Mouse germline cysts contain a fusome-like structure that mediates oocyte development
- Howard Hughes Medical Institute Research Laboratories, Carnegie Institution for Science, United States
Figures
Figure 1 with 9 supplements
Mouse pre-meiotic primordial germ cells (PGCs) contain a ‘fusome’.
(A) PGCs and early germline cysts from E9.5-E12.5 ovaries. EMA (red), DAPI (blue). Boxed regions magnified at right (R) (arrows, EMA granules). (B) EMA granule asymmetry in an E11.5 2-cell cyst: Yellow cells represent a lineage-labeled 2-cell cyst marked with both YFP (green, lineage) and DDX4 (red). (B′) Boxed region showing EMA granules (white triangles). Graph at R: EMA granule volumes consistently differ between daughter cells in 2-cell cysts. N=16. (B″) Varying volumes of daughter cells within E12.5 4-cell lineage-labeled cysts. EMA granules (white triangles), EMA (red). Graph at R: EMA volume asymmetry in 4-cell cyst, N=18; (C) Rosette formation in E13.5 ovary and E13.5 testis (C’). GCNA (germ cell nuclei, green), EMA (fusome, red; outline, dotted white). Graph at R: % of female (blue) and male (red) cysts with branched fusomes indicative of rosette formation (N=26 for each). (D) Ring canal abundance in fusome-enriched cells. A E13.5 lineage-labeled (YFP, green) cyst, fusome (EMA, red; outline, dotted white), and ring canals (TEX14, yellow). (D’) Zoomed image (boxed region in D) showing branched region with enriched fusome (white triangle) containing multiple ring canals. Graph at R: Ring canal number vs. fusome enrichment (≥10 μm³). N=54. (E-E″) EM of an E14.5 cyst in rosette configuration showing a Golgi-rich fusome spanning an intercellular bridge (E’’). (E') EM of an E11.5 PGC with a Golgi-enriched region (red outline) and likely EMA granule (compare to 1A-B). (F-F') E11.5 germ cells with EMA granules (EMA, red) co-stained with the Golgi markers F. (GM130, green) or F’ (Rab11a1, green). (G) Co-staining of Wheat germ agglutinin - WGA (red) and EMA (green) in E11.5 germ cells. (G’) WGA (red) staining of rosette fusome in E13.5 ovary: GCNA (nuclei, green). (H) Schematic of rosette formation in 4-cell cyst. (I) Plot showing EMA staining loss in germ cells after E13.5. (N=15 per stage). Student’s t-test was used for each graph in Figure 1. (***p<0.001). Scale bars: 5 μm (A, F, F′), 10 μm (B-B″, C-C′, D′, G-G′), 20 μm (D), 2 μm (E).
Figure 1—figure supplement 1
EMA/Lectin-stained aggregate (Mouse fusome) distribution in pre-meiotic primordial germ cells (PGCs).
(A) Immunostaining of E9.5-E12.5 ovary for germ cell specific marker EMA (Red) and DAPI (Blue) (B) Volume rendering of EMA aggregate (White arrows) within lineage labeled cyst (Green, YFP) using Imaris software. (C) Random 3-D sampling using Imaris for E13.5 ovary showing branched mouse fusome structure within germ cells GCNA (green) and EMA (red). (D) Representative image depicting sampling of E13.5 male gonad stained for EMA (Red), DAPI (Blue) and GCNA (Green). (E) Images of different E13.5 ovary stained for EMA (Red), GCNA (Green), Tex14 (Yellow), and DAPI (Blue). The central region with enriched fusome is often associated with higher number of ring canals. (F) E11.5 ovary stained for WGA (Red) and YFP (Green) as lineage labeling 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 Mouse Fusome structure (dotted lines). Scale bar. 100 μm (A), 20 μm (B), 10 μm (D–F).
Figure 1—figure supplement 2
EMA/Lectin-stained aggregate (Mouse fusome) distribution in pre-meiotic primordial germ cells (PGCs).
