Fig. 3

Fig. 3 G2/M arrest and increased apoptosis contribute to HSPC exhaustion in tubgcp6 mutants.
A Triple immunofluorescence staining of cd41:eGFP (green), BrdU (red), and pH3 (blue) in the CHT region showed reduced proliferation and mitotic arrest of HSPCs in tubgcp6 mutants at 3 dpf. White arrows indicated BrdU-positive cd41:eGFP^low HSPCs, and orange arrows indicated pH3-positive cd41:eGFP^low HSPCs. Scale bars: 50 μm. B Quantification of cell numbers of cd41:eGFP^low HSPCs, percentage of BrdU-positive cd41:eGFP^low HSPCs, and percentage of BrdU-negative but pH3-positive cd41:eGFP^low HSPCs in siblings and tubgcp6 mutants. (mean ± SD; n > 10; Student’s t test and nonparametric Mann–Whitney test, *p < 0.05, **p < 0.01, ****p < 0.0001). Ccd41:eGFP^low HSPCs from siblings and tubgcp6 mutants were stained with Hoechst33342 and subjected to flow cytometry for cell cycle analysis, which showed an increased percentage of G2/M phase of HSPCs in tubgcp6 mutants. The y-axis indicated cell counts, and the x-axis indicated DNA content. Gating strategies were provided in Supplementary Fig. 7. D Statistical data of the percentages at each phase from (C). (mean ± SD; n = 3 independent experiments; Student’s t test, ns p > 0.05, *p < 0.05, **p < 0.01). E Double staining of cd41:eGFP (green), TUNEL (red) in the CHT region showed increased apoptosis of HSPCs in tubgcp6 mutants at 3 dpf. Yellow arrows indicated TUNEL-positive cd41:eGFP^low HSPCs. Scale bars: 50 μm. F Percentage of co-stained TUNEL-positive cd41:eGFP^low HSPCs in siblings and tubgcp6 mutants. (mean ± SD; n ≥ 10; nonparametric Mann–Whitney test, *p < 0.05). G Triple immunofluorescence staining of coro1a:eGFP (green), α-tubulin (red), and DAPI (blue) demonstrated an abnormal mitotic spindle in tubgcp6 mutants at 4 dpf. The white dashed line indicates the mitotic cell. Scale bars: 5 μm. H Quantification of the percentage of mitotic spindles in siblings and tubgcp6 mutants from (G) (Fisher’s exact test, n > 10, ****p < 0.0001). Data shown were pooled from three independent experiments. I Loss of Tubgcp6 disrupts mitotic progression by impairing γ-TuRC-mediated microtubule nucleation, leading to abnormal spindle formation and subsequent dysregulation and depletion of HSPCs (Created with BioGDP.com⁹⁰).