Interactions between VEGFR inhibitor and sprouting in <i>sema3fb</i> mutants.

Abstract

A) A model of signaling pathways that regulate angiogenic sprouting, highlighting key genes controlling tip and stalk cell identity. B) Quantification of Tg(kdrl:mCherry) transgene expression levels in wild type and sema3fbca305 ISAs at 30hpf. N = 3: wild type (n = 13 embryos, average of 6500 a.u.) and sema3fbca305 (n = 14 embryos, average of 8400 a.u.). T-test with Welches correction, *p = 0.0186, a.u. = arbitrary unit of intensity. C). B) Fluorescent HCR in situ for vegfr2 and sflt1 RNA transcripts in whole-mount wild type and embryos sema3fbca305 embryos fixed at 30 hpf. D) Whole-mount Immunostaining for phosphoERK (pERK) in WT and sema3fbca305 embryos fixed at 30 hpf. E) Lateral confocal images of the trunk vasculature Tg(kdrl:mCherry) (white) in embryos treated with 0.5 μM SU5416 from 20hpf-30hpf. DLAV gaps (blue asterisks) and ISA truncated sprouts (yellow dashed line at the level of horizontal myoseptum are indicated. Scale bar, 100 μm. F) Quantification of ISA sprout length in 30 hpf embryos treated with 0.5 μM SU5416, N = 1: WT + DMSO (25 ISAs, 5 embryos, mean of 107±8 μm), WT + 0.5μM SU5416 (25 ISAs, 5 embryos, mean of 50±14 μm), sema3fbca305 + DMSO (30 ISAs, 6 embryos, mean of 82±17 μm), and sema3fbca305 +0.5μM SU5416 (30 ISAs, 6 embryos, mean of 82±19 μm). G) Percentage of ISA sprouts connected at DLAV in 30 hpf embryos treated with 0.5 μM SU5416, N = 1: WT + DMSO (25 ISA-DLAV, 5 embryos, mean 78% of ISA-DLAV/embryo), WT + 0.5 μM SU5416 (25 ISA-DLAV, 5 embryos, mean of 78%), sema3fbca305 + DMSO (30 ISA-DLAV, 6 embryos, mean of 51%), and sema3fbca305 + 0.5 μM SU5416 (30 ISA-DLAV, 6 embryos, mean of 82±19%). Error bars = ±SD. H) Lateral confocal images of the trunk vasculature Tg(kdrl:mCherry) (white) in embryos treated with low doses of DMH4 μM SU5416 from 20hpf-30hpf. ISA truncated sprouts (yellow dashed line at the level of horizontal myoseptum are indicated. Scale bar, 50 μm. I) Quantification of length of ISA sprouts in 30 hpf embryos treated with 15 μM DMH4, J) Quantification of length of ISA sprouts in 30 hpf embryos treated with 25 μM DMH4. I-J) N = 1; WT+ DMSO n = 3, 15 ISAs, average (ave.) 102±9 μm; WT+ μM DMH4 n = 2, 10 ISA, ave. 17±17 μm; WT+ 25 μM DMH4 n = 3, 15 ISAs, ave. 7±8 μm. sema3fbca305 + DMSO = 3, 15 ISA, ave. 87±18 μm, sema3fbca305 + 15 μM DMH4 n = 2, 10 ISA, ave. 40±16 μm; sema3fbca305 + 25 μM DMH4 n = 3,15 ISA, 32±13 μm. One-Way ANOVA Tukey’s multiple comparisons test, * means ¬¬p = 0.012, *****p = (TIF)</p