(H) Co-localization of Golgi and fusome. E11.5 gonad stained for Golgi markers Gm-130 /Gs28 (green) along with EMA (red) and DAPI (blue). (I-I’) Brefieldin A (BFA; Golgi inhibitor) treatment of gonad in vitro and recovery without Brefieldin A is shown. (I) Images for Untreated/BFA treated/Recovered E11.5 gonad stained for DDX4 (red), Gm-130 (green) and DAPI (blue) validating successful reversible inhibition of Golgi formation by BFA I’ is gonad stained for EMA (red), Gm-130 (green), and DAPI (blue) depicting Golgi-dependent formation of fusome. (J and J’) E11.5 gonad stained for general fucosylation specific lectins, AAL/LCA (green), DAPI (blue), and fusome marker EMA (red). (K and K’) Localization of endoplasmic reticulum (ER) markers in vicinity to fusome is validated by staining ER-specific Sec63 or Calnexin (green), DAPI (blue), and EMA (red). (L) Electron microscopy of E11.5 gonad depicting Golgi clusters (Dotted red lines, labeled as Fusome) near intercellular bridges (IB marked by red solid arrows). Scale bar. 10 μm (in H-K), 2 μm (L).
Figure 1—video 1
EMA staining disappearance from germ cell membrane in E13.5 ovary and acquiring a continuous branched appearance within the germline cyst.
E13.5 ovary stained with EMA (red), germ cell nuclear marker GCNA (green).
Figure 1—video 2
Successful single-cell lineage labeling of germ cells.
E12.5 ovary stained for EMA (red), DAPI (blue), and lineage labeling shown via YFP (green).
Figure 1—video 3
Visualization of branched central EMA-stained fusome.
E13.5 ovary stained for EMA (red), germ cell nuclear marker GCNA (green), and DAPI (blue).
Figure 1—video 4
Visualization of germ cell cluster specifically with branched fusome.
E13.5 ovary stained for EMA (red) and germ cell nuclear marker GCNA (green).
Figure 1—video 5
Successful Golgi formation within germ cells of in vitro cultured fetal gonads.
Untreated E11.5 gonads cultured for 6 hr show intact Golgi structures within DDX4+ germ cells. DDX4 (red), Gm-130 (green), and DAPI (blue).
Figure 1—video 6
Validation of Brefeldin A (BFA) effect on Golgi formation.
E11.5 gonads cultured for 6 hr in the presence of BFA display a complete loss of detectable Golgi structures, confirming the efficacy of BFA treatment. DDX4 (red), Gm-130 (green), and DAPI (blue).
Figure 1—video 7
Reversibility of Brefeldin A (BFA) treatment.
Following 6 hr of BFA exposure, withdrawal of the drug results in the successful restoration of Golgi structures post 24 h in E11.5 gonads, demonstrating reversible inhibition. DDX4 (red), Gm-130 (green), and DAPI (blue).
Figure 2 with 8 supplements
Stabilized spindle microtubules mediate fusome asymmetry and cyst breakage.
(A) Pericentric fusome localization in E11.5 germ cells. The early fusome (EMA granule) associates with centrosomes (dashed arrowheads) in E11.5 germ cells: EMA (red), centrosomes PCNT (Pericentrin, green). Left column shows PCNT and DAPI alone. (A′) Summary. (B) Fusome (EMA) behavior during indicated stages of the cyst cell cycle. (B’) Diagrams summarize behavior at the listed mitotic stages deduced by AcTub (Acetylated Tubulin) staining. (C, C') Symmetric early telophase fusome. (D,D') Asymmetric fusome segregation during late cytokinesis. (B–D) EMA, acetylated tubulin (AcTub) and DAPI. (E-E’’) Three lineage-marked (YFP) E12.5 8-cell cysts in early interphase stained to reveal microtubules (AcTub) and fusome (EMA). The absence of spindle remnants in one cyst region (gap in AcTub) predicts future cyst breakage (blue dashed line, summary at R only of YFP+ cells). The E’’ cyst has already broken into 2-cell and 6-cell cyst derivatives. (F) Frequency distribution predicted cyst breakage products by size based on 15 lineage-labeled cysts analyzed as in E (7-cell: 3; 8-cell: 8; 9-cell: 1; 10-cell: 3). Binomial test (see text) compared observed 6-cell cyst production frequency (13/15) to prediction for single random junction breakage of an 8-cell cyst. (****p<0.0001). (G) Model of cyst production and breakage into four 6-cell cysts and 4 2-cell cysts. Scale bars: 5 μm (B), 10 μm (A, C–E).
