Fig. 9-S1

CAMSAP2 plays a role in sprouting angiogenesis in vivo.

(A) RT-PCR analysis of Tg(fli1a:eGFP) embryos injected with a splice-blocking morpholino targeting the exon2/intron2 boundary in Camsap2b (black box), a control or no morpholino (-) with primers (arrows) allowing amplification of distinct spliced species. A shorter amplicon is expected if exon2 is skipped or partially deleted. The same amplification was done with no cDNA (H₂O) or with samples that were not treated with reverse transcriptase (NoRT). (B) qPCR analysis of CAMSAP2b exon2 mRNA expression in embryos injected with control or CAMSAP2b morpholinos; results are expressed relative to the control after normalization to ELFA housekeeping gene, n = 3 different primer pairs used in triplicate. (C) Live images of 48 hpf Tg(fli1a:eGFP) embryos injected with control or CAMSAP2b morpholinos showing the trunk vasculature; arrows point to abnormal venous sprouts. (D) Quantification of the number of secondary sprouts in control or CAMSAP2b-inactivated embryos at 34 hpf, n = 64 and 56 embryos in three independent experiments. and at 41 hpf, n = 42 and 41 embryos in two independent experiments. At 41 hpf, the secondary sprouts that have fused with the neighboring primary intersegmental vessel were distinguished among the total secondary sprouts. (E) Live confocal images (Z-maximum projections) of 48 hpf Tg(Fli1ep:Lifeact-EGFP) embryos injected with control or CAMSAP2b morpholinos; asterisks show venous sprouts forming parachordal lymphangioblast in control and CAMSAP2b morphant embryo. (F) Quantification of the number of secondary sprouts at 36 hpf in embryos injected with control or CAMSAP2b morpholinos, or co-injected with CAMSAP2b morpholinos and RNA coding for a morpholino-insensitive mutant of human CAMSAP2, n = 42, 42 and 30 embryos in two independent experiments. (G) Live confocal images (Z-maximum projections) of 48 hpf Tg(Fli1ep:Lifeact-EGFP) embryos injected with control or CAMSAP2 morpholinos showing caudal vein plexus morphology. The plot shows the number of avascular loops in the caudal vein plexus in both conditions, n = 35 and 41 embryos in three independent experiments. Data are shown using box plots; Student’s paired two-tailed t-test (B), Mann-Whitney U test (D,F,G): ***p<0.001, **p<0.01, *p<0.05.