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Fig. 2. Dicer down-regulation affects retinal cell lamination, delays the exit from the cell cycle and pro- motes cell death. (A) Comparison be- tween morphant (mo) and wild type (wt) eyes (arrows) at st. 42. Embryos were injected with 10 nl of 125 nM Xdcr-Mo1 in one dorsal cell at 4 cell-stage embryos. Injected embryos were traced and selected by GFP mRNA coinjection (300 ng, not shown). (B-D) Nuclear Hoechst staining of wt (B) and morphant (C,D) eye sections. PE: pig- mented epithelium, ONL: outer nuclear layer, INL: inner nucler layer, GCL: ganglion cell layer. Although lens and PE morphology does not appear to be affected in morphants, the thickness of morphant PE is clearly reduced com- pared to control, as shown in box in (B) (control) and (C,D) (mild and severe morphant phenotypes, respectively). (E-G) BrdU labelling index (LI), obtained by the analysis of wt and mo embryos after 8 h BrdU incorporation. (G) Sta- tistical analysis of BrdU-positive cells (red in E,F, and Supplementary Fig. 3) in the central aspect of retinal sections (delimited by dashed lines in E,F). GFP traces Xdcr-Mo1 injected cells. A sig- nificant LI increase (triple asterisk: p <0.001, student ttest) was observed in morphants compared to wt. Error bars show s.e.m., n: number of cells. (H) Statistical analysis of apoptotic TUNEL-positive cells (Supplementary Fig. 4) in mo and wt retinas (n= 69 and 482, respectively) at different stages. Mo showed a significantly higher num- ber of apoptotic cells/section than wt (double asterisk: p < 0.01, single asterisk p < 0.05). Error bars: s.e.m. (I-N) In situ hybridisation of Xenopus cell- type specific markers (Decembrini et al., 2006) on st. 42 mo and wt retinal sections: Xirbp (photoreceptors, PHC), Xhermes (ganglion cells, GC), Xprox1
(horizontal cells, HC). Although expressing the specific markers, retinal cells are not properly layered in mo (J,L,M) compared to wt (I,L,N). |
