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 ) 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 ).

Article Snippet: Antibody , Tex14 , Proteintech , 18351–1-AP, RRID: AB_10641992 , IF (1:400).

Techniques: Labeling, Staining

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 ) 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 ).

Article Snippet: Antibody , Tex14 , Proteintech , 18351–1-AP, RRID: AB_10641992 , IF (1:400).

Techniques: Immunostaining, Marker, Labeling, Software, Sampling, Staining

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: Antibody , Tex14 , Proteintech , 18351–1-AP, RRID: AB_10641992 , IF (1:400).

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: Antibody , Tex14 , Proteintech , 18351–1-AP, RRID: AB_10641992 , IF (1:400).

Techniques: Staining