Fig. S8

Morpholino efficiency and off-target cell death measurements in angpt1^e2i2 and angpt2^e2i2 morphants. (A) RT-PCR of a fragment surrounding the exon 2-intron 2 boundary from angpt1^e2i2 morphants injected with 3.2 ng of angpt1^{e2i2 MO} showing a shift in size from the predominant isoform (arrowhead) to that of higher and lower molecular weights (asterisk) in comparison to uninjected control embryos. (B) RT-PCR of a fragment surrounding the exon 2-intron 2 boundary from angpt2^e2i2 morphants injected with 1.25 ng angpt2^{e2i2 MO} (the dose used to rescue hemorrhage in igu^fo10a mutants). Morpholino injection results in a shift from the wild type band in uninjected controls (arrowhead) to the presence of five additional bands of differing sizes representing different mis-spliced transcripts (asterisk) and reduced presence of a wild type sized band. (C-E) Off-target morpholino-induced cell death assessment using acridine orange. Epifluorescent images of uninjected embryos (C) incubated in acridine orange displaying basal levels of apoptosis. Angpt2^e2i2 (D) and angpt2^ATG (E) morphants injected with 1.25 ng or 1.3 ng of morpholino, respectively, display increased levels of apoptosis in the head region. This increase in apoptosis does not likely influence our rescue results since we would expect increased neural death to exacerbate hemorrhage, not rescue it. In A and B, “M” denotes New England Biolabs 100bp ladder. Scale bars in C-E are 500 μm.