooth line marks the DAPI stained region during Anaphase depicting clear separation of Nuclei. (C) E10.5 gonad stained for EMA (Red) and Ac. Tub. (Green) Dotted line marks the newly formed fusome as spindle remnant. (D) Visham asymmetry arises during cytokinesis depicted in E11.5 ovary stained for EMA (Red) (E) In-vitro cultured untreated gonad and treated gonad (microtubule inhibitor Ciliobrevin D and cold treatment) stained for Ac. Tub (Green), EMA (Red) and DAPI (Blue). Quantification showing % germ cells with Visham in Untreated versus Celioberivin D/Cold treated E11.5 gonad. (F) Lineage labelled 4-cell cyst stained at E12.5 for YFP (Green), EMA (Red) and Ac. Tub (Grey). (F’) 3D modelling of lineage labelled cyst at E12.5. Video depicts animation video generated using Imaris software to create uniform spheres in green to position the labelled 8-cells within cyst and surface rendering of Visham in White within lineage labelled germ cells (See Video S2F). Scale bar: 5 μm (A-B), 10 μm (C-F).

Pard3 gene expression in E12.5-E13.5 gonad
(A) Z-stack video of E12.5 gonad stained for EMA (Green), PARD3 (Red) and DAPI (Blue) (See Video S3A). (B) Z-stack video of E13.5 ovary (♀, See Video S3B1) and E13.5 testis (♂, See Video S3B2) stained for EMA (Green), PARD3 (Red) and GCNA (Blue). (C) E13.5 ovaries stained for GCNA (Blue), EMA (Green) and PARD3 (Red). (D) Lineage labelled E13.5 ovary stained for YFP (Green), GCNA (Blue), PARD3 (Red) and EMA (Grey). (E) Quinacrine (Male chromosome specific stain, Red) and DAPI (Green) staining of male vs female fetal tail samples (F) Feature plot and UMAP plot depicting Xist expression within E10.5 and E11.5 gonad followed by bioinformatic segregation of female gonadal germ cells (XX only) to avoid Quinacrine false-negative by segregating the cells expressing significant amount of XX specific genes (eg: Xist, Ddx3x, Utx) depicted in UMAP plot as XX only cells. (G) Cluster of germ cells from E12, E14, E16, E18, P1 and P5 of previously published data (NCBI: Niu and Spradling, 2020) were used. E10, E11 and E15 (10X genomics) ScRNA seq was performed and germ cells isolated bioinformatically to create E10.5-P5 merged germ cell data to comprehensively look at gene expression across developmental stages. The number of cells used to create merged dataset is shown. (H) Feature plot validating all the cells express germ cell specific markers (Dppa3/DDx4) (I) Pre-meiotic/Meiotic gene expression analysis depicted by feature plot for various developmental stages within merged dataset. (J) Stack violin plot depicting Golgi-UPR pathway associated gene expression pattern across E10.5 to P5 meiotic germ cell and nurse cell developmental stages. Scale bar= 20 μm (A,B-B, D) and 10 μm (C), 100 μm (E).

Pard3 gene expression in E12.5-E13.5 gonad
(A) Z-stack video of E12.5 gonad stained for EMA (Green), PARD3 (Red) and DAPI (Blue) (See Video S3A). (B) Z-stack video of E13.5 ovary (♀, See Video S3B1) and E13.5 testis (♂, See Video S3B2) stained for EMA (Green), PARD3 (Red) and GCNA (Blue). (C) E13.5 ovaries stained for GCNA (Blue), EMA (Green) and PARD3 (Red). (D) Lineage labelled E13.5 ovary stained for YFP (Green), GCNA (Blue), PARD3 (Red) and EMA (Grey). (E) Quinacrine (Male chromosome specific stain, Red) and DAPI (Green) staining of male vs female fetal tail samples (F) Feature plot and UMAP plot depicting Xist expression within E10.5 and E11.5 gonad followed by bioinformatic segregation of female gonadal germ cells (XX only) to avoid Quinacrine false-negative by segregating the cells expressing significant amount of XX specific genes (eg: Xist, Ddx3x, Utx) depicted in UMAP plot as XX only cells. (G) Cluster of germ cells from E12, E14, E16, E18, P1 and P5 of previously published data (NCBI: Niu and Spradling, 2020) were used. E10, E11 and E15 (10X genomics) ScRNA seq was performed and germ cells isolated bioinformatically to create E10.5-P5 merged germ cell data to comprehensively look at gene expression across developmental stages. The number of cells used to create merged dataset is shown. (H) Feature plot validating all the cells express germ cell specific markers (Dppa3/DDx4) (I) Pre-meiotic/Meiotic gene expression analysis depicted by feature plot for various developmental stages within merged dataset. (J) Stack violin plot depicting Golgi-UPR pathway associated gene expression pattern across E10.5 to P5 meiotic germ cell and nurse cell developmental stages. Scale bar= 20 μm (A,B-B, D) and 10 μm (C), 100 μm (E).

