Figure 3
Figure 3. CDS2 deficiency blocks VEGFA signaling transduction. (A) cds2 knockdown results in decreased arterial and increased venous marker expression in zebrafish embryos. Wild-type zebrafish embryos were injected at the 1- to 2-cell stage with either control morpholino (ctrl; 6 pg), low-dose (1.5 pg) vegfaa morpholino, full-dose (1.8 pg) cds2 morpholino, or full-dose (6 pg) vegfaa morpholino. Trunk tissues were collected at 24 hours after fertilization (hpf) and used for quantitative RT-PCR with primers amplifying either arterial marker dll4, venous marker flt4 (vegfr3), or β-actin (internal control). Red columns show dll4 expression, and blue columns show flt4 expression; both were normalized to levels in control morpholino-injected animals. (B) Low-dose vegfa morphants phenocopy cds2 morphants. Quantitation of the intersegmental vessel (ISV) phenotypes of 48-hpf Tg(fli-EGFP)y1 wild-type zebrafish injected as in panel A. The bars show percentages of ISVs that have failed to sprout (blue), ISVs that have grown only up to the horizontal myoseptum half way up the trunk (red), and ISVs that have grown all the way to the dorsal trunk to form the dorsal longitudinal anastomotic vessel (DLAV; yellow). The number of embryos (n = 50) counted for each injection is shown at the top of each column, with 10 trunk segments counted per embryo (for a total of 500 segments). (C) CDS2 deficit causes reduced endothelial cell proliferation. Tg(fli-nEGFP)y7 transgenic zebrafish embryos were injected with morpholinos as in panel A. The average number of endothelial cells present in the 3 posterior-most trunk segments was measured in each of 10 embryos at 26 hpf. (D) Reduced VEGFA signaling in cds2 and vegfa morphants. Wild-type zebrafish embryos injected as in panel A were collected at 24 hpf for trunk tissue harvest and immunoanalysis with the use of anti-total ERK1/2 and anti–phospho-ERK1/2 antibodies. The level of activated phospho-ERK is normalized to total ERK level. ERK activation level in control embryos was set to 100%. Ctrl indicates control; CDS, CDP-diacylglycerol synthetase; and WT, wild-type.

CDS2 deficiency blocks VEGFA signaling transduction. (A) cds2 knockdown results in decreased arterial and increased venous marker expression in zebrafish embryos. Wild-type zebrafish embryos were injected at the 1- to 2-cell stage with either control morpholino (ctrl; 6 pg), low-dose (1.5 pg) vegfaa morpholino, full-dose (1.8 pg) cds2 morpholino, or full-dose (6 pg) vegfaa morpholino. Trunk tissues were collected at 24 hours after fertilization (hpf) and used for quantitative RT-PCR with primers amplifying either arterial marker dll4, venous marker flt4 (vegfr3), or β-actin (internal control). Red columns show dll4 expression, and blue columns show flt4 expression; both were normalized to levels in control morpholino-injected animals. (B) Low-dose vegfa morphants phenocopy cds2 morphants. Quantitation of the intersegmental vessel (ISV) phenotypes of 48-hpf Tg(fli-EGFP)y1 wild-type zebrafish injected as in panel A. The bars show percentages of ISVs that have failed to sprout (blue), ISVs that have grown only up to the horizontal myoseptum half way up the trunk (red), and ISVs that have grown all the way to the dorsal trunk to form the dorsal longitudinal anastomotic vessel (DLAV; yellow). The number of embryos (n = 50) counted for each injection is shown at the top of each column, with 10 trunk segments counted per embryo (for a total of 500 segments). (C) CDS2 deficit causes reduced endothelial cell proliferation. Tg(fli-nEGFP)y7 transgenic zebrafish embryos were injected with morpholinos as in panel A. The average number of endothelial cells present in the 3 posterior-most trunk segments was measured in each of 10 embryos at 26 hpf. (D) Reduced VEGFA signaling in cds2 and vegfa morphants. Wild-type zebrafish embryos injected as in panel A were collected at 24 hpf for trunk tissue harvest and immunoanalysis with the use of anti-total ERK1/2 and anti–phospho-ERK1/2 antibodies. The level of activated phospho-ERK is normalized to total ERK level. ERK activation level in control embryos was set to 100%. Ctrl indicates control; CDS, CDP-diacylglycerol synthetase; and WT, wild-type.

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