Figure 7. Ablation of nuclear germ granules reduces cell divisions in PGCs.

(A,B) Oskar∆NLS/+ PGCs (A) form nuclear germ granules (white arrows) while Oskar∆NLS PGCs do not (B). Granules were immunostained against Osk (red). DAPI-stained DNA is shown in blue and nuclear lamin is shown in white. (C,D) Number of embryos with small (C) and hollow (D) nuclear germ granules stained with α-Osk in Oskar∆NLS and Oskar∆NLS/+ PGCs. In both the total number of embryos counted is written above the graph bars. (E,F) Cross-section of the posterior pole of an embryo with Oskar∆NLS/+ (A) and Oskar∆NLS (B) maternal genotype immunostained against Vasa (green) and DAPI (white). G Number of PGCs in 41 Oskar∆NLS/+ and 42 Oskar∆NLS embryos. Mean ± STDEV is plotted (statistical significance: unpaired t-test, p<0.001). (H) PGC buds in 15 Oskar∆NLS/+ and 14 Oskar∆NLS embryos. Mean ± STDEV is plotted (statistical significance: unpaired t-test). (I,J) Quantification of PGC divisions in live Oskar∆NLS/+ (I) and Oskar∆NLS (J) embryos. The emergence of the first PGC bud represented time 0 and the cellularization of somatic nuclei at NC 14 represented the end of the experiment. The number of Vasa:GFP associated HiS2Av:mRFP-stained nuclei was counted in three dimensions at every time point (red, green, orange and violet circles). Lines represent an estimated progression among successive cell divisions. Percent of dividing Vasa:GFP +nuclei is marked on each graph (Figure 7—video 1 and 2). Note, that in live observation, the total number of PGCs was higher than in fixed embryos (compare G with I and J). We attribute this to the fact that we counted all nuclei with even a small amount of Vasa:GFP associated, which may not have cellularized and become PGCs and thus would not have been counted as PGCs in fixed tissue. (K) Western blot of Oskar protein in Oskar∆NLS and Oskar∆NLS/+ embryos. α-tubulin was used for normalization control. L Total amount of Vasa germ plasm fluorescence in Oskar∆NLS and Oskar∆NLS/+ embryos marked with an antibody against Vasa. Mean total fluorescent levels ± STDEV of eight (Oskar∆NLS/+) and 13 (Oskar∆NLS) embryos per genotype are shown (statistical significance: two-tailed t-test). (M,N) Early embryos (M) and late embryos (N) hybridized with nos (red) and CycB (green) smFISH probes. DAPI-stained nuclei are shown in blue. O Quantification of localized mRNA levels hybridized with CycB, nos and gcl smFISH probes. Mean total fluorescent levels ± STDEV of four CycB and nos-stained embryos per genotype are shown and mean total fluorescent levels ± STDEV of four (Oskar∆NLS/+) and five (Oskar∆NLS) gcl-stained embryos are shown (statistical significance: two-tailed t-test). Scale bar in A,B is 2 μm and in E,F,M,N is 10 μm.

Figure 7—figure supplement 1. Ablation of nuclear germ granules reduces cell divisions in PGCs.

(A) Alleles containing deletions of the Osk NLS1 created by separate CRISPR/Cas9. (B-D) Antibody against Oskar (red) non-specifically stains background in Drosophila embryos. Somatic nuclei of embryos laid by homozygous Osk∆NLS mothers (B), their heterozygous siblings (C) or laid by mothers that do not express Oskar (∆Osk: osk54/Df(3R)Pxt103), D) all contain small red speckles when stained with anti-Oskar antibody, which most likely respond to unspecific binding of the polyclonal Osk antibody used. Nuclear lamin is shown in white, F-actin in green and DAPI in blue. (E, F) Oskar∆NLS PGCs do not form Vasa:GFP-labeled nuclear germ granules (E) while Oskar∆NLS/+PGCs do (white arrows) (F). (G) Early (i) and late (ii) Oskar∆NLS/+ and Oskar∆NLS embryos hybridized with gcl (red) smFISH probes and stained with DAPI (blue) to label the nuclei. (H) Embryo hatch rate. Numbers above the bars represent an average number of eggs counted per plate ± STDEV (n = 3). I Nuclei of the Oskar∆NLS and Oskar∆NLS/+ PGCs are transcriptionally silent, while their surrounding somatic nuclei are not. Transcriptionally active RNA polymerase II is detected with an antibody that detects phosphorylated Ser2 residue in the C-terminal Repeat Domain (CTD) of PolII (red), Vasa antibody (green) marked PGCs and DAPI (blue) stained the nuclei. J Oskar∆NLS embryos over-expressing gcl mRNA (P(EP)/mat-α gal4; Oskar∆NLS) do not accumulate WT (P(EP)/mat-α gal4; Oskar∆NLS/+) levels of PGCs. Mean ± STDEV of the number of PGCs per embryo are shown where eight embryos per genotype were quantified (statistical significance: two-tailed t-test). Note that over-expression of gcl increases the number of PGCs in both genotypes relative to WT embryos (Figure 7G), as anticipated(Jongens et al., 1994), yet Oskar∆NLS mutant embryos still produce 29% fewer PGCs compared to Oskar∆NLS/+ siblings. (K-M) S2R+ cells transfected with Short mCherry:Osk divide like untransfected S2R+ cells. Transfected and DAPI-stained cells were sorted as mCherry+ (6.32% of total cells) and mCherry- cells (92.2% of total cells) (K). Binning of cells into G1, S and G2/M cell cycle phases revealed that mCherry- (L) and mCherry+ (M) cells cycled with similar rates. Scale bar in B-D is 5 μm and in E-G,I is 10 μm.

Figure 7—video 1. PGC division in live Oskar∆NLS/+ embryos.

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DOI: 10.7554/eLife.37949.028

Figure 7—video 2. PGC division in live Oskar∆NLS/Oskar∆NLS embryos.

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DOI: 10.7554/eLife.37949.029