Validation of female Dazl mutant phenotype, MACS and Activity assays.
(A) Expected absence of Dazl protein in Dazl mutant gonad is shown by staining of E18.5 WT and Dazl-/-gonad for Dazl (Red), GCNA (Green) and DAPI (Grey). (B) E12.5 WT and Dazl^-/- gonad stained for DNMT3a (Red), EMA (Green) and DAPI (Grey) (C) Fetal tail genotyping for ScRNA sequencing: Gel electrophoresis showing the standard Dazl genotyping PCR assay by Jackson (Stock No: 035880 Protocol 40585) with expected results: Wild type (192 bp), Heterozygous mutant (300 and 192 bp) and Homozygous mutant (300 bp). (D) Feature plot depicting positive Xist Expression in ScRNA seq data of E11.5 and E12.5 Dazl mutant fetal gonad, thus concluding them as female samples. (E) Stackviolin Plot depicting pluripotency gene expression pattern in E11.5-E12.5 WT vs Dazl-/-gonad. (F) Stackviolin Plot depicting Xbp1 targets gene expression pattern in E1.5 WT vs Dazl-/-gonad (G) Staining of both unbound SSEA1(-ve) and the bound fraction consisting of SSEA1(+ve) cells for DDX4 (Magenta) and DAPI (Grey). (H) Gel electrophoresis showing the PCR amplification of housekeeping gene GAPDH and Germ cell specific marker DDX4 in SSEA1(-ve) and SSEA1 (+ve) germ cells from E11.5 and E12.5 ovary. The amplicon size is indicated on left. (I) Proof of functioning of Xbp1 assay is shown by staining of both mixture of SSEA1(-ve) and SSEA1(+ve) cells for GCNA (Green), DAPI (Grey) and Xbp1(Red) (J) Proof of function of proteasome activity assay using Trypsin as technical positive control. The proteasome activity assay was shown for two different assays (I and II) with technical duplicates at three different Trypsin concentrations. Mean fluorescent intensity in arbitrary units is shown obtained using plate reader. Scale bar: 20 μm (A), 10 μm (B), 100 μm (G and I).

Visham enrichment within destined medullary Oocytes in WT vs Dazl mutant.
(A-B) Zoomed out E17.5 ovary covering large span of tissue stained with DAPI (Blue), WGA (Red), GCNA (Green) and Tex14 (Yellow). White dotted line is zoomed in and shown separately in main figure (Refer to main Fig 5A and A’). (C) Video created using Imari software showing E17.5 ovary stained for GCNA (Green), Tex14 (Yellow) and WGA (Red) followed by surface rendering of WGA aggregate (continuous one) associated with ring canals. (See Video S5C) (D) Video of E18.5 female gonad stained with DAPI (Blue), GCNA (Green) and WGA (Red) depicts the cortex and medullary region where medullary region showing distinct enriched WGA aggregate compared to surrounding small nurse cells. (See Video S5D) (D’) One of the z-stack form E18.5 ovary Video in E stained with DAPI, GCNA and WGA depicts the medullary big Oocyte-like cells showing distinct enriched WGA aggregate compared to surrounding small nurse cells. E) Z-stack video of E18.5 WT, Dazl +/-and Dazl-/-gonad stained for GCNA Green) and WGA (Red). (See Videos S5E1 to S5E3). (F) Z-stack video of P0 WT, Dazl +/- and Dazl-/- gonad stained for GCNA(Green) and PARD3 (Red). (See S5 F1 to F3) (G) E18.5 WT and Dazl +/- gonad stained for Mitotracker (Red) and GCNA (Green). Scale bar: 20 μm (A-B and G), 50 μm (D-E), 100 μm (F).

Visham enrichment within destined medullary Oocytes in WT vs Dazl mutant.
(A-B) Zoomed out E17.5 ovary covering large span of tissue stained with DAPI (Blue), WGA (Red), GCNA (Green) and Tex14 (Yellow). White dotted line is zoomed in and shown separately in main figure (Refer to main Fig 5A and A’). (C) Video created using Imari software showing E17.5 ovary stained for GCNA (Green), Tex14 (Yellow) and WGA (Red) followed by surface rendering of WGA aggregate (continuous one) associated with ring canals. (See Video S5C) (D) Video of E18.5 female gonad stained with DAPI (Blue), GCNA (Green) and WGA (Red) depicts the cortex and medullary region where medullary region showing distinct enriched WGA aggregate compared to surrounding small nurse cells. (See Video S5D) (D’) One of the z-stack form E18.5 ovary Video in E stained with DAPI, GCNA and WGA depicts the medullary big Oocyte-like cells showing distinct enriched WGA aggregate compared to surrounding small nurse cells. E) Z-stack video of E18.5 WT, Dazl +/-and Dazl-/-gonad stained for GCNA Green) and WGA (Red). (See Videos S5E1 to S5E3). (F) Z-stack video of P0 WT, Dazl +/- and Dazl-/- gonad stained for GCNA(Green) and PARD3 (Red). (See S5 F1 to F3) (G) E18.5 WT and Dazl +/- gonad stained for Mitotracker (Red) and GCNA (Green). Scale bar: 20 μm (A-B and G), 50 μm (D-E), 100 μm (F).

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