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Dll4 and Notch signalling couples sprouting angiogenesis and artery formation

Nature Cell Biology volume 19, pages 915–927 (2017)Cite this article

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Abstract

Endothelial sprouting and proliferation are tightly coordinated processes mediating the formation of new blood vessels during physiological and pathological angiogenesis. Endothelial tip cells lead sprouts and are thought to suppress tip-like behaviour in adjacent stalk endothelial cells by activating Notch. Here, we show with genetic experiments in postnatal mice that the level of active Notch signalling is more important than the direct Dll4-mediated cell–cell communication between endothelial cells. We identify endothelial expression of VEGF-A and of the chemokine receptor CXCR4 as key processes controlling Notch-dependent vessel growth. Surprisingly, genetic experiments targeting endothelial tip cells in vivo reveal that they retain their function without Dll4 and are also not replaced by adjacent, Dll4-positive cells. Instead, activation of Notch directs tip-derived endothelial cells into developing arteries and thereby establishes that Dll4–Notch signalling couples sprouting angiogenesis and artery formation.

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Figure 1: Notch-dependent changes in endothelial CXCR4 and VEGF-A.
Figure 2: Role of endothelial VEGF-A in Notch-controlled angiogenesis.
Figure 3: Control of retinal angiogenesis by endothelial CXCR4.
Figure 4: CXCR4 is required for Notch-controlled endothelial sprouting.
Figure 5: Control of Notch- and VEGF-A-mediated-processes in vitro and in vivo.
Figure 6: Genetic tracking and manipulation of tip cells in vivo.
Figure 7: Control of tip cell behaviour by Dll4 and Jag1.
Figure 8: Notch activation directs tip cell progeny into arteries.

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Acknowledgements

We thank G. de Luxán for help with the active NICD immunostaining protocol and H. J. Fehling (Institute of Immunology, University Clinics Ulm, Germany) for providing ROSA26RFP mice. The Max Planck Society, the University of Münster, the Cells in Motion (CiM) graduate school, the DFG Research Unit 2325 and the DFG cluster of excellence ‘Cells in Motion’ have supported this study.

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Authors and Affiliations

  1. Department of Tissue Morphogenesis, Max Planck Institute for Molecular Biomedicine, and University of Münster, Faculty of Medicine, Röntgenstrasse 20, D-48149, Münster, Germany

    Mara E. Pitulescu, Inga Schmidt, Tobiah Antoine, Frank Berkenfeld, Hongryeol Park, Manuel Ehling, Daniel Biljes, Susana F. Rocha, Urs H. Langen & Ralf H. Adams

  2. Institute of Biochemistry, University of Giessen, Friedrichstrasse 24, D-35392, Giessen, Germany

    Benedetto Daniele Giaimo, Francesca Ferrante & Tilman Borggrefe

  3. Max Planck Institute for Molecular Biomedicine, Flow Cytometry Unit, Röntgenstrasse 20, D-48149, Münster, Germany

    Martin Stehling

  4. Laboratory of Stem Cell Biology and Developmental Immunology, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, 1-3 Yamada-oka, Suita, 565-0871, Osaka, Japan

    Takashi Nagasawa

  5. University of California San Diego Medical Center, 9500 Gilman Drive, La Jolla, California, 92093, USA

    Napoleone Ferrara

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  1. Mara E. Pitulescu
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Contributions

M.E.P., S.F.R. and R.H.A. designed the study. M.E.P., I.S., T.A. and F.B. performed experiments, B.D.G., F.F. and T.B. performed and analysed ChIP experiments, M.E. initiated the NICD rescue experiments, D.B. performed the R26R-Confetti clonal expansion and part of the RbpjiΔTC experiments, H.P. and U.H.L. contributed with image acquisition and quantification, M.S. performed the flow cytometry sorting of retinal ECs, S.F.R., T.N. and N.F. generated mutant mice, and M.E.P. and R.H.A. wrote the manuscript.

