rabbit anti g3bp1  (Cell Signaling Technology Inc)

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: E18.5 Dazl + /- ovary stained for GCNA in green and wheat germ agglutinin (WGA) in red.

Article Snippet: Mouse strains used - C57BL/6, CAG-cre/ERT2 mice, R26R-EYFP (as described in ) and Dazl +/- (Strain #035880 from JACKSON laboratories).

Techniques:

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: P0 Dazl + /- ovary stained for GCNA in green and Pard3 in red.

Article Snippet: Mouse strains used - C57BL/6, CAG-cre/ERT2 mice, R26R-EYFP (as described in ) and Dazl +/- (Strain #035880 from JACKSON laboratories).

Techniques:

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: E18.5 Dazl -/- ovary stained for GCNA in green and wheat germ agglutinin (WGA) in red.

Article Snippet: Mouse strains used - C57BL/6, CAG-cre/ERT2 mice, R26R-EYFP (as described in ) and Dazl +/- (Strain #035880 from JACKSON laboratories).

Techniques:

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: P0 Dazl -/- ovary stained for GCNA in green and Pard3 in red.

Article Snippet: Mouse strains used - C57BL/6, CAG-cre/ERT2 mice, R26R-EYFP (as described in ) and Dazl +/- (Strain #035880 from JACKSON laboratories).

Techniques:

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: ( 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 ).

Article Snippet: Mouse strains used - C57BL/6, CAG-cre/ERT2 mice, R26R-EYFP (as described in ) and Dazl +/- (Strain #035880 from JACKSON laboratories).

Techniques: Mutagenesis, Staining, Sequencing, Nucleic Acid Electrophoresis, Expressing, Gene Expression, Amplification, Marker, Activity Assay, Positive Control

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: E18.5 Dazl + /- ovary stained for GCNA in green and wheat germ agglutinin (WGA) in red.

Article Snippet: Genetic reagent ( Mus musculus ) , Dazl- 1L -/+ , Jackson Laboratory , RRID: IMSR_JAX:035880 , .

Techniques:

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: P0 Dazl + /- ovary stained for GCNA in green and Pard3 in red.

Article Snippet: Genetic reagent ( Mus musculus ) , Dazl- 1L -/+ , Jackson Laboratory , RRID: IMSR_JAX:035880 , .

Techniques:

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: E18.5 Dazl -/- ovary stained for GCNA in green and wheat germ agglutinin (WGA) in red.

Article Snippet: Genetic reagent ( Mus musculus ) , Dazl- 1L -/+ , Jackson Laboratory , RRID: IMSR_JAX:035880 , .

Techniques:

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: P0 Dazl -/- ovary stained for GCNA in green and Pard3 in red.

Article Snippet: Genetic reagent ( Mus musculus ) , Dazl- 1L -/+ , Jackson Laboratory , RRID: IMSR_JAX:035880 , .

Techniques:

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: ( 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 ).

Article Snippet: Genetic reagent ( Mus musculus ) , Dazl- 1L -/+ , Jackson Laboratory , RRID: IMSR_JAX:035880 , .

Techniques: Mutagenesis, Staining, Sequencing, Nucleic Acid Electrophoresis, Expressing, Gene Expression, Amplification, Marker, Activity Assay, Positive Control

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: ( 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).

Article Snippet: Genetic reagent ( Mus musculus ) , Dazl- 1L -/+ , Jackson Laboratory , RRID: IMSR_JAX:035880 , .

Techniques: Mutagenesis, Biomarker Discovery, Microscopy, Labeling, Fluorescence, Activity Assay, Staining, Marker

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: ( 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 ).

Article Snippet: Genetic reagent ( Mus musculus ) , Dazl- 1L -/+ , Jackson Laboratory , RRID: IMSR_JAX:035880 , .

Techniques: Staining, Single Cell, Labeling, Mutagenesis, Marker, Electron Microscopy

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: ( 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 ). ( 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 ).

Article Snippet: Genetic reagent ( Mus musculus ) , Dazl- 1L -/+ , Jackson Laboratory , RRID: IMSR_JAX:035880 , .

Techniques: Staining