Figure 2
Figure 2. VEGF induction promotes VP, which is increased by PLCβ3 knockdown. Microangiography was performed on 3-dpf zebrafish with red-permeabilizing tracer and green ISV marker (A,B,D,F-G). (A) Control and VEGF-induced zebrafish were imaged in real time at the indicated time points. Three-dimensional rotating images are shown in supplemental Videos 1 and 2. (B) Basal, acute, and chronic (0, 1, and 3 VEGF inductions, respectively) VP was assessed at 3 dpf and representative images are shown. (C) MO-mediated knockdown of c-src was confirmed by immunoblotting in 3-dpf zebrafish with α-tubulin as a loading control. Densitometric analysis revealed 72% knockdown of c-src. (D) Representative images of extravasated Texas Red-dextran are shown in control; VEGF-induced, control MO-injected; and VEGF-induced, c-src MO–injected 3-dpf zebrafish. (E) Control MO- and PLCβ3 MO–injected 3-dpf zebrafish cDNA was used for PCR to demonstrate a molar ratio of 39% wild-type (WT) to 61% PLCβ3 MO PCR product. (F) Control; VEGF-induced, control MO–injected; and VEGF-induced, PLCβ3 MO–injected zebrafish were imaged in real time and representative images are shown. (G) Surface projection representation of confocal live imaging performed at the indicated times on a set of similarly treated zebrafish. Pink arrows indicate tracer extravasated directly from ISVs; white arrows, tracer within diagonal and horizontal lymphatic vessels. See supplemental Videos 3 through 5 for live fluorescence microscopy and supplemental Videos 6 through 8 for real-time surface projection confocal imaging. (H) Quantitation of confocal time-lapse imaging. Open squares indicate controls (n = 4); closed circles, VEGF-induced, control MO (n = 4); and open diamonds, VEGF-induced, PLCβ3 MO (n = 5). *P < .05 for control versus VEGF-induced, control MO or VEGF-induced, control MO versus VEGF-induced, PLCβ3 MO. Error bars represent SD. Images depicted in panels A, B, D, and F were obtained using a Zeiss Apitome microscope equipped with a Fluar 5×, 0.25 numerical aperture lens at room temperature. Images shown in panel G were acquired using a Zeiss LSM 780 confocal microscope equipped with an LD Plan Neofluar 40×, 0.6 numerical aperture lens at room temperature.

VEGF induction promotes VP, which is increased by PLCβ3 knockdown. Microangiography was performed on 3-dpf zebrafish with red-permeabilizing tracer and green ISV marker (A,B,D,F-G). (A) Control and VEGF-induced zebrafish were imaged in real time at the indicated time points. Three-dimensional rotating images are shown in supplemental Videos 1 and 2. (B) Basal, acute, and chronic (0, 1, and 3 VEGF inductions, respectively) VP was assessed at 3 dpf and representative images are shown. (C) MO-mediated knockdown of c-src was confirmed by immunoblotting in 3-dpf zebrafish with α-tubulin as a loading control. Densitometric analysis revealed 72% knockdown of c-src. (D) Representative images of extravasated Texas Red-dextran are shown in control; VEGF-induced, control MO-injected; and VEGF-induced, c-src MO–injected 3-dpf zebrafish. (E) Control MO- and PLCβ3 MO–injected 3-dpf zebrafish cDNA was used for PCR to demonstrate a molar ratio of 39% wild-type (WT) to 61% PLCβ3 MO PCR product. (F) Control; VEGF-induced, control MO–injected; and VEGF-induced, PLCβ3 MO–injected zebrafish were imaged in real time and representative images are shown. (G) Surface projection representation of confocal live imaging performed at the indicated times on a set of similarly treated zebrafish. Pink arrows indicate tracer extravasated directly from ISVs; white arrows, tracer within diagonal and horizontal lymphatic vessels. See supplemental Videos 3 through 5 for live fluorescence microscopy and supplemental Videos 6 through 8 for real-time surface projection confocal imaging. (H) Quantitation of confocal time-lapse imaging. Open squares indicate controls (n = 4); closed circles, VEGF-induced, control MO (n = 4); and open diamonds, VEGF-induced, PLCβ3 MO (n = 5). *P < .05 for control versus VEGF-induced, control MO or VEGF-induced, control MO versus VEGF-induced, PLCβ3 MO. Error bars represent SD. Images depicted in panels A, B, D, and F were obtained using a Zeiss Apitome microscope equipped with a Fluar 5×, 0.25 numerical aperture lens at room temperature. Images shown in panel G were acquired using a Zeiss LSM 780 confocal microscope equipped with an LD Plan Neofluar 40×, 0.6 numerical aperture lens at room temperature.

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