Figure 2—figure supplement 1
Validating microtubule-dependent fusome formation and its distribution during cyst fragmentation.
(A) E11.5-E12.5 ovary stained for EMA (red), Pericentrin (PCNT, green), AcTub (gray) and DAPI (blue). Arrows mark duplicated centrosomes during interphase. Dotted circles mark centrosomes. Smooth line marks the DAPI-stained region during anaphase depicting clear separation of Nuclei. (B) E10.5 gonad stained for EMA (red) and AcTub (green). Dotted line marks the newly formed fusome as spindle remnant. (C) Fusome asymmetry arises during cytokinesis; depicted in E11.5 ovary stained for EMA (red). (D) E10.5 primordial germ cells (PGCs) stained for AcTub (green) and EMA (red) showing movement of EMA-stained vesicles on microtubules (E) In vitro cultured untreated gonad and treated gonad (microtubule inhibitor ciliobrevin D or cold treatment) stained for AcTub (green), EMA (red), and DAPI (blue). Quantification showing % germ cells with fusome in untreated versus celiobrevin D/cold treated E11.5 gonad. (Student’s t-test: p-value **<0.01) (F) Lineage labeled 4-cell cyst stained at E12.5 for YFP (green), EMA (red) and AcTub (gray). (G) 3D modelling of lineage labeled 10-cell cyst at E12.5. (H) YFP-labeled cells (green spots) and surface-rendered EMA (gray surface) within lineage labeled 8-cell cyst analyzed using Imaris. Yellow arrows pointing to the fusome surface within the nearest germ cell to the putative cleavage site. Scale bar: 5 μm (A–B), 10 μm (C–F).
Figure 2—video 1
Fusome localization during interphase stage.
E11.5 gonadal tissue stained for EMA (red), AcTub (green) and DAPI (blue).
Figure 2—video 2
Fusome localization during the mitosis stage.
E11.5 gonadal tissue stained for EMA (red), AcTub (green) and DAPI (blue).
Figure 2—video 3
Fusome localization during the anaphase stage.
E11.5 gonadal tissue stained for EMA (red), AcTub (green) and DAPI (blue).
Figure 2—video 4
Fusome localization during the telophase stage.
E11.5 gonadal tissue stained for EMA (red), AcTub (green) and DAPI (blue).
Figure 2—video 5
Fusome localization during the late telophase stage.
E11.5 gonadal tissue stained for EMA (red), AcTub (green) and DAPI (blue).
Figure 2—video 6
3D visualization of fusome and acetylated tubulin connections within lineage labeled 8-cell cyst.
Animation video generated using Imaris software to create spherical spots (green) to position the cells within lineage labeled 8-cell cyst with surface rendered EMA (white) and AcTub (red).
Figure 2—video 7
3D visualization of fusome and acetylated tubulin connections within lineage labeled 10-cell cyst.
E12.5 Ovary; Animation video generated by Imaris; Surface rendering of EMA-stained fusome (red) within acetylated tubulin (white) connected lineage labeled 10-cell germline-cyst (YFP in green spherical spots).
Figure 3 with 5 supplements
Mouse fusome associates with Pard3 and apical polarity.
(A–B) Pard3 associates with fusome as observed in E11.5-E13.5; Gonad stained for Pard3 (red), EMA (green) and DAPI (blue) (A, A') and after rosette formation at E13.5 (B, B'). (C-C') Ring canals (RACGAP, yellow) localize within the Pard3+ (red) apical domain in germ cells (GCNA, green). (D) A lineage-labeled E13.5 cyst (YFP, green); channels below show enrichment of Pard3 (red) with enriched fusome (EMA, gray). Graph: Quantification of Pard3 stained area colocalizing with large- ≥ 20 μm2 and small <20 μm2 fusome within lineage labeled cyst (Student’s t-test, N=13; ***p<0.001). (E) Xbp1 (green) enrichment in EMA (red) granule of E11.5 PGC. (F–H) scRNA-seq of E10.5-P5 gonad. UMAP of re-clustered germ cells at various stages (F), UMAP (G) UMI Feature Plot; NC = nurse cells. (H): UMAP with clusters labeled in ascending order of meiotic development. pre-meiotic (Pre-M), leptotene (Lp), zygotene (Zy), pachytene (Pa), diplotene (Dp), dictyate (Dc). (I-I′) Bar plots: (I) Xbp1, Xbp1-target expression plots. (I') Genes orthologous to fusome components. Scale bars: 10 μm (A–C, E), 20 μm (D).
