spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online October 24, 2008
doi: 10.1242/10.1242/dev.022475

Development 135, 3755-3764 (2008)
Published by The Company of Biologists 2008
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in Development
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, Y. H.
Right arrow Articles by Wang, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, Y. H.
Right arrow Articles by Wang, R. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Artery and vein size is balanced by Notch and ephrin B2/EphB4 during angiogenesis

Yung Hae Kim, Huiqing Hu, Salvador Guevara-Gallardo, Michael T. Y. Lam*, Shun-Yin Fong{dagger} and Rong A. Wang{ddagger}

Laboratory for Accelerated Vascular Research, Division of Vascular Surgery, Department of Surgery, University of California, San Francisco, CA 94143, USA.


Figure 1
View larger version (58K):
[in this window]
[in a new window]

  		Fig. 1. EC-specific gain-of-function allele of Notch4 elicits DA enlargement and CV underdevelopment. (A,B) Whole-mount CD31 staining shows enlarged DA and underdeveloped CV in the trunk region of embryos expressing int3 in ECs at E9.0 (18 ss). Arrows, DA; arrowheads, anterior CV (ACV). (C,D) Higher magnifications of A, B, respectively. Arrows and white brackets, DA; arrowheads and blue brackets, ACV. (E,F) CD31 staining (red) of cross-sections of A, B, respectively, confirms enlarged DA and underdeveloped CV in embryos expressing EC-specific int3. Arrows, DA; arrowheads, ACV. (G,H) Quantitative analysis of EC distribution. Total ECs, including those in the DA, primordial CV and capillaries, were counted from cross-sections of the anterior region of E8.75 (15-16 ss) embryos. A total of 3328 and 3334 ECs were counted in control and mutant embryos, respectively. Total EC number between mutant and control is comparable (n=5, P=0.94). The proportion of ECs in DA (da, red) to primordial ACV including capillaries (p-acv, blue) is significantly increased (n=5; *P=0.02) in mutants (H) when compared with controls (G). (I,J) Whole-mount lacZ staining of the Tie2-lacZ reporter identifies head vessels at E10.5. Females were treated with tetracycline water (500 µg/ml) until E7.5, and embryos were collected at E10.5. Internal carotid arteries (yellow arrows) are enlarged, and head veins are reduced (yellow arrowheads) in embryos expressing EC-specific int3 (J). Scale bars: 600 µm in B; 200 µm in D,F,J.

 

Figure 2
View larger version (91K):
[in this window]
[in a new window]

  		Fig. 2. Notch4 gain-of-function mutation causes enlargement of DA prior to SMC recruitment and leads to vascular shunting. (A,B) Whole-mount lacZ staining for the arterial marker ephrin B2-tauLacZ in E9.5 embryos reveals an enlarged DA (bracket) in the mutant (B). (C,D) Whole-mount lacZ staining for the venous marker EphB4-tauLacZ in E9.5 embryos shows reduced staining in the CV (black arrowheads) in the mutant (D). Common CV (CCV), blue arrowheads. In A-D, anterior is upwards, dorsal is rightwards. (E,F) Vasculature of E9.5 (24 ss) embryos as revealed by ink injection. In the control (E), ink filled the DA (black arrows) evenly from the heart to the tip of the tail. In the mutant (F), ink leaked into head vessels (blue arrow) and the venous compartment (arrowheads), and failed to reach the tip of the tail (red arrow). (G,H) CD31 (green) and SM{alpha}A (red) staining of E9.0 (18 ss) embryos. DA enlargement in the mutant (H) occurs before recruitment of SMCs to the vessel walls. Scale bars: 200 µm in B,D,H; 800 µm in F. **, heart in A,B,E-H.

 

Figure 3
View larger version (72K):
[in this window]
[in a new window]

  		Fig. 3. Notch4 gain-of-function mutation promotes arterial marker expression in venous ECs. (A) CD31 (green) and BrdU (red) staining of DA cross-sections in E9.0 (13-15 ss) embryos prior to any apparent gross abnormalities in the mutant. Arrows, BrdU-labeled ECs. (B) Rate of BrdU incorporation in DA ECs at 13-15 ss suggests int3 does not affect proliferation in the enlarged DA. Data were analyzed by t-test and results are reported as mean±s.d. (n=3). A total of 2320 and 2074 ECs were counted in control and mutant embryos, respectively. (C,D) Cross-sections of E9.0 (16 ss) embryos expressing Efnb2-tauLacZ. lacZ-stained sections (blue) through the heart were counterstained with Eosin (pink). Ephrin B2 is expressed in the ACV (arrowheads) in the mutant (D) but not in the control (C). Arrows, DA. (E,F) CCV of 15 ss embryos stained for expression of Efnb2-tauLacZ (red) and EphB4 (green). Both markers are co-expressed in the mutant EC (F). (G,H) Cross-sections of 15 ss embryos after Dll4 in situ hybridization staining. Dll4 is expressed in the ACV (black arrowheads) and the CCV (blue arrowheads) in the mutant (H). Note the atretic anterior DA on the right side of the mutant, one of the occasional cases where the DA on one side was small. Scale bars: 10 µm in A; 25 µm in D,F; 100 µm in H.

