Targeting efficacy and specificity of syndecan-2 morpholinos. Messenger RNA synthesized from the syndecan-2 5′UTR-GFP fusion construct was coinjected with syndecan-2 MO1 or UROD MO into zebrafish embryos. GFP expression was assessed at 24 hpf. (A-D) GFP expression was analyzed under the bp-GFP filterset (original magnification, × 2.5). (A) Embryos injected with 120 pg syndecan-2 5′UTR-GFP RNA. (B) Embryos injected with 120 pg syndecan-2 5′UTR-GFP RNA and 5 ng syndecan-2 MO1. (C) Embryos injected with 120 pg syndecan-2 5′UTR-GFP RNA and 5 ng UROD MO. (D) Uninjected wild-type embryos. (E) Quantitation of GFP activity. (F) Embryos were injected with syndecan-2 MO1, syndecan-2 d4 MO1, or syndecan-2 MO3. Injections of syndecan-2 MO1 or MO3 resulted in a high penetrance of vascular phenotypes. In contrast, a strong attenuation of vascular phenotypes was observed in embryos injected with d4 MO1. (G-J) In situ hybridization (original magnification, × 10). (G) In situ analysis of flk-1 expression in a wild-type embryo. (H) flk-1 expression in a syndecan-2 MO–injected embryo, showing a characteristic lack of intersegmental expression. (I) Embryo coinjected with syndecan-2 MO and a mixed solution of zebrafish syndecan-2 and EGFP expression constructs, showing partial sprouts of intersegmental vessels (arrowheads). (J) Embryo coinjected with syndecan-2 MO and EGFP expression construct. No new intersegmental expression was observed. (K) Results from 6 independent rescue experiments. The group coinjected with syndecan-2 MO and syndecan-2 DNA showed a significant increase in the number of embryos showing intersegmental expression of flk-1, compared with the group injected with the syndecan-2 MO only. (L) Results from 4 injection experiments, showing no effect on flk-1 expression by the control DNA (EGFP). Error bars indicate standard errors.