Figure 3—figure supplement 1
Pard3 gene expression in E12.5-E13.5 gonad.
(A) E13.5 ovaries stained for GCNA (blue), EMA (green) and PARD3 (red). (B) Lineage labeled E13.5 ovary stained for YFP (green), GCNA (blue), PARD3 (red), and EMA (gray). (C) Zoomed images of E13.5 gonad stained for RACGAP (green) and Pard3 (red). Scale bar = 10 μm (A and B), 20 μm (C).
Figure 3—figure supplement 2
Validation of ScRNA-seq analysis to show mouse fusome association with Golgi-UPR pathway.
(D) Quinacrine (Y chromosome specific stain, red) and DAPI (green) staining of male vs female fetal tail samples (D’) 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 negatives by segregating the cells expressing significant amount of XX specific genes (eg: Xist, Ddx3x, Utx) depicted in UMAP plot as XX only cells. (E) E10, E11 and E15 (10 X genomics) scRNA-seq was performed and germ cells isolated bioinformatically. A cluster of germ cells from E12, E14, E16, E18, P1 and P5 of previously published data (NCBI: Niu and Spradling, 2020) were used 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 the merged dataset is shown. (F) Feature plot validating that all the cells express germ cell-specific markers (Dppa3/Ddx4) in a stage-dependent manner (G) Pre-meiotic/Meiotic gene expression analysis depicted by feature plot for various developmental stages within the merged dataset. (H) Stack violin plot depicting Golgi-unfolded protein response (UPR) pathway-associated gene expression pattern across E10.5 to P5 meiotic germ cells and nurse cells across various developmental stages. Scale bar = 100 μm (D).
Figure 3—video 1
Pard3 staining pattern within pre-meiotic germline cyst.
E12.5 Ovary stained for PARD3 in Red, EMA in Green and DAPI in Blue.
Figure 3—video 2
Pard3 and fusome as a continuous branched structure within female germline cyst.
E13.5 Ovary stained for GCNA in Blue, PARD3 in Red and EMA in Green.
Figure 3—video 3
Pard3 and fusome as discontinuous, separate structures within male germline cysts.
E13.5 Testis stained for GCNA in Blue, PARD3 in Red and EMA in Green.
Figure 4 with 1 supplement
Unfolded protein response (UPR) genes are active during cyst formation and controlled by Dazl.
(A) Dnmt3a and EMA levels at E12.5. Dnmt3a levels are reduced in wild-type (WT) compared to Dazl-/- germ cells. Graph - Dnmt3a fluorescent levels within germ cells as normalized with somatic cells in WT versus Dazl mutant gonad. (N=10 tissues; **p<0.05). (B) Ring canals are smaller and defective in E13.5 Dazl-/-cysts compared to WT. (N=44; **p<0.05). (C) scRNA-seq of E11.5 and E12.5 WT and Dazl-/- gonad germ cells. UMAP. Germ cell clusters overlapped at E11.5 and segregated at E12 of WT and Dazl-/-. (C’) Xbp1, Xbp1 targets, and fusome orthologs in WT vs Dazl-/- germ cells. (D) Validation of IRE1-Xbp1 assay: Ovarian cells visualized by fluorescent microscopy showing GCNA labeled bigger germ cells with higher Xbp1 fluorescence than smaller somatic 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. (D’; 6 experiments: ~32 mice, ≥5 mice, and ≥20 ovaries per experiment, D”-D”’; 3 experiments: ~40 mice, ≥5 mice, and ≥25 ovaries per experiments, *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001) (E-E″) Proteasome activity comparing SSEA1+germ vs SSEA1− somatic cells at E11.5 and WT vs Dazl-/- germ cells at E12.5. (N=3 biological assays with ~35–60 E11.5 ovary per assay and ~25–28 E12.5 ovaries were used per assay. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001) (F) Golgi fragmentation in E12.5 Dazl-/- germ cells stained with golgi marker Gs28 (red), EMA (green) and DAPI (blue). Graph: germ cell percent with fragmented Golgi in wild-type versus Dazl mutant mouse gonad (Student’s t-test: N=16, ***p<0.005) (F’) Failure of E13.5 Dazl-/- germ cells to form EMA (gray) rosettes or enrich Pard3 (red). (G) Dazl-/- effects on fusome, Golgi and Pard3. (H) Proposed function of fusome-mediated regulation of ERAD-UPR proteostasis. Scale bar: 10 μm (except zoomed in 2 μm).
