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First published online 11 June 2008
doi: 10.1242/dev.020131

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c-myc in the hematopoietic lineage is crucial for its angiogenic function in the mouse embryo

Chen He^1,*,, Huiqing Hu^1,*, Rickmer Braren^1,*,, Shun-Yin Fong^1,, Andreas Trumpp², Timothy R. Carlson^1,¶ and Rong A. Wang^1,**

¹ Pacific Vascular Research Laboratory, Division of Vascular Surgery, Departments of Surgery and Anatomy, University of California, San Francisco, CA 94143, USA.
² Genetics and Stem Cell Laboratory, Swiss Institute for Experimental Cancer Research, Ch. des Boveresses 155, CH-1066, Epalinges, Switzerland and Ecole Polytechnique Fédérale de Lausanne (EPFL), School of Life Sciences, CH-1015 Lausanne, Switzerland.

View larger version (122K): [in this window] [in a new window]   Fig. 1. c-myc null and Sox2-Cre-mediated c-myc deletion result in anemia, vascular defects and lethality. (A-H) Anti-CD31-stained whole-mount embryos (A,B,E,F) and yolk sacs (C,D,G,H) dissected at 8.75 dpc (A-D) or 10.25 dpc (E-H). (I,J) Cross-sections (5 µm) of anti-CD31-stained whole-mount embryos. (K-N) Live embryos dissected at 11.5 dpc. (O-T) Anti-CD31-stained whole-mount yolk sacs (O,P) and heads (Q-T) at 11.5 dpc. Note the less developed vessels with reduced caliber (yolk sac) and number of branches (head) in the mutant animal. Da, dorsal aorta; ISV, intersomitic vessel; pp, primitive plexus; vv, vitelline vessels; CA, carotid artery; HV, primary head veins; CV, cardinal vein; nt, neural tube; Ph, pharynx. Scale bars: 200 µm.

View larger version (74K): [in this window] [in a new window]   Fig. 2. Tie2-Cre-mediated c-myc deletion results in anemia, vascular defects and lethality. (A-F) Live embryos in yolk sac dissected at 9.5 dpc (A,B), 10.5 dpc (C,D) or 11.5 dpc (E,F). (G-L) Anti-CD31-stained whole-mount yolk sacs (G,H) and heads (I-L) at 11.5 dpc. Vasculature in the mutant is less developed with reduced caliber and number of branches. Scale bar: 200 µm. (M,N) Efficient c-Myc deletion by Tie2-Cre did not increase N-Myc expression in 10.5 dpc primary ECs. The percentage of c-Myc-positive ECs is indicated in M. The percentage of N-Myc positive ECs is shown in N. `n' represents the number of cells counted in each assay. (O) Peripheral blood cell counts at 10.5 dpc from control (n=19) and Tie2-Cre;c-mycflox/- mutant (n=7) embryos. The numbers above the brackets show the fold decrease in the mutants compared with controls. *P<0.01; **P<0.05 by Student's t-test.

View larger version (111K): [in this window] [in a new window]   Fig. 3. Tie2-Cre-mediated c-myc deletion did not affect allantoic or placental vasculature but diminished hemogenic endothelial cells in the aorta. (A,B) Allantoic explants stained for CD31 show no obvious difference between the control and mutant. (C,D) Placental vasculature at 11.5 dpc revealed by Tie2-lacZ reporter assay shows no obvious differences between the control and mutant. Arrowheads indicate the blood vessels. (E,F) H&E stained cross-section of dorsal aorta shows hemogenic endothelial cells (arrows) in the control but not in the mutant. Scale bars: 50 µm.

View larger version (62K): [in this window] [in a new window]   Fig. 4. Vav-iCre-mediated c-myc deletion results in cytopenia, vascular defects and lethality. (A,B) Live embryos at 11.5 dpc. (C-H) Anti-CD31-stained whole-mount yolk sacs (C,D) and heads (E-H) at 11.5 dpc. CA, internal carotid arteries; HA, primary head veins. Scale bars: 200 µm. (I) Peripheral blood cell counts of control (n=16) and Vav-iCre;c-mycflox/- mutant (n=8) embryos. The numbers above brackets show the fold decrease in the mutants compared with controls. *P<0.01 and **P<0.05 by Student's t-test.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Tie1-Cre-mediated c-myc deletion results in partial survival. Peripheral blood cell counts from control (n=20) and Tie1-Cre;c-mycflox/- mutant (n=6) embryos. The numbers above brackets show the fold decrease in the mutants over controls. *P<0.01 and **P<0.05 by Student's t-test.

View larger version (29K): [in this window] [in a new window]   Fig. 6. c-myc-deficient ECs exhibit no detectable defects in proliferation, survival, morphology, motility or tube formation. (A) BrdU+ ECs in the dorsal aortae. Cross-sections were stained with anti-CD31 and anti-BrdU antibodies. (B) Fold increases in apoptosis in yolk sac ECs and non-ECs. Cross-sections of Tie2-Cre;c-mycflox/- (at 10.5 dpc) and Vav-iCre;c-mycflox/- (at 11.5 dpc) mutant (black bars) and control (white bars) yolk sacs were stained with anti-CD31 and for TUNEL. TUNEL+ CD31+ or TUNEL+ CD31- cells were quantified. **P<0.05; *P<0.01 by Student's t-test. Data were collected from four pairs of embryos from two different litters. (C,D) Phalloidin (green), anti-CD31 (red) and DAPI (blue) stained ECs isolated from 10.5 dpc embryos. (E-G) Random movement of cultured ECs from 10.5 dpc embryos monitored by timelapse microscopy. Representative EC migration paths are shown (E) and quantitative analysis demonstrated no significant difference in either the net-path of cell migration (F, P=0.083) or in the average migration speed (G, P=0.090) in two independent experiments. The ANOVA two-tailed test was used in the statistical analysis. (H-K) No detectable defects in EC tube formation. mycflox/flox ECs were purified from adult vena cava. Cells 48 hours after infection with GFP fusion Cre-expressing adenovirus AdCreGFP or GFP expression control virus AdGFP were plated on Matrigel, and cultures were photographed 20 hours later (H,I). The results from four independent experiments were analyzed by two-tailed t-test (J, P=0.1916). Green, GFP; red, DiI-AcLDL labeled ECs. Genomic DNA PCR analysis demonstrates the deletion of c-myc floxed sequence by AdCreGFP but not AdGFP (K). Scale bar: 10 µm in C,D; 200 µm in H,I.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Expression of proangiogenic factors in Vav-iCre;c-mycflox/- and Tie2-Cre;c-mycflox/- mutant embryos. (A) mRNA levels of proangiogenic factors in Vav-iCre;c-mycflox/- at 11.25 dpc. Real-time PCR was performed using total RNA from five mutants and 15 control embryos (Vav-iCre;c-mycflox/+, c-myc+/- or Vav-iCre;c-myc+/-). The statistical significance of the differences in means between controls (relative expression=1) and mutants are listed by their P values. (B) Increase in VEGF protein levels by ELISA in control and Tie2-Cre;c-mycflox/- embryos (n=5). VEGF levels in the Tie2-Cre mutant embryos were significantly elevated (*P<0.01).

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