(
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.