Figure 4—figure supplement 1
Validation of female Dazl mutant phenotype, magnetic-activated cell sorting (MACS) and Activity assays.
(A) Expected absence of Dazl protein in Dazl mutant gonad is shown by staining of E18.5 wild-type (WT) and Dazl-/- gonad for Dazl (red), GCNA (green), and DAPI (gray). (B) E12.5 WT and Dazl-/- gonad stained for DNMT3a (red), EMA (green), and DAPI (gray) (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) Stack violin plot depicting pluripotency gene expression pattern in E11.5-E12.5 WT vs Dazl-/- gonad. (F) Stack violin plot depicting Xbp1 targets gene and Drosophila fusome ortholog gene expression pattern in E11.5 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 (gray). (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 the left. (I) Proof of functioning of the Xbp1 assay is shown by staining of both mixture of SSEA1(-ve) and SSEA1(+ve) cells for GCNA (green), DAPI (gray), and Xbp1 (red) (J) Proof of function of the 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 a plate reader. Scale bar: 20 μm (A), 10 μm (B), 100 μm (G and I).
Figure 5 with 9 supplements
Fusome and Pard3 associate with endoplasmic reticulum (ER) and mitochondria prior to Balbiani body formation.
(A) E17.5 ovary stained for WGA, GCNA, and TEX14. (A’) E17.5 ovary stained for GCNA, RACGAP, and PARD3; Graph: Fusome volume and Pard3 Stained area versus ring canal number N=65 (Fusome volume; N=51 (Pard3); ANOVA, ***p<0.005, ****p<0.0001). (B-B′) E18.5 ovary shows WGA-Fusome/PARD3 enrichment in large medullary oocytes vs smaller nurse cells; line: medulla/cortex boundary; dotted circle: large medullary oocytes; white dotted area: small nurse cells. The area marked as a white dotted rectangle is shown as a zoomed inset (white arrow). Black arrow in inset: WGA stained fusome; Graph compares fusome volume and Pard3 stained area versus Germ cell nucleus diameter (N=54 (WGA), N=37(PARD3); Student’s paired t-test, *p<0.05, ****p<0.001). (C) Single cell lineage labeled E18.5 ovary stained for YFP, DAPI, WGA, and GCNA Graph: Within single-cell lineage-labeled E18.5 ovary-Fusome volume difference according to germ cell nucleus size (N=10; ****p<0.0001). (D) Single cell lineage labeled E18.5 ovary stained for YFP, PARD3, and GCNA Graph: Within single-cell lineage-labeled E18.5 ovary- difference in PARD3 stained area according to germ cell nucleus size (N=10; ***p<0.005). (G-G′) Dazl +/- E18.5 ovary- Fusome (WGA) and Pard3 enrichment failure in medullary oocytes (GCNA). Graph: Fusome volume in potential oocytes, i.e., bigger germ cells with nucleus diameter d ≥12 μm in wild-type versus Dazl +/- mutant F-F″. Organelle enrichment analysis: E18.5 (WT-F-F’, and Dazl +/- ovary F”) stained for WGA, mitochondrial marker ATP5a and GCNA (F and F”). (F’) - Electron microscopy (EM) image of Golgi-rich Fusome (arrow) surrounded by mitochondria. (G-G′) Endoplasmic reticulum (ER)-mitochondria association in E18.5 WT ovary: G-EM image of ER tubules (arrow) wrapping mitochondria and G’- E18.5 WT ovary- GCNA, ER, and Mitochondria tracker staining. Scale bars: 20 μm (A-E, G-G′, F,F”, G′), 5 μm (B-, B′- right most inset panel), 0.5 μm (EM images F′, G).
