Figure 3—figure supplement 2.

Figure 3—figure supplement 2. Phenotypic analysis of dt-runx1 expressing mutants: evidence for apicobasal polarity of hemogenic cells.

30–32 hpf and 48 - 55hpf embryos obtained from outcrossed Tg(Kdrl:mKate2-podxl2) X Tg(kdrl:Gal4;UAS:RFP;4xNR:dt-runx1-eGFP) fishes were imaged in the trunk (AGM) region using spinning disk confocal microscopy (single z-planes). (A) Two aortic segments imaged in control sibling embryos (not expressing the dt-runx1 mutant, hence negative for the cleaved eGFP). Typical HE cells are outlined in the white boxes (with the luminal and abluminal membranes clearly separated from each other owing to reduction of their surface area (in comparison to flat aortic cells) and elongation in the antero-posterior axis). Note the absence of enrichment of mKate2-podxl2 in luminal membranes in comparison to basal membranes (magenta arrows). (B) Two aortic segments in dt-runx1 expressing embryos. Images are depicting typical HE cells (in boxes) from hemogenic regions (as in A). Note the enrichment of mKate2-podxl2 in apical/luminal membranes (magenta arrows) in comparison to basal membranes (green arrows). Green: cytosolic eGFP released from the cleavage of dt-runx1-eGFP. Note that because of mosaicism, HE cell 2 does not express mkate2-podxl2. Scale bars = 20 µm. (C) Evolution of an EHT cell extracted from a 7 hr time-lapse sequence (0–420 min, starting from around 40hpf) and showing significant changes in its morphology throughout emergence. Note the sub-luminal and cytosolic localization of pools of Podxl2 (notably at t=30 min, magenta arrows) suggesting enhanced trafficking of the protein and relative instability of apical polarity, consistently with apparent fluctuation of the luminal membrane surface contacting the aortic lumen (green arrows, in particular at t=240 min). At t=420 min, the cell has emerged and Podxl2 containing membranes remain in close contact with the membrane contacting the aortic floor. Scale bar = 10 µm.