Phenotype of BG morphant embryos. Representative examples of Tg(fli:EGFP)y1 embryos, that were injected with 7 ng of mo-BG02 (b-d), or its control mismatched morpholino mo-BG02-mis (a, see Supporting Information Table S1 for nucleotide sequences) illustrating the main macroscopic features of “mild”, “moderate” and “severe” morphants at 30 hpf. Note that in d, three embryos are shown in the picture. a′-d′: The same embryos shown in a-d showing GFP fluorescence to illustrate vascular defects. Arrows point to DLAV in control, and white asterisks indicate examples of defective ISVs without DLV connections. Bracket in control indicates caudal vein plexus (CVP). a′′-d′′: merged images. Snapshots of a representative time-lapse imaging experiment, showing vascular development in control and mo-BG02 injected Tg(fli:EGFP)y1 embryos (e). Images correspond to lateral views of live embryos, at the anterior limit of the CVP, around somites 15-16th. Upper panel corresponds to control (mo-BG02-mis) and lower panel to a mild zBG morphant, injected with 7 ng of mo-BG02. All images were taken with two-photon confocal microscope as described in materials and methods, and correspond to Supporting Information movie S1 and movie S2. Pictures were extracted from the time-lapse series at the approximated times indicated. Dotted line indicates the dorsal aspect where the DLAV forms. Red arrowheads in control (upper panel, 24-32 hpf), indicate sprouts from the CVP. White arrows in mo-BG02 (lower panel 28 hpf) indicate morphant ISV sprouts that reached the dorsal aspect. Yellow arrows (lower panel 36-40 hpf) indicate aberrant connections between adjacent ISVs. Note that the developing CVP in mo-BG02-injected is largely devoid of the ventral sprouts and varicosities found in control, resulting in a dysmorphic CVP at 40hpf.
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