Figure 5—figure supplement 1
Fusome enrichment within destined medullary oocytes in wild-type (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). The white dotted line is zoomed in and shown separately in the main figure (Refer to main Figure 5A and A’). (C) 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. (D) E18.5 WT and Dazl+/- gonad stained for Mitotracker (red) and GCNA (green). Scale bar: 20 μm (A–B), 50 μm (C), 100 μm (F).
Figure 5—video 1
Fusome enrichment proportionate to number of ring canals.
3D surface rendering of E17.5 ovary: WGA-stained fusome in red enriched within region spanning ring canal stained by Tex14 in yellow and GCNA-stained germ cells are shown in green.
Figure 5—video 2
Medullary (Wave 1) oocytes start to appear bigger in volume by E18.5 and is often surrounded by smaller nurse cells.
E18.5 ovary stained with wheat germ agglutinin (WGA) (red), GCNA (green), and DAPI (blue).
Figure 5—video 3
Fusome enrichment in early medullary oocyte.
E18.5 wild-type ovary stained for GCNA in green and wheat germ agglutinin (WGA) in red.
Figure 5—video 4
Pard3 enrichment in early medullary oocyte.
P0 wild-type ovary stained for GCNA in green and Pard3 in red.
Figure 5—video 5
Dazl heterozygous mutation causes reduction in fusome enrichment within early medullary oocyte.
E18.5 Dazl +/-ovary stained for GCNA in green and wheat germ agglutinin (WGA) in red.
Figure 5—video 6
Dazl heterozygous mutation causes reduction in Pard3 enrichment within early medullary oocyte.
P0 Dazl +/-ovary stained for GCNA in green and Pard3 in red.
Figure 5—video 7
Absence of big medullary oocytes and no enriched fusome in Dazl homozygous mutant females.
E18.5 Dazl -/- ovary stained for GCNA in green and wheat germ agglutinin (WGA) in red.
Figure 5—video 8
Absence of big medullary oocytes and no enriched Pard3 in Dazl homozygous mutant females.
P0 Dazl -/- ovary stained for GCNA in green and Pard3 in red.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | Dazl | NCBI | https://www.ncbi.nlm.nih.gov/Dazl | |
| Gene (Mus musculus) | Pard3 | NCBI | https://www.ncbi.nlm.nih.gov/Pard3 | |
| Gene (Mus musculus) | Xbp1 | NCBI | https://www.ncbi.nlm.nih.gov/Xbp1 | |
| Strain, strain background (Mus musculus) | C57BL/6 J | Jackson Laboratory | 000664; RRID:IMSR_JAX:000664 | |
| Genetic reagent (Mus musculus) | CAG-Cre-ER | Jackson Laboratory | 004682, RRID:IMSR_JAX:004682 | |
| Genetic reagent (Mus musculus) | Dazl-1L -/+ | Jackson Laboratory | RRID:IMSR_JAX:035880 | |
| Genetic reagent (Mus musculus) | R26R-EYFP | Jackson Laboratory | 006148; RRID:IMSR_JAX:006148 | |
| Biological sample (Mus musculus) | fetal ovary | |||
| Antibody | EMA | DSHB | EMA-1 RRID:AB_531885 | IF (1:1) Culture supernatant |
| Antibody | GCNA | Abcam | ab82527 RRID:AB_1659152 | IF (1:400) |
| Antibody | DDX4 | Abcam/R&D systems | ab13840 RRID:AB_443012/ab27591 RRID:AB_11139638/AF2030 RRID:AB_2277369 | IF (1:400) |
