Expression profile of s1p₁ during zebrafish embryonic development. (A-G) In situ hybridization for s1p₁ in zebrafish embryos at 24 hpf (A,A2), 30 hpf (B,B2) and 52 hpf (C,C2). A2, B2 and C2 show magnifications of the tail area shown in A, B and C, respectively. NT, neural tube. (D) Lateral view of the tail at 24 hpf. (E) Cross-section from the rectangular area marked in D. (F) Cross-section of Tg(fli-egfp)^y1 zebrafish embryo counter-immunostained with anti-GFP antibody to mark blood vessels. (G) Magnification of F; both the dorsal aorta (DA) and the caudal vein plexus (CVP) show s1p₁ expression. Scale bars: in A-C2, 200 μm; in D-F, 50 μm; in G, 20 μm.

s1p₁ knockdown in zebrafish causes defects in blood vessel formation. Tg(fli-egfp)^y1 zebrafish embryos were injected with a control MO and a translation-blocking s1p₁ MO and analyzed at 3 dpf. (A,B) Bright-field images of control and s1p₁ MO embryos. Arrows in B indicate hindbrain malformations, pericardial edema and heart defects observed in the mutant. (C) Fluorescence microscopy of control and s1p₁ MO embryos at 24 hpf. Right-hand panel shows magnifications of the boxed areas on the left. (D) Measurements of ISV length (corresponding to the boxed area in C) in control and s1p₁ MO embryos (n=8 WT and 8 MO embryos, P<0.0001, error bars represent s.e.m.). (E) Quantification of ISV location relative to the midline (n=10 WT and 10 MO embryos; 6-8 ISVs from each embryo were analyzed, P<0.05, error bars represent s.e.m.). Scale bars: in C, left panel, 200 μm; in C, right panel, 100 μm.

s1p₁ MO embryos exhibit remodeling defects in the ISV and CVP. (A) Schematic showing the anatomical location of the areas shown in B and C. (B,C) Fluorescence microscopy of the ISV (B) and CVP (C) regions in WT and s1p₁ MO Tg(fli-egfp)^y1 zebrafish embryos at 24 and 48 hpf. (B2) Graph showing comparable filopodia numbers in ISV of WT and s1p₁ MO zebrafish (n_wt=11, n_s1p1MO=16, P=0.92). (C2) Graph showing comparison of the intercapillary area in the CVP between WT and s1p₁ MO zebrafish (n_wt=7, n_{s1p1 MO}=7, P=0.002, error bars represent s.e.m.). Scale bars: in B, 10 μm; in C, 20 μm.

Excessive filopodia formation leads to aberrant vascular remodeling in s1p₁ MO zebrafish and S1P₁^-/- mouse embryos. (A) Sequential confocal microscopy images showing lateral views of the CVPs from WT and s1p₁ MO Tg(fli-egfp)^y1 zebrafish embryos. Circles demarcate two sprouts, marked as 1 and 2, in WT and s1p₁ MO embryos that extend filopodia (marked with asterisks). This results in the formation of a capillary loop in the WT and in over-sprouting and fusion in the mutant. For visualization of the entire sequence, see supplementary material Movies 1 and 2. (B) Immunofluorescent staining with anti-CD31 of whole-mount forelimbs from E9.5 WT (a,b) and S1P₁^-/- (c,d) mice; b and d show magnifications of a and c, respectively. Asterisks indicate excessive filopodia in S1P₁^-/- limbs, compared with WT limbs. Scale bars: in a,c, 50 μm; in b,d, 20 μm.

Both Vegfa-dependent and -independent effects of S1p₁ on vascular development in zebrafish. Fluorescence microscopy of 48 hpf control and s1p₁ MO zebrafish embryos. Left panel: SIV of untreated (control), DMSO-treated and 5 μM SU5416-treated embryos; right panel: CVP of untreated (control), DMSO-treated and 5 μM-SU5416 treated embryos.

Rescue of the vascular phenotype by s1p1 mRNA injection. (A) Fluorescence microscope images of control (upper panel), s1p1 MO (middle) and s1p1 mRNA-injected morphant (lower panel) zebrafish embryos at 24 (left) and 48 hpf (right). (B) Quantification of ISV location relative to the midline. Both mRNA-injected (rescue experiment) and non-injected s1p₁ MO zebrafish were compared with control embryos (n=6 WT, 6 MO and 6 mRNA-injected embryos, 6-8 ISVs from each embryo were analyzed; s1p₁ MO: P<0.05; rescue experiment: P_{below midline} -0.74, P_{above midline}=0.74; error bars represent s.e.m.) (C) Comparison of the intercapillary area in the CVP between WT, slp₁MO and mRNA-injected zebrafish (n=6 in all groups; P_{s1p1 MO}=0.002, P_Rescue=0.39, error bars represent s.e.m.).

S1p1 knockdown in zebrafish causes defects in blood vessel formation. (A-C) Fluorescence microscopy of control (A) and slp₁ MO (B,C) embryos at 3 dpf. Arrows indicate subintestinal vessel (SIV), parachordal vein and caudal vein plexus. SIVs in the slp₁ MO embryo exhibit two phenotypes, namely dilated vessels (B) and ectopic sprouting (C). Scale bar: 100 μm.