Figure 7—figure supplement 7.

MO interference with ArhGEF11 exon 38 splicing leads to the accumulation of CD41 positive cells in the aortic floor and negatively impacts on the generation of hematopoietic precursors.

(A, B) Representative images of AGM region of double transgenic Tg(CD41:eGFP; Kdrl:nls-mKate2) embryos, injected with control (A) or ArhGEF11 exon 38 splicing morpholino (B) at 48–55hpf. The two top panels show maximum z-projection of aortic segments (top panels with the CD41:eGFP signal in green and the Kdrl:nls-mKate2 signal in magenta, bottom panel only with the CD41:eGFP signal in white). The three bottom panels show single z-planes extracted from the above z-projections. Automatically segmented GFP-positive cells are outlined in white. Magenta arrows point at double positives (GFPlow/kdrl + cells), corresponding to hemogenic/EHT cells. Blue arrows point at single positive (GFPlow/kdrl- cells) HSPC precursors. Green arrows point at circulating thrombocytes that were automatically eliminated from the segmentation. The asterisk labels a non-moving thrombocyte (GFPhigh/kdrl- cells). The aortic floor is outlined by the white dashed line. Scale bars = 10 µm. (C) GFP and mKate2 fluorescence intensity was automatically extracted for each segmented cell and normalized to background. Vertical and horizontal dashed lines on the plot and on the zoomed plot represent the threshold selected for the classification of mKate2+ versus mKate2- (1.05, horizontal line) and eGFPhigh versus eGFPlow (1.4, vertical line). (D) Hemogenic/EHT cell count. (E) HSPC precursors cell count. For (D and E) Statistical tests: two-sided unpaired two samples Wilcoxon test, all p-values are displayed. Analysis was carried out on n=8 control and n=6 mutant embryos.