Figure 6.
Figure 6. Genetic interaction between VEGF and syndecan-2 in vivo. (A-B) In situ hybridization. (A) Cross section of a wild-type embryo at 22 hpf, showing VEGF-A expression in medial somitic mesoderm (purple staining within the area as indicated by the red dashed lines). DA indicates dorsal aorta. (B) Cross section of a wild-type embryo at 22 hpf, showing syndecan-2 expression in the mesenchyme (black dashed lines) surrounding the dorsal aorta, ventral to the somitic mesoderm. Note that the region of VEGF-A expression is adjacent to the mesenchyme where syndecan-2 is expressed. (C) Summary of microangiography analysis. Low doses of syndecan-2 MO and VEGF-A MO synergize to generate embryos with vascular defects (compare column 3 with columns 1 and 2). There is no interaction between VEGF-A MO and the 4-base mismatch syndecan-2 MO (d4). The total number of embryos analyzed from 2 independent experiments is indicated. (D-F) Microangiography analysis. (D) Microangiogram of a wild type at 48 hpf. (E) Microangiogram of an embryo coinjected with syndecan-2 MO and VEGF-A MO, showing aberrant sprouting of intersegmental vessels (arrowheads) and abnormal formation of blood vessels in the tail region (arrows). (F) Microangiogram of an embryo coinjected with syndecan-2 MO and VEGF-A MO with no trunk angiogenesis (arrowheads) and reduced tail vessels (arrows). (G) Summary of in situ analysis, using ectopic expression of flk-1 to assess the extent of neovascularization in injected embryos. (H-J) In situ hybridization. (H) flk-1 expression in a wild-type embryo. (I) flk-1 expression in an embryo injected with 5 pg VEGF expression construct, labeling ectopic vessels (arrowheads). (J) flk-1 expression in an embryo coinjected with 5 pg VEGF-165 expression construct and syndecan-2 MO, demonstrating no induction of new vessels. (K) Summary of in situ analysis, assessing ectopic expression of flk-1. (L-N) In situ hybridization. (L) flk-1 expression in a wild-type embryo. (M) flk-1 expression in an embryo injected with a low dose of VEGF-165 DNA, indicating no induced new vessels. (N) flk-1 expression in an embryo coinjected with VEGF-165 DNA and syndecan-2 DNA with ectopic vessels (arrowheads). Original magnification, × 10 (A-B, H-J, L-N); × 5 (D-F). Error bars indicate SE.

Genetic interaction between VEGF and syndecan-2 in vivo. (A-B) In situ hybridization. (A) Cross section of a wild-type embryo at 22 hpf, showing VEGF-A expression in medial somitic mesoderm (purple staining within the area as indicated by the red dashed lines). DA indicates dorsal aorta. (B) Cross section of a wild-type embryo at 22 hpf, showing syndecan-2 expression in the mesenchyme (black dashed lines) surrounding the dorsal aorta, ventral to the somitic mesoderm. Note that the region of VEGF-A expression is adjacent to the mesenchyme where syndecan-2 is expressed. (C) Summary of microangiography analysis. Low doses of syndecan-2 MO and VEGF-A MO synergize to generate embryos with vascular defects (compare column 3 with columns 1 and 2). There is no interaction between VEGF-A MO and the 4-base mismatch syndecan-2 MO (d4). The total number of embryos analyzed from 2 independent experiments is indicated. (D-F) Microangiography analysis. (D) Microangiogram of a wild type at 48 hpf. (E) Microangiogram of an embryo coinjected with syndecan-2 MO and VEGF-A MO, showing aberrant sprouting of intersegmental vessels (arrowheads) and abnormal formation of blood vessels in the tail region (arrows). (F) Microangiogram of an embryo coinjected with syndecan-2 MO and VEGF-A MO with no trunk angiogenesis (arrowheads) and reduced tail vessels (arrows). (G) Summary of in situ analysis, using ectopic expression of flk-1 to assess the extent of neovascularization in injected embryos. (H-J) In situ hybridization. (H) flk-1 expression in a wild-type embryo. (I) flk-1 expression in an embryo injected with 5 pg VEGF expression construct, labeling ectopic vessels (arrowheads). (J) flk-1 expression in an embryo coinjected with 5 pg VEGF-165 expression construct and syndecan-2 MO, demonstrating no induction of new vessels. (K) Summary of in situ analysis, assessing ectopic expression of flk-1. (L-N) In situ hybridization. (L) flk-1 expression in a wild-type embryo. (M) flk-1 expression in an embryo injected with a low dose of VEGF-165 DNA, indicating no induced new vessels. (N) flk-1 expression in an embryo coinjected with VEGF-165 DNA and syndecan-2 DNA with ectopic vessels (arrowheads). Original magnification, × 10 (A-B, H-J, L-N); × 5 (D-F). Error bars indicate SE.

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