| Antibody | SSEA1 | DSHB | MC-480 RRID:AB_528475 | IF (1:1) Culture supernatant |
| Antibody | Gm-130 | BD Biosciences/Novus Biologicals | 610822, RRID:AB_398141/ NBP2-53420, RRID:AB_2916095 | IF (1:200) |
| Antibody | Rab9 | Thermo Fisher | MA5-31997, RRID:AB_2809291 | IF (1:200) |
| Antibody | Tex14 | Proteintech | 18351–1-AP, RRID:AB_10641992 | IF (1:400) |
| Antibody | GFP/YFP | Aves Labs | GFP-1020, RRID:AB_10000240 | IF (1:600) |
| Antibody | Dazl | BioRad Labs/GeneTex | MCA2336, RRID:AB_2292585/GTX89448, RRID:AB_10722773 | IF (1:100) |
| Antibody | Lysotracker deep | Thermo Fisher | L7528 | IF (1:600) |
| Antibody | Pard3 | Novus Biologicals | NBP1-88861, RRID:AB_11056253 | IF (1:200) |
| Antibody | LAMP1 | Cell Signaling Technology | 9091, RRID:AB_2687579 | IF (1:200) |
| Antibody | Xbp1 | Abcam | ab37152, RRID:AB_778939 | IF (1:200) |
| Antibody | Gs28 | BD Biosciences | 61184, RRID:AB_398718 | IF (1:200) |
| Antibody | Sec63 | Thermo Fisher | PA5-100180, RRID:AB_2815710 | IF (1:200) |
| Antibody | Calnexin | Abcam | ab219644, RRID:AB_3732991 | IF (1:200) |
| Antibody | Gs28 | Proteintech | CL555-16106, RRID:AB_2919629 | IF (1:200) |
| Antibody | Acetyl-α-Tubulin (Lys40) | Cell Signaling. Tech | 5335, RRID:AB_10544694 | IF (1:600) |
| Antibody | Alpha Tubulin (acetyl K40) | Abcam | ab289875, AB_3733017 | IF (1:100) |
| Antibody | Pericentrin | Abcam | ab4448, RRID:AB_304461 ab28144, RRID:AB_2160664 | IF (1:200) |
| Antibody | Rac GAP1 Antibody (A-6) | Santa Cruz Biotechnology, Inc | sc-271110, RRID:AB_10611939 | IF (1:200) |
| Antibody | Dnmt3a | Cell Signaling. Tech | 3598, RRID:AB_2277449 | IF (1:200) |
| Antibody | ATP5A | Abcam | ab14748, RRID:AB_301447 | IF (1:200) |
| Sequence-based reagent | Uba1_Forward | PCR Primers | McFarlane et al., 2013 | 5ʹ-TGGTCTGGACCCAAACGCTGTCCACA-3ʹ |
| Sequence-based reagent | Uba1_Reverse | PCR Primers | McFarlane et al., 2013 | 5ʹ-GGCAGCAGCCATCACATAATCCAGATG-3ʹ, |
| Sequence-based reagent | Sly_Forward | PCR Primers | McFarlane et al., 2013 | 5’-GATGATTTGAGTGGAAATGTGAGGTA-3’ |
| Sequence-based reagent | Sly_Reverse | PCR Primers | McFarlane et al., 2013 | 5’-CTTATGTTTATAGGCATGCACCATGTA-3’ |
| Sequence-based reagent | Zfy_Forward | PCR Primers | McFarlane et al., 2013 | 5’-GACTAGACATGTCTTAACATCTGTCC-3’ |
| Sequence-based reagent | Zfy_Reverse | PCR Primers | McFarlane et al., 2013 | 5’-CCTATTGCATGGACTGCAGCTTATG-3’ |
| Sequence-based reagent | Ddx4 Forward | PCR Primers | Gao et al., 2011 | 5'-GAGATTGCCTTCAGTACCTATGTG-3' |
| Sequence-based reagent | Ddx4 Reverse | PCR Primers | Gao et al., 2011 | 5'-GTGCTTGCCCTGGTAATTCT-3' |
| Sequence-based reagent | Gapdh Forward | PCR Primers | Wang et al., 2011 | 5'-GGTGAAGCAGGCATCTGAGGG-3' |
| Sequence-based reagent | Gapdh Reverse | PCR Primers | Wang et al., 2011 | 5'-GGTGGGTGGTCCAGGGTT-3' |
| Sequence-based reagent | Dazl Common | PCR Primers | JAX Protocol (Strain #035880, Primer# 56340) | 5'-GAC ATT ACT AAG AAA ACA GCA GTG G-3' |
