Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 2. ccbe1, vegfc and vegfr3 genetically interact in double and triple heterozygous animals. (A-H) Confocal projections of Tg(fli1a:EGFP; kdr- l:mCherry) show grossly unaltered overall morphology and blood vasculature in ccbe1hu3613 (B), vegfr3hu4602 (E) and vegfchu5055 (F) mutants compared with wild type (A). The TD (C, arrows) is absent in ccbe1hu3613 (D), vegfr3 hu4602 (G) and vegfc hu5055 (H) mutants (asterisks). (I-K) ccbe1, vegfc and vegfr3 genetically interact in double heterozygote embryos, which display lymphatic defects. Offspring from vegfc+/−and vegfr3+/−carriers give rise to 28% of embryos (n=28/99) with a TD length of ≤50%. This population is significantly enriched (71%; n=20/28; P<0.0001) in double heterozygotes (I). Similarly in ccbe1+/−and vegfr3+/−

Article Snippet: Western blotting was performed with antibodies targeting VEGFC (R&D Systems, BAF752) or CCBE1 (Abcam, ab101967).

Techniques:

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 3. Phenotypes driven by ectopic Vegfc/Vegfr3 signaling are suppressed in ccbe1-deficient embryos. (A) At 72 hpf, dll4 morphants display an arterial hyperbranching phenotype (arrow) driven by increased Vegfc/Vegfr3 signaling in the transgenic Tg(fli1a:EGFP) line. This phenotype was suppressed in ccbe1hu3613 mutants. Eighty-one per cent of MO-dll4 injected embryos displaying wild-type or mild phenotypes were ccbe1 mutants (n=17/21), whereas the population displaying the most severe phenotype was mainly composed of wild-type or heterozygous siblings (83%; n=139/168). (B) In dll4 morphants, arteries are sensitized to increased vegfc expression during primary sprouting. Arteries in MO-dll4, vegfc mRNA-injected embryos display aberrant, ectopic turning (arrow) as early as 30 hpf. Embryos from ccbe1 carrier incrosses, injected with 100 ng vegfc mRNA and 5 ng MO-dll4, were sorted into the phenotypic categories ‘wild type’ and ‘severe’. Genotyping revealed that 70% of the embryos displaying wild-type morphology were ccbe1 mutants (n=19/27). By contrast, the population affected by the most severe phenotype was composed of 83% wild-type or heterozygous siblings (n=122/147). (C) Confocal projections of Tg(fli1a:EGFP; flt1:tomato; hsp70l:Gal;4XUAS:vegfc) embryos show that endothelial cells in heat-shocked embryos display aberrant ectopic branching at 72 hpf. The ectopic endothelial cells are venous derived (flt1:tomato negative, arrow in Ciii). Heat-shocked embryos that were injected with 2.5 ng of MO-ccbe1 do not show this phenotype (asterisks). Scoring of the number of aberrant vISVs per heat-shocked embryo showed a significant rescue (0.12 in MO-ccbe1 injected n=22, versus 4.76 in uninjected controls n=25; P<0.0001) of the phenotype.

Article Snippet: Western blotting was performed with antibodies targeting VEGFC (R&D Systems, BAF752) or CCBE1 (Abcam, ab101967).

