Knockdown of tubgcp6 disrupts HSPCs division decision.
A Selected frames of time-lapse imaging (1 frame/4 min) from 32–56 hpf were a merge of kdrl:mCherry+ endothelial cells (red) and cmyb:eGFP+ HSPCs (green) in the control group injecting NC MO and tubgcp6 knockdown group injecting tubgcp6 MO. Time was hours: minutes post-fertilization. The yellow arrows indicated daughter cells retained within the endothelial niche, while the blue arrows indicated those out of the endothelial niche. Scale bar: 20 μm. B Representative lineage tree analyses of cmyb+ HSPCs tracked in parallel time-lapse movies in the NC MO injected group and tubgcp6 knockdown group from 32–56 hpf. Each line represented a continuously tracked HSPCs from the moment it entered the CHT field of view until it exited. Branch points represented distinct cell divisions. The yellow dots indicate the daughter cell remaining in the endothelial niche, while the blue arrows indicate those out of the endothelial niche. C Quantification of the percentage of HSPC division decisions tracked in the time-lapse movies. In the NC MO injected group, 71 divisions were recorded across 14 imaged embryos, including 38 symmetric divisions (19 remain in/remain in; 19 migrate out/migrate out) and 33 asymmetric divisions (remain in/migrate out); In the tubgcp6 knockdown group, 56 divisions were observed across 14 imaged embryos, comprising 40 symmetric divisions (11 remain in/remain in; 29 migrate out/migrate out) and 16 asymmetric divisions (remain in/migrate out). (Fisher’s exact test, *p < 0.05). D Representative time-lapse frames showing dividing cmyb+ HSPCs (green) in cd41:numb-mCherry (red) injected embryos, displaying patterns of asymmetric and symmetric Numb-mCherry inheritance. Scale bar: 10 μm. E Quantification of Numb-mCherry fluorescence in cmyb+ HSPC daughter cells showing symmetric (left) and asymmetric (right) segregation during division from (D). The ratio of fluorescence between sister cells immediately after division was shown. F Quantification of Numb inheritance patterns in dividing cmyb+ HSPCs. In the control group (NC MO injection), 29 divisions were observed, consisting of 21 symmetric and 8 asymmetric inheritance. In the tubgcp6 knockdown group, 23 divisions were analyzed, comprising 22 symmetric and 1 asymmetric inheritance. (Fisher’s exact test, *p < 0.05). All data were pooled from three independent experiments.
|
