A WISH of notch3 viewed dorsally at 13 hpf (left) and laterally in the trunk at 19 (middle) and 24 hpf (right). Black arrowheads denote somitic expression; red arrowheads denote PLM expression at 13 hpf and endothelial expression at 19 and 24 hpf.
B, C WISH of the HSPC marker runx1 at 26 hpf (B) and cmyb at 36 hpf (C) on uninjected and notch3 morphants. Arrowheads indicate HSPCs in the DA.
D, E Confocal fluorescence microscopy images of transgene reporter expression in cmyb:GFP; kdrl:RFP trunk region at 48 hpf (D) and rag2:GFP at 4 dpf (E) transgenics uninjected or with notch3 morpholino injected. Arrowheads in cmyb:GFP; kdrl:RFP embryos indicate double-positive HSPCs, and dotted lines in rag2:GFP embryos outline the thymic lobes where GFP+ lymphoid cells should reside.
F Enumeration of cmyb:GFP+; kdrl:RFP+ cells in the floor of the DA at 48 hpf. Bars represent mean ± SEM of double-positive cells for uninjected (n = 12) and notch3 morphants (n = 20). P = 2.3 × 10−15.