De novo centriole biogenesis is partially impaired in unfertilized eggs overexpressing Plk4 after depletion of γ-tubulin. (A) Maximum-intensity z projections of fluorescence from unfertilized eggs overexpressing Plk4 alone (control) or together with RNAi against γ-tubulin 23C, γ-tubulin 37C, or a combination of both. Eggs were stained against Bld10 (cyan), Ana1 (yellow) and tyrosinated α-tubulin (magenta). Centrioles were identified by colocalization of spot-like signals from at least two of the three reporters. Inset squares in each fluorescence channel are shown at higher magnification on the right (scale bar, 3 µm). Orange asterisks reveal putative meiotic defects, previously described to occur in oocytes from γ-tubulin 37C mutant females (Tavosanis et al., 1997). In these example images, the control shows signal spots in all channels, while in RNAi conditions, some reporter signals were present (white square) and others absent (orange square) in the fewer centrosome-like dots observed. Note that in the double knock-down condition (γ-tubulin 23C+37C) we did not detect any signal from tyrosinated α-tubulin despite the presence of some centrosome-like dots bearing centriolar reporters (two examples are shown, i and ii). (B) Quantification of eggs with centrioles depleted of γ-tubulin 37C alone, or in combination with depletion of γ-tubulin 23C. The presence of centrioles was scored by the concomitant detection of at least two centrosomal reporters. n = 30 eggs (control); n = 49 eggs (γ-tubulin 23C); n = 47 eggs (γ-tubulin 37C); n = 54 eggs (γ-tubulin 23C + 37C). ns (not significant), P ≥ 0.05; **, P < 0.01; *, P < 0.001 (Pearson’s χ2 test, and two-proportions z test on pooled data). α-Tub, α-tubulin.