Corresponding authors

Correspondence to Mara E. Pitulescu or Ralf H. Adams.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Effect of Notch signalling on CXCR4 and VEGF-A in retinal ECs.

(a) Isolectin B4 (IB4, white) stained retinas of Dll4iΔEC/ΔEC, Dll1iΔEC/+ Dll4iΔEC/ΔEC, Dll1iΔEC/ΔEC Dll4iΔEC/ΔEC and control mice. Circles indicate similar vessel outgrowth. (b) Quantitation of retinal vessel progression (n = 10 control, 8 Dll4iΔEC/ΔEC, 14 Dll1iΔEC/+ Dll4iΔEC/ΔEC and 6 Dll1iΔEC/ΔEC Dll4iΔEC/ΔEC retinas) and EC area per field (n = 9 control, 5 Dll4iΔEC/ΔEC, 14 Dll1iΔEC/+ Dll4iΔEC/ΔEC and 6 Dll1iΔEC/ΔEC Dll4iΔEC/ΔEC retinas). Data represent mean ± s.e.m. P values, one-way ANOVA with Tukey’s multiple comparison post-hoc test. (c) Dll4 (white) and IB4 (green) whole-mount staining of control, Dll4iΔEC/ΔEC and Dll4iΔEC/ΔEC NICDiOEC/OEC retinas. Endothelial Dll4 protein can be seen in control but not in mutant samples (arrowheads). (d) Quantitative analysis of VEGF-A immunosignal in IB4-labelled vessels and total retinas of the indicated mutants at P6 (n = 8 control, 5 Dll4iΔEC/ΔEC, 6 Dll4iΔEC/ΔEC NICDiOEC/+ and 5 Dll4iΔEC/ΔEC NICDiOEC/OEC retinas). Increased VEGF-A levels in Dll4iΔEC/ΔEC ECs were normalized after expression of active Notch. Data represent mean ± s.e.m. P values, one-way ANOVA with Tukey’s multiple comparison post-hoc test. (e) Representative images of VEGF-A protein in Dll4iΔEC/ΔEC NICDiOEC/OEC retinas. ECs were visualized by IB4. Images on the right and in centre depict higher magnifications of insets. Rescue efficiency correlates with endothelial VEGF-A expression (arrowheads). (f) RT-qPCR analysis of Hes1, Hey1 and Efnb2 transcripts in sorted retinal ECs of the indicated mutants and littermate controls (n = 5 control, 6 Dll4iΔEC/ΔEC, 3 Dll4iΔEC/ΔEC NICDiOEC/+ and 3 Dll4iΔEC/ΔEC NICDiOEC/OEC mice from two independent tamoxifen injections). Notch target gene upregulation in Dll4 iΔEC/ΔEC NICDiOEC/+ ECs was reduced close to control expression in Dll4iΔEC/ΔEC NICDiOEC/OEC ECs. Data represent mean ± s.e.m. P values, one-way ANOVA with Tukey’s multiple comparison post-hoc test. Comparisons where P value was not shown are not significant. (g) Elevated ESM1 (blue/white) in IB4-stained (green) Dll4iΔEC/ΔEC ECs relative to control littermates. Arrowheads mark ESM1 + sprouts. ESM1 immunostaining behind the angiogenic front was greatly reduced in Dll4iΔEC/ΔEC NICDiOEC/OEC double mutant retinas (arrows).

Supplementary Figure 2 Endothelial VEGF signalling in Notch-dependent angiogenesis.