Techniques: Transgenic Assay, Injection, Expressing, Derivative Assay

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 4. Vegfr3-dependent Erk signaling requires ccbe1 during the induction of secondary sprouting in zebrafish. (A) Analysis of phospho-Erk (P-Erk) expression in 32 hpf embryos. P-Erk (green) and fli1a:EGFP (white) images (lateral view) show P-Erk detected broadly in whole-mount and cross-sectioned (right-hand panels, merge upper, P-Erk lower) control embryos. Signal was increased in Vegfc-induced (dll4 MO + vegfc mRNA-injected) embryos in the posterior cardinal vein (n=8/8; Vegfc-induced embryos all showed ectopic expression in the ventral wall of the PCV). Cross section merged channel images shown in a and b, P-Erk only in c and d. Treatment with the Erk inhibitor PD98059 led to a reduction in all P-Erk staining. Arrows indicate P-Erk expression in the dorsal PCV. DA (dorsal aorta) and PCV (posterior cardinal vein) are indicated. (B) Comparison of P-Erk staining in control uninjected (left), MO-vegfr3 and MO-ccbe1 embryos. Upper panels are merged images and lower P-Erk only, viewed laterally (left) and cross-sectioned (right). Cross sections (right) are from separate embryos. Arrows indicate P-Erk expression in the dorsal PCV. DA and PCV are indicated. (C) Quantification of P-Erk-positive cells in the cardinal vein located in the dorsal compared with ventral wall (left-hand graph). Scores through individual sections of z-stack images from 12 control embryos, scored laterally across three somites in the trunk. Quantification of P-Erk-positive cells in the cardinal vein in control and MO-injected conditions (right-hand graph) (scores from n=10 control embryos, n=13 MO-vegfr3-injected and n=15 MO-ccbe1-injected embryos). (D) Immunoprecipitation (IP) and western blot (IB) detection of phosphorylated Vegfr3 at 32 hpf in wild type and in ccbe1, vegfr3, vegfc morphant and vegfc mRNA-injected embryos. The level of phosphorylated Vegfr3 is markedly reduced in ccbe1, vegfr3 and vegfc morphants, but is increased in vegfc-mRNA injected (500 ng) embryos compared with wild type (D, upper blot, IP for phospho-Vegfr3 and IB detection with phospho-Vegfr3). Loading controls were: the IgG light chain [IgG(l)] present in all blots after IP (D, middle blot), and Myosin to monitor protein input in IPs (D, lower blot). Quantification of Vegfr3 phosphorylation (relative to the loading control) based on three independent experiments is shown in right-hand panel. The decrease in MO-ccbe1 compared with uninjected controls is statistically significant (P<0.05). (E) qPCR analysis of the expression of ccbe1, vegfr3, vegfc, kdr and kdrl in uninjected control and MO-ccbe1-, MO-vegfc-, and MO-vegfr3-injected embryos. Error bars represent s.d. (C) or s.e.m. (D,E).

Article Snippet: Western blotting was performed with antibodies targeting VEGFC (R&D Systems, BAF752) or CCBE1 (Abcam, ab101967).

Techniques: Expressing, Control, Injection, Staining, Comparison, Immunoprecipitation, Western Blot, Phospho-proteomics

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 5. Ccbe1 enhances Vegfc-driven sprouting and regulates levels of bioactive VEGFC in vitro. (A) Confocal projections at 32 hpf of Tg(shh:ccbe1), Tg(shh:vegfc) and Tg(shh:ccbe1;shh:vegfc) in a Tg(fli1a:EGFP) background. Co- overexpression of ccbe1 and vegfc in the floorplate leads to aberrant ectopic turning of the ISVs at 32 hpf (upper panels; n=32/36; P<0.0001). At 48 hpf, ccbe1 overexpression in the floorplate does not result in any phenotype, whereas vegfc-overexpressing animals display hyperbranching of the ISVs, and enhanced endothelial cell accumulation at the horizontal myoseptum (arrowhead). ccbe1 and vegfc co-overexpression in the floorplate also leads to hyperbranching ISVs, and to a marked accumulation of endothelial cells at dorsal aspects of the embryo (arrow). (B) Western blot of 293EBNA-1 cells (stably expressing VEGFC) indicate that CCBE1 is detected in the lysate of cells transfected with CCBE1 plasmid, but not in controls. (C) An increase in the levels of all forms of VEGFC is detected in the medium of CCBE1- transfected cells, compared with control cells. The mature form of VEGFC (detected at ~23 kDa) is predominant. (C′) Relative intensity (split axis) of the different processed forms of VEGFC presented in C based on multiple exposures. Note the saturation of the mature form in C. (D) qPCR showing that CCBE1 transfection does not affect VEGFC mRNA levels in vitro in 293EBNA-1 cells stably expressing VEGF-C (D, left panel). Consistent with this, in zebrafish embryos the injection of vegfc or ccbe1 mRNA does not affect the endogenous levels of the other (D, right panel). Error bars represent s.e.m.

Article Snippet: Western blotting was performed with antibodies targeting VEGFC (R&D Systems, BAF752) or CCBE1 (Abcam, ab101967).