 

Figure 4
View larger version (58K):
[in this window]
[in a new window]

  		Fig. 4. Notch1 loss-of-function mutation elicits smaller DA and enlarged CV regions. (A,B) CD31 (red) and EphB4 (green) staining of cross-sections of E9.0 (15 ss) embryos shows remnant DA (arrows) and expanded primordial ACV areas (arrowheads) in the mutant (B). Note the increase in EphB4-expressing ECs in the primordial ACVs and DA areas (B). (C,D) Quantitative analysis of EC distribution. Total ECs, including those in the DA, CV and capillaries, were counted from the cross-sections of the anterior region of E8.75 (12-15 ss) embryos. A total of 1336 and 1385 ECs were counted in control and mutant embryos, respectively. Total EC number between mutants and controls is comparable (n=4, P=0.52). The proportion of ECs in the DA (da, red) over primordial ACVs including capillaries (p-acv, blue) is significantly decreased (n=4; **, P=0.0007) in the mutants (D), compared with the controls (C). (E,F) Whole-mount CD31 staining shows reduced DA (arrows) and enlarged CV (arrowheads) in E8.75 (13 ss) embryos with Tie1-cre-mediated deletion of Notch1. (G,H) Higher magnifications of E, F, respectively. Arrows and white brackets, DA; arrowheads and blue brackets, ACV. Scale bars: 200 µm.

 

Figure 5
View larger version (51K):
[in this window]
[in a new window]

  		Fig. 5. Efnb2 loss-of-function mutation elicits enlarged DA and underdeveloped CV, resembling Notch gain-of-function mutant morphology. (A,B) Whole-mount CD31 staining of E9.0 (17 ss) embryos. Arrows, DA; arrowheads, CV. (C,D) Higher magnifications of A, B, respectively. Note the enlarged DA (arrows and white brackets) and underdeveloped CV (arrowheads and blue brackets) in the Efnb2-deficient embryo (B,D). (E,F) Quantitative analysis of EC distribution. Total ECs, including those in the DA, CV and capillaries, were counted from the cross-sections of the anterior region of E8.75 (15-17 ss) embryos. A total of 2122 and 1714 ECs were counted in control and mutant embryos, respectively. Total EC number between mutants and controls is decreased (n=3, P=0.02). The proportion of ECs in DA (da, red) over primordial ACVs (p-acv, blue) is significantly increased (n=3; *P=0.02) in mutants (F), when compared with controls (E). (G,H) CD31 (red) and EphB4 (green) staining of cross-sections of E8.75 (15 ss) embryos. EphB4+ ECs are present in the DA (arrow) of the enlarged Efnb2-deficient DA (H). (I,J) Whole-mount CD31 staining shows enlarged DA and reduced CV in E8.75 (16 ss) embryos with Tie1-cre-mediated deletion of Efnb2 (J). (K,L) Higher magnifications of I, J, respectively. Arrows and white brackets, DA; Arrowheads and blue brackets, ACV. Scale bars: 200 µm.

 

Figure 6
View larger version (72K):
[in this window]
[in a new window]

  		Fig. 6. Ephb4 loss-of-function mutation elicits DA enlargement and CV underdevelopment resembling Efnb2 loss-of-function and Notch gain-of-function mutant morphology. (A,B) Whole-mount CD31 staining of E9.5 (20 ss) embryos. (C,D) Higher magnifications of A, B, respectively. Note the enlarged DA (arrows and white brackets) and underdeveloped ACV (arrowheads and blue brackets) in the Ephb4-deficient embryo (B,D). (E,F) CD31 (red) and ephrin B2-H2BGFP (green) staining of E9.5 (22 ss) embryo cross-sections. The enlarged mutant DA contains ephrin B2- ECs (F, black arrowheads) not seen in the control (E). Nuclei were stained with DAPI (blue). (G,H) CD31 (red) and EphB4-tauLacZ (green) staining of E9.5 (20 ss) embryo cross-sections. Note that the enlarged mutant DA (arrow) contains EphB4-tauLacZ+ ECs (H) not seen in the control (G). Scale bars: 200 µm in B,D; 100 µm in F,H.

 

Figure 7
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 7. Notch and ephrin-B2/EphB4 pathways regulate the balanced anterior DA and CV morphogenesis. (A) Summary of DA and CV phenotypes at ~E9.0. In wild type, all ECs in the DA express ephrin B2 (red) and all ECs in the CV express EphB4 (blue). In the gain-of-function Notch mutants, the DA is enlarged whereas the CV is reduced, and cells in the CV, in addition to the DA, express ephrin B2. Some CV cells co-express ephrin B2 and EphB4 (purple striped). The ratio of arterial to venous ECs is increased. In the loss-of-function Notch mutant, the DA is reduced while the CV is enlarged, and some ECs in the DA, in addition to the CV, express EphB4. The ratio of arterial to venous ECs is reduced. In both loss-of-function Efnb2 and Ephb4 mutants, the DA is enlarged, whereas the CV is reduced. The enlarged DA bears some ECs with venous identity, ephrin B2- and EphB4+ (blue). The CV size is reduced and its ECs express EphB4. (B) Proposed model depicts the Notch and ephrin B2/EphB4 pathways as molecular regulators in the balanced growth of the DA and CV. Alterations in the size of one type of vessel are accompanied by reciprocal changes in the other. Notch signaling controls this equilibrium by promoting arterial differentiation, thereby dictating the ratio of arterial to venous ECs. Ephrin B2/EphB4 signaling regulates this balance by sorting differential ECs into the respective vessels.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2008