(a,b) Representative overview (a) and confocal images (b) of IB4-stained VegfaiΔEC/+ and control retinas treated with vehicle or with DAPT. Circles indicate vessel outgrowth in control. (c) Representative overview pictures of IB4-stained VegfaiΔEC/ΔEC and control retinas treated with vehicle or with DAPT. Circles indicate vessel outgrowth in control. (d,e) Confocal images of IB4 (green), ESM1 (blue) and ERG (red) stained vehicle (d) and DAPT-treated (e) VegfaiΔEC/ΔEC and control P6 retinas. Note residual ESM1 staining (arrowheads) in VegfaiΔEC/ΔEC EC sprouts but impaired DAPT-induced upregulation in the plexus (arrows) relative to control. (f) Confocal images of IB4 (green), VEGF-A (blue) and CXCR4 (red) staining of P6 retina. Note correlation (arrowheads) between CXCR4 and VEGF-A immunosignals.

Supplementary Figure 3 Role of endothelial CXCR4 in retinal angiogenesis.

(a) Representative overview confocal images showing IB4-labelled Cxcr4iΔEC/ΔEC and Cxcr4iΔEC/KO retinas together with the corresponding controls. Circles indicate vessel outgrowth in the control. (b) Combined ERG (EC nuclei; red), IB4 (green) and EdU (blue) staining shows reduced ERG + EdU + EC numbers in Cxcr4iΔEC/ΔEC retinal vessels compared to littermate control. EdU pulse-labelling was done 2 h prior to analysis. (c) Representative confocal images of CXCR4 (red/white) and IB4 (green) stained Cxcr4iΔEC/KO and control retinas. Arrowheads mark sprouts at the angiogenic front. (d) ESM1 (white) and IB4 (green) staining of Cxcr4iΔEC/ΔEC and control retinas. ESM1 expression is not overtly changed. (e) Quantitative analysis of EC proliferation for Cxcr4iΔEC/ΔEC and control littermates (n = 7 control and 8Cxcr4iΔEC/ΔEC retinas from two independent tamoxifen injections). Graphs indicate reduction of Cxcr4iΔEC/ΔEC ERG + EdU + cells per EC area at the angiogenic front relative to control, while the number of ERG + cells was not significantly changed because of the impaired outgrowth. Data represent mean ± s.e.m. P values, two-tailed unpaired t-test. (f) Quantitative analysis of vascular parameters for Cxcr4iΔEC/KO, Cxcr4KO/+ and control retinas: retinal vessel progression, EC area, branching points (n = 7 control, 10 Cxcr4KO/+ and 6 Cxcr4iΔEC/KO retinas), and sprouts (n = 5 control and 6 Cxcr4iΔEC/KO retinas). Note absence of significant differences between Cxcr4KO/+ heterozygotes and controls. Data represent mean ± s.e.m. P values, two-tailed unpaired t-test and one-way ANOVA with Tukey’s multiple comparison post-hoc test. (g) RT-qPCR analysis for Cxcr4 (n = 6 control and 7 Cxcr4iΔEC/ΔEC mice), Vegfa(n = 6 control and 12 Cxcr4iΔEC/ΔEC mice) and Esm1 (n = 5 control and 7 Cxcr4iΔEC/ΔEC mice) in sorted Cxcr4iΔEC/ΔEC and control retinal ECs. Data represent mean ± s.e.m. P values, two-tailed unpaired t-test. (h) RT-qPCR expression analysis of the Notch pathway components Dll4 (n = 7 control and 9 Cxcr4iΔEC/ΔEC mice), Jag1 (n = 8 control and 12 Cxcr4iΔEC/ΔEC mice) Hey1 (n = 4 control and 10 Cxcr4iΔEC/ΔEC mice) and Hes1 (n = 5 control and 12 Cxcr4iΔEC/ΔEC mice) in sorted Cxcr4iΔEC/ΔEC and control retinal ECs. Data represent mean ± s.e.m.P values, two-tailed unpaired t-test.

Supplementary Figure 4 Role of CXCR4 in Notch-controlled angiogenesis.

