Figure 2

RpS5b mutant ovaries have numerous developmental defects that can be rescued by germline expression of RpS5a or RpS5b. (a) Western blot demonstrating that RpS5b is undetectable in RpS5b homozygotes and present at reduced levels in RpS5b heterozygotes, confirming the loss-of-function nature of the mutation. (b,c) Oogenesis does not proceed beyond stage 8 in RpS5b ovaries, at which point apoptosis is induced, as measured by increased levels of activated caspase-3 (C3). α-Orb is used to label the oocyte. (d–h) Various defects observed in RpS5b ovaries: (d) an extra round of germ cell division; (e) a compound egg chamber partially separated by follicle cells, (f) oocyte duplication in a single egg chamber; (g) mis-localized oocyte; (h) multiple layers of follicle cells at the posterior of the egg chamber (white arrow). (i–l) Alterations in the microtubule cytoskeleton in RpS5b oocytes, as measured by immunostaining against (i,j) α-Tubulin, or (k,l) α -Dynein heavy chain. Note the aberrant accumulation of α-Tubulin around the oocyte in (j), and the focus of Dynein in the centre of the oocyte in (l). (m,n) Distribution of Osk and Grk in (m) wildtype and (n) RpS5b oocytes, showing that deployment of these proteins is disrupted in the mutant. (o,p) Analysis of RpS5b germline clones, showing similar defects as found in the mutant, but more extreme overproliferation of follicle cells. (q) Western blot showing RpS5b expression in RpS5b mutant and germline clones. The residual expression in the germline clones is somatic, as is also apparent in (o,p). (r) Western blot comparing RpS5a expression in 0–2 h embryos collected from wildtype females and those expressing shRNA targeting RpS5a driven by the germline-specific promoter nos, showing the efficacy of knockdown. (s) Analysis of ovaries from females expressing shRNA targeting RpS5a driven by the germline-specific promoter nos, showing normal patterning. (t) Brightfield images of whole ovaries showing that RpS5a germline knockdown produces no phenotype but worsens the RpS5b mutant phenotype. (u–w) RpS5b mutant ovaries (u) without a transgene as control or expressing transgenic (v) RpS5b or (w) RpS5a under the control of the nos promoter. Normal oogenesis is restored in both cases. (x) Western blot of lysates from 0–2 h embryos collected from wildtype (WT), nos > RpS5a; RpS5b (NG4–5a; S5b) and nos > Rps5b; RpS5b (NG4-5b; S5b) females, confirming high-level expression from the transgenes. (y) Graph showing hatching rates of embryos from females of the genotypes indicated, demonstrating that either RpS5a or RpS5b can fully rescue the fertility of RpS5b females when expressed in germline.