Techniques: In Vitro, Over Expression, Western Blot, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Control, Injection

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 6. Ectopic expression of mature VEGFC rescues secondary sprouting in ccbe1 morphants. (A) Confocal projections of Tg(fli1a:EGFP) at 54 hpf. Knock down of ccbe1 or vegfr3 leads to a loss of PLs at the horizontal myoseptum (arrowheads and asterisks). Ectopic expression of the mature form of VEGFC strongly rescues PL formation in ccbe1 morphants but not in vegfr3 morphants. Arrows indicate hyperbranched ISVs. (B) Quantification of PL formation at 54 hpf. In wild type, 98% (n=54/55) of embryos develop PLs, whereas in MO- ccbe1-injected embryos <4% (n=2/52) do. PL development is rescued to 74% (n=29/39) in ccbe1 morphants transiently overexpressing ΔNΔCVEGFC (P<0.0001). This rescue was never observed in vegfr3 morphants with all embryos devoid of PLs (n=23/23). (C) Quantification of ISV hypersprouting at 54 hpf. ISV hypersprouting was observed in wild-type embryos (93%; n=40/43), with mild reductions in ccbe1 morphants (79%; n=31/39) and vegfr3 morphants (65%; n=15/23) after ΔNΔCVEGFC overexpression.

Article Snippet: Western blotting was performed with antibodies targeting VEGFC (R&D Systems, BAF752) or CCBE1 (Abcam, ab101967).

Techniques: Expressing, Knockdown, Injection, Over Expression

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 7. Ccbe1 activates Vegfc to induce Vegfr3 signaling. Proposed model for coordination of angiogenesis by Ccbe1, Vegfc and Vegfr3 in the developing embryo. Vegfc is produced in a largely inactive full-length form that is processed and released from the cell surface/ECM in a Ccbe1- dependent manner to generate the mature, highly active form. Downstream, arteries respond in a manner dampened by Dll4-dependent suppression of Vegfr3 signaling (Hogan et al., 2009b), whereas Vegfr3 signaling in veins induces secondary angiogenesis, which produces both intersegmental veins and lymphatic vascular precursor cells.

Article Snippet: Western blotting was performed with antibodies targeting VEGFC (R&D Systems, BAF752) or CCBE1 (Abcam, ab101967).

Techniques: Produced

Journal: Circulation Research

Article Title: Distinct Pathways Regulate Expression of Cardiac Electrical and Mechanical Junction Proteins in Response to Stretch

doi: 10.1161/01.res.0000178788.76568.8a

Figure Lengend Snippet: Figure 3. Effects of stretch, exogenous VEGF, and stretch in the presence of an anti-VEGF antibody (StrVEGF Ab) on expression of Cx43 and adhesion junc- tion proteins. n4 for each condition. *P0.05 compared with control.

Article Snippet: In other experiments, cultures were incubated with 1 or more reagents including recombinant human VEGF165 (165-aa isoform of human VEGF; R&D Systems); the src kinase antagonist, 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolol[3,4d]pyrimidine (PP2) (1 nmol/L); monoclonal blocking antibodies against 1-integrins (clone Ha2/5; BP Pharmingen) or 3-integrins (clone 2C9G2; BD Pharmingen) (50 g/mL for each); or a monoclonal blocking antibody against VEGF (clone 26503; R&D Systems).

Techniques: Expressing, Control

Journal: Circulation Research

Article Title: Distinct Pathways Regulate Expression of Cardiac Electrical and Mechanical Junction Proteins in Response to Stretch

doi: 10.1161/01.res.0000178788.76568.8a

Figure Lengend Snippet: Figure 6. A, Effects of stretch on expres- sion of Cx43 and mechanical junction proteins in cells infected with virus expressing GFP-FRNK (FRNK) or GFP alone (Adv-GFP). n6 for Cx43 and N-cadherin; n2 or 3 for plakoglobin and desmoplakin. *P0.001, †P0.05 com- pared with control in each group. B, Effects of stretch-conditioned medium (CM) and exogenous VEGF in cells previ- ously infected with adenovirus express- ing GFP-FRNK (FRNK). n4 for each condition. *P0.001.

Article Snippet: In other experiments, cultures were incubated with 1 or more reagents including recombinant human VEGF165 (165-aa isoform of human VEGF; R&D Systems); the src kinase antagonist, 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolol[3,4d]pyrimidine (PP2) (1 nmol/L); monoclonal blocking antibodies against 1-integrins (clone Ha2/5; BP Pharmingen) or 3-integrins (clone 2C9G2; BD Pharmingen) (50 g/mL for each); or a monoclonal blocking antibody against VEGF (clone 26503; R&D Systems).

Techniques: Infection, Virus, Expressing, Control