roundabout4 is essential for angiogenesis in vivo

Abstract

Stereotypical patterns of vascular and neuronal networks suggest that specific genetic programs tightly control path determination and, consequently, angiogenesis and axon-guidance mechanisms. Our study focuses on one member of the roundabout family of receptors, which traditionally mediate repulsion from the midline. Here, we characterize a fourth member of this family, roundabout4 (robo4), which is the predominant roundabout (robo) that is expressed in embryonic zebrafish vasculature. Gene knockdown and overexpression approaches show that robo4 is essential for coordinated symmetric and directed sprouting of intersomitic vessels and provide mechanistic insights into this process. Also, human robo4 gene functionally compensates for loss of robo4 gene function, suggesting evolutionary conservation. This article reports an endothelial-specific function for a robo gene in vertebrates in vivo.

Fig. 1.

Expression patterns of robo4 in development. A montage of robo4 whole-mount in situ staining is shown, with 16 (A), 19 (B), 20 (C), 23 (D), 24 (E), and 20 (F) som. (G) High power of F.(H) High power of I.(I) A 22-som telencephalon (t), hindbrain (hb), and neural tube (nt). J is 28-29 hpf. Arrowhead in G indicates expression in angioblasts; asterisk in H shows ISV expression. robo4 (K), G-RCFP (L), DAPI (M), and merge (N) show two-color staining of frozen 20-μm anterior trunk sections of vegfr2-G-RCFP 25-hpf embryos. The arrowhead and asterisk in K-N indicate ISV and axial vessels, respectively. Images were taken with a ×20 (A-E), ×10 (F, I, and J), ×40 (G and H), and ×20 (K-N) lens with ×2.5 zoom.

Fig. 2.

Splice MO targeting and endothelial marker analysis of MO-injected embryos. (A) The exon-intron genomic structure from exon 7-12 is shown. Splice MO1 and MO2 target the predicted TM domains. Total RNA was extracted from uninjected (lane 1), MO (lane 2; MO1, upper gel; MO2, lower gel), and msMO (lane 3; msMO1, upper gel; msMO2, lower gel) embryos at 24 hpf, and RT-PCR was performed. Black, gray, and dotted arrows indicate the alternative transcripts that were generated in MO-targeted embryos (lane 2), the black arrowhead indicates a common untargeted bands in lanes 1 and 3, and the gray arrowhead points to 1.5-kb band in lane 2. E, exon. (B) Structural details of predicted protein products generated from aberrant splicing are shown. Hexagons, pentagons, white bar, and black bars represent IgG, FN, TM, and CC domains, respectively. Double staining with flk digoxigenin RNA (blue) and Ab to znp-1 (C-E) (brown) or acetylated tubulin (F and G) in the trunk region of 22-24 som is shown. (C-G) Magnified images of the trunk region, showing WT (C and F), MO1-injected (8 ng) (D and G), and MO2-injected (8 ng) (E) embryos. Asterisk indicates location of ISV. (H) Quantification of embryos with ISV defects. WT (I), MO1 (J), and msMO1 (K) show in situ staining for cdh5 marker at 24 hpf. Anterior is to the left.

Fig. 3.

robo4 overexpression results in misdirected or truncated flk⁺ ISV from DA. WT (A), YFP (B), and robo4 (C) RNA-injected embryos at 22-24 som are shown. (D) Quantitation of overexpression is shown for WT (n = 36), YFP (n = 29), and robo4 (n = 28). Anterior is to the left.

Fig. 4.

robo4 gene knockdown affects flk⁺ ISV and rescue of defects by hrobo4. Splice MOs alone (12 ng) or in combination with hrobo4 RNA (150 pg) were microinjected into Tg(vegfr2: G-RCFP)^y10 zebrafish embryos at the one-cell stage, and images were taken at 22-24 som and 48 hpf. From left to right, images are MO, msMO, and MO+hrobo4-injected embryos. A-C and G-I show embryos at 22-24 som, whereas D-F and J-L show embryos at 48 hpf. (A and D) MO1 (n = 31). (G and J) MO2 (n = 28). (B and E) msMO1 (n = 31). (H and K) msMO2 (n = 54). (C and F) MO1+hrobo4 (n = 39). (I and L) MO2+hrobo4 (n = 46). (M) Quantitation for rescue experiments with hrobo4 (A-L). (N) Quantitation of embryos injected for WT (n = 124), MO2 (n = 119), MO2+robo1 (n = 82), MO2+robo2 (n = 115), and MO2+robo4 (n = 91). (O) Quantitation comparison of overexpression experiment for WT (n = 118), hrobo4 (n = 71), and robo4 (n = 91) RNA-injected (150 pg) embryos. A defective embryo was counted as three or more missing ISV. Anterior is to the left.