Figure 2—figure supplement 2.

Evolution of non-polarized HE cells throughout emergence.

Tg(Kdrl:Gal4;UAS:RFP;4xNR:eGFP-podxl2) embryo imaged using spinning disk confocal microscopy. Images were obtained from discontinuous time-lapse sequences covering a period of 13 hr (from 35 – to – 48 hpf). Successive phases of the evolution of HE cells are visible, from a non-polarized status (with the accumulation of Podxl2-containing intra-cytosolic vesicles as well as cell division) to post-emergence EHT cells remaining beneath the aortic floor. The top panel is a z-projection of 69 consecutive z-sections interspaced by 0.3 µm; green (eGFP-podxl2) and red (soluble RFP, in magenta) channels are shown; 4 individual HE cells (1–4, green arrows)) are marked, with cells 1 and 4 more advanced in the process of emergence. All the other panels are z-sections focused on the aortic floor, allowing visualizing the progression of the EHT, in particular at 35 hpf (t=0), a timing point at which the separation between the luminal and basal membranes are equally labeled with eGFP-podxl2, attesting for the absence of apicobasal polarity (see cells 1 and 4, see also cell 1 at t=2.5 hr and cells 3’ and 3’’ at t=5 hr). Note, at t=2.5 and 5.0 hr, the presence of intracytoplasmic eGFP-podxl2-labeled vesicles inside HE cells, suggesting vesicular transport (white arrowheads, to be compared with images Figure 2—figure supplement 1, at 30 hpf). At 48 hpf (bottom panel), HE cells have emerged and remain, for some of them, in close contact with the aortic floor (after having performed mitosis (notably for HE cell 2 [the division is visualized at 5 hr] and for HE cell 3, each one giving rise to cells 2’ - 2’’ and 3’ - 3’’ respectively, all containing residual eGFP-podxl2 containing membranes as EHT signature). Scale bar = 80 µm.