| Sequence-based reagent | Dazl WT reverse | PCR Primers | JAX Protocol (Primer# 56341) | 5'-TTC TGC ACA TCC ACG TCA TT-3' |
| Sequence-based reagent | Dazl Mut Reverse | PCR Primers | JAX Protocol (Primer# 56342) | 5'-ATC CCT CCC TTT AGG GCT CA-3' |
| Chemical compound, drug | AAL | Vector Labs Inc | FL-1391–1 | IF (1:200) |
| Chemical compound, drug | LCA | Vector Labs Inc | FL-1041–5 | IF (1:200) |
| Chemical compound, drug | WGA | Thermo Fisher | W7024 | IF (1:1000) |
| Chemical compound, drug | Paraformaldehyde | Electron Microscopy Sci. | 15714 | Concentration 4% |
| Chemical compound, drug | Corn Oil | Sigma Chemical | C8267 | |
| Chemical compound, drug | Tween 20 | Sigma Chemical | P1379 | |
| Chemical compound, drug | Triton X | Sigma Chemical | X100 | |
| Chemical compound, drug | Mounting media | Vector Labs Inc. | H-1000 | |
| Chemical compound, drug | Trypsin-EDTA (0.25%) | Fisher | 25200056 | |
| Chemical compound, drug | Fetal Bovine serum | Sigma Chemical | F2442 | chemical compound, drug |
| Chemical compound, drug | Bovine serum albumin | Sigma Chemical | A4503 | chemical compound, drug |
| Chemical compound, drug | SSEA1 (CD15) microbeads | Miltenyi Biotech | 130-094-530 | chemical compound, drug |
| Chemical compound, drug | DMEM/F-12 | Fischer | 11320–033 | chemical compound, drug |
| Chemical compound, drug | Celiobrevin D | Millipore | 250401 | chemical compound, drug |
| Chemical compound, drug | Tamoxifen | Sigma Chemical | T5648 | |
| Commercial assay or kit | Mitotracker /Deep | Thermo Fischer | M7514/M22426 | IF (1:10000) |
| Commercial assay or kit | ER-Tracker / | Thermo Fischer | E34251/E34250 | IF (1:10000) |
| Commercial assay or kit | Kappa fast hot start ready-mix | KAPPA Biosystems | KK5608 | |
| Commercial assay or kit | KAPPA express extract | KAPPA Biosystems | KR0383-v4.16 | |
| Commercial assay or kits | IRE1-Xbp1 Assay | Montana Molecular | U0921G | |
| Commercial assay or kit | Proteasome Activity | Amplite | 13456 | |
| Commercial assay or kit | Superscript IV cells direct cDNA synthesis kit | Fischer | 11750510 | |
| Software, algorithm | IMARIS | Oxford Instruments (formerly Bitplane) | Version: 10.2, RRID:SCR_007370 | IMARIS |
| Software, algorithm | SEURAT | Satija Lab | Version: 5.1.0, RRID:SCR_016341 | SEURAT |
| Software, algorithm | CELL RANGER PIPELINE | 10 X Genomics | Version: 6.0.1/8.0.1, RRID:SCR_017344 | CELL RANGER PIPELINE |
| Software, algorithm | GRAPHPAD PRISM | GraphPad Software | Version: 10.5.0, RRID:SCR_002798 | GRAPHPAD PRISM |
| Other | MACS MS separation columns | Miltenyi Biotech | 130-042-201 | MACS MS separation columns |
| Other | Cell culture inserts | Millipore | PICM01250 | Cell culture inserts |
| Other | Cell strainer | Sigma | CLS431752 | Cell strainer |
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Mouse germline cysts contain a fusome-like structure that mediates oocyte development
eLife 14:RP109358.
https://doi.org/10.7554/eLife.109358.3
