Figure 3—figure supplement 1.

Expression of dt-runx1 triggers the accumulation of myb + cells in the aortic floor.

(A) Representative image (Imaris 3D-rendering) of RNA-scope in situ hybridization for the hematopoietic marker myb in 48–50hpf Tg(Kdrl:eGFP) control embryos (left) and 48–50hpf Tg(Kdrl:Gal4;UAS:RFP;4xNR:dt-runx1-eGFP) mutant embryos (right). Top row: 3D rendering without segmentation. The aorta is outlined with the white dashed lines. Center top row: Magnification of aortic segments (outlined with white boxes on the top rows). Cyan arrows: hemogenic cells (comprising characterized elongated hemogenic cells as well as EHT undergoing cells). Magenta arrows: hematopoietic cells in the sub-aortic space. Center bottom row: GFP + cells segmentation (white cellular outline) and RNAscope signal segmentation (magenta spots). Bottom row: Manual cell classification post segmentation. Aortic roof cells (virtually all negative for myb) are displayed in green, hemogenic and EHT cells in cyan and sub-aortic hematopoietic cells in magenta (all expressing myb). Scale bars = 10 µm. (B) Quantitative analysis of the dt-runx1 expressing mutant phenotype. Left: endothelial roof cell count (green segmented cells on panel (A)). Right: hemogenic/EHT cell count (cyan segmented cells on panel (A)). (C) myb-positive cell count in roof endothelial cells left, corresponding to green cells in panel (A), hemogenic/EHT cells (middle, corresponding to cyan cells in panel (A)) and hematopoietic cells in the sub-aortic region (right, corresponding to magenta cells in panel (A)). (D) Number of segmented RNA-scope myb dots per segmented roof cells (left panel) and hemogenic/EHT cells (right panel). For panels (B, C and D), statistical comparisons have been performed using two-sided unpaired Wilcoxon tests, all p-values are displayed. Analysis was carried out on n=4 control embryos and n=3 mutant embryos, with 2 aortic segments per embryo.