Fig. 5.

Endothelial cells lose haemogenic potential upon cell cycle block. a Immunofluorescence staining showing the expression of CDH5 and RUNX1 over time. At EHT D3, media were supplemented either with 0.1% DMSO (CTRL) or with 0.1 μg/ml Nocodazole (Noc). After 48 h, at EHT D5, cells were washed and media refreshed every other day. After cell cycle block is released, cells reprise proliferation, but RUNX1 expression is gradually lost, with positive cells representing very rare events at EHT D12. Haematopoietic clusters are outlined in CTRL and consist of RUNX1+ cells that are mainly round shaped and grouped in clusters. These clusters can also be recognised in the CDH5-stained panel (green), in which they display a typical round morphology, as opposed to the surrounding elongated endothelial cells. Finally, these clusters can also be recognised in the DAPI (grey) panel, in which they are characterised by the typical dense nuclear morphology and brighter appearance compared to endothelial cells. In Nocodazole-treated cultures, all these features are not present: the few RUNX1+ cells are more scattered and not grouped in clusters; they mark elongated endothelial cells rather than round cells; these elongated cells are CDH5 positive (green panel) and thus clearly endothelial; finally, their nuclei do not appear to be denser or brighter than the surrounding cells, as was the case in haematopoietic clusters found in control. Scale bar is 50 μm. b Forty-eight hours after Nocodazole release, at EHT D7, cells were collected and cultured in a CFU assay to assess their differentiation potential. Data are mean ± SEM of 3 independent experiments, with statistical significance compared to control as *P < 0.5, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-tailed unpaired t test. Degrees of freedom = 4; t values for the distinct colonies are BFU-E = 5.803, CFU-E = 4.02, CFU-GM = 6.2, CFU-G = 3.625, CFU-M = 7.407, and CFU-GEMM = 5.547. c Diagram summarising the role of cell cycle progression during EHT