Figure 4

Figure 4

Prpf31 deficiency causes abnormal spindle structure and mitotic arrest. (A) Double staining of EDU (S-phase cells) and pH3 (M-phase cells) in the retinal sections of WT siblings and prpf31^−/− mutants at 36 and 48 hpf. The number of M-phase cells were significantly increased in the prpf31^−/− mutants compared with siblings, suggesting that RPCs may be arrested in M phase. White arrows, overlapping signals of EDU and pH3. Scale bars: left, 100 μm; right, 10 μm. (B) Quantification of EDU⁺, pH3⁺ and EDU⁺ / pH3⁺ cells shown in (A). n = 6 for each panel. (C) In vivo imaging of the H2A-mCherry labeled chromosomes showed the mitotic progression of RPCs at 36 and 48 hpf. The time point of nuclear envelope breakdown (NEBD) was set as the start of mitosis. Scale bar, 10 μm. (D, E) Quantification of the time from NEBD to anaphase in RPCs at 36 and 48 hpf. 15 cells from more than five embryos were observed for each group. For abnormally divided cells, the longest observation time was 150 min. Scale bar, 10 μm. (F) The spindle and nuclei of RPCs from mutants and WT siblings were stained with anti-α-tubulin (green) antibody and PI (red), respectively. The different types of spindle anomalies are displayed in the panels (Abnormal 1–4). Scale bar, 10 μm. (G) Quantitative analysis of the RPCs numbers in each of the phases of mitosis in sibling and prpf31 mutant embryos at 36 and 48 hpf.