Artery and vein size is balanced by Notch and ephrin B2/EphB4 during angiogenesis - PubMed (original) (raw)

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

Yung Hae Kim et al. Development. 2008 Nov.

Abstract

A mutual coordination of size between developing arteries and veins is essential for establishing proper connections between these vessels and, ultimately, a functional vasculature; however, the cellular and molecular regulation of this parity is not understood. Here, we demonstrate that the size of the developing dorsal aorta and cardinal vein is reciprocally balanced. Mouse embryos carrying gain-of-function Notch alleles show enlarged aortae and underdeveloped cardinal veins, whereas those with loss-of-function mutations show small aortae and large cardinal veins. Notch does not affect the overall number of endothelial cells but balances the proportion of arterial to venous endothelial cells, thereby modulating the relative sizes of both vessel types. Loss of ephrin B2 or its receptor EphB4 also leads to enlarged aortae and underdeveloped cardinal veins; however, endothelial cells with venous identity are mislocalized in the aorta, suggesting that ephrin B2/EphB4 signaling functions distinctly from Notch by sorting arterial and venous endothelial cells into their respective vessels. Our findings provide mechanistic insight into the processes underlying artery and vein size equilibration during angiogenesis.

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Figures

Figure 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 (18ss). Arrows, DA; arrowheads, anterior CV (ACV). C, D, Higher magnifications of panels 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 (15–16ss) 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), as compared to 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, 600μm (B) 200 μm (D, F, J).

Figure 1. EC-specific gain-of-function allele of Notch4 elicits DA enlargement and CV underdevelopment

Figure 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. A–D, Anterior is up, dorsal is right. E, F, Vasculature of e9.5 (24ss) 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 SMαA (red) staining of e9.0 (18ss) embryos. DA enlargement in the mutant (H) occurs before recruitment of SMCs to the vessel walls. Scale bars, 200 μm (B, D, H); 800 μm (F).

Figure 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 (13–15ss) embryos prior to any apparent gross abnormalities in the mutant. Arrows, BrdU-labeled ECs. B, Rate of BrdU incorporation in DA ECs at 13–15ss suggests int3 does not affect proliferation in the enlarged DA. Data were analyzed by _t_-test and results are reported as mean±SD (_N_=3). A total of 2320 and 2074 ECs were counted in control and mutant embryos, respectively. C, D, Cross-sections of e9.0 (16ss) embryos expressing ephrin-B2-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 15ss embryos stained for expression of ephrin-B2-tauLacZ (red) and EphB4 (green). Both markers are co-expressed in the mutant EC (F). G, H, Cross-sections of 15ss 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 (A); 25 μm (D, F); 100 μm (H).

Figure 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 (15ss) 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 (12–15ss) 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 to the controls (C). E, F, Whole-mount CD31 staining shows reduced DA (arrows) and enlarged CV (arrowheads) in e8.75 (13ss) embryos with Tie1-cre-mediated deletion of Notch1. G, H, Higher magnifications of panels E, F, respectively. Arrows and white brackets, DA; arrowheads and blue brackets, ACV. Scale bars, 200 μm.

Figure 5. ephrin-B2 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 (17ss) embryos. Arrows, DA; arrowheads, CV. C, D, Higher magnifications of panels A, B, respectively. Note the enlarged DA (arrows and white brackets) and underdeveloped CV (arrowheads and blue brackets) in the ephrin-B2 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 (15–17ss) 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), as compared to controls (E). G, H, CD31 (red) and EphB4 (green) staining of cross-sections of e8.75 (15ss) embryos. EphB4+ ECs are present in the DA (arrow) of the enlarged ephrin-B2 deficient DA (H). I, J, Whole-mount CD31 staining shows enlarged DA and reduced CV in e8.75 (16ss) embryos with Tie1-cre-mediated deletion of ephrin-B2. K, L, Higher magnifications of panels I, J, respectively. Arrows and white brackets, DA; Arrowheads and blue brackets, ACV. Scale bars, 200 μm.

Figure 5. ephrin-B2 loss-of-function mutation elicits enlarged DA and underdeveloped CV, resembling Notch gain-of-function mutant morphology

Figure 6. EphB4 loss-of-function mutation elicits DA enlargement and CV underdevelopment resembling ephrin-B2 loss-of-function and Notch gain-of-function mutant morphology

A, B, Whole-mount CD31 staining of e9.5 (20ss) embryos. C, D, Higher magnifications of panels 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 (22ss) embryo cross-sections. The enlarged mutant DA contains ephrin-B2− ECs (F, open 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 (20ss) 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 (B, D); 100 μm (F, H).

Figure 7. Notch and ephrin-B2/EphB4 pathways regulate the balanced anterior DA and CV morphogenesis

A, Summary of DA and CV phenotypes at approximately 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 while 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). 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 ephrin-B2 and EphB4 mutants, the DA is enlarged, while 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.

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Artery and vein size is balanced by Notch and ephrin B2/EphB4 during angiogenesis - PubMed (original) (raw)