(a) Representative overview images showing DAPT-treated Cxcr4iΔEC/ΔEC and control retinas. Circles indicate vessel outgrowth in control. (b) Confocal images showing vehicle- or DAPT-treated Cxcr4iΔEC/ΔEC and control retinas. Note the decrease sprouting (arrowheads) in DAPT-treated Cxcr4iΔEC/ΔEC compared to control littermates. (c) Overview images of IB4-stained untreated or AMD3100-treated Dll4iΔEC/ΔEC retinas. Circles indicate vessel outgrowth in control (d) (e) Representative overview pictures. (d) and high magnification confocal images (e) of IB4-stained Cxcr4iΔEC/KO RbpjiΔEC/ΔEC and Cxcr4iΔEC/+ RbpjiΔEC/ΔEC retinas. Sprouting (arrowheads) was reduced in Cxcr4iΔEC/KO RbpjiΔEC/ΔEC retinas compared to Cxcr4iΔEC/+ RbpjiΔEC/ΔEC control retinas. (f) Representative confocal images of IB4-stained Cxcr4iΔEC/ΔEC Dll4iΔEC/ΔEC and Dll4iΔEC/ΔEC retinas. Sprouting (arrowheads) was reduced in Cxcr4iΔEC/ΔEC Dll4iΔEC/ΔEC retinas relative to Dll4iΔEC/ΔEC control retinas. (g) Quantitative analysis of vascular parameters for Cxcr4iΔEC/ΔEC Dll4iΔEC/ΔEC, Dll4iΔEC/ΔEC and control (Cre negative) retinas: retinal vessel progression, EC area and sprouting (n = 8 control, 6 Dll4iΔEC/ΔEC and 6 Cxcr4iΔEC/ΔEC Dll4iΔEC/ΔEC retinas). Note significant differences in sprouting but not in vessel progression and EC area between Cxcr4iΔEC/ΔEC Dll4iΔEC/ΔEC and Dll4iΔEC/ΔEC retinas. Data represent mean ± s.e.m. P values, one-way ANOVA with Tukey’s multiple comparison post-hoc test.

Supplementary Figure 5 Notch and VEGF-A-controlled processes.

(a) ChIP analysis of modified and total H3 at the Hes1, Vegfa, Esm1 and Cxcr4 loci in MS1 cells. A region located on chromosome X (Chr. X) was used as control. Shown is mean ± SD of 3 independent experiments for H3K4me1, H3K4me3 and total H3 and 2 independent experiments, measured twice each, for H3K9ac and H3K27me3 ([NS] not significant, [∗]P < 0.05, [∗∗] P < 0.01, [∗∗∗] P < 0.001, two-tailed unpaired t-test). Statistics source data for are shown in Supplementary Table 3. (b) Representation of regions (red) analysed by ChIP with distance in kilobases (kb) relative to transcriptional start sites (black arrows). (c) RT-qPCR analysis of the indicated genes in MS1 cells treated with DMSO or DAPT for 3 h (n = 6 DMSO-treated MS1 and 6 DAPT-treated MS1; individual experiments in triplicate). Data represent mean ± s.e.m. P values, two-tailed unpaired t-test. (d) Elevated VEGF-A (white) in the P7 RbpjiΔEC/ΔEC vascular plexus (arrows) and adjacent avascular tissue (arrowheads). Images are representative of three mice analysed. (e) RT-qPCR analysis of Esm1 (n = 6 control and 8 NICDiOEC/OEC mice), Vegfa (n = 5 control and 9 NICDiOEC/OEC mice), Cxcr4 (n = 6 control and 10 NICDiOEC/OEC mice) and Dll4 (n = 6 control and 9 NICD iOEC/OEC mice) in sorted NICDiOEC/OEC and control retinal ECs. Data represent mean ± s.e.m.P values, two-tailed unpaired t-test. (f) RT-qPCR analysis of Vegfr2 (n = 6 control and 10 Flk1iΔEC/KO mice), Vegfa (n = 6 control and 6 Flk1iΔEC/KO mice), Cxcr4 (n = 6 control and 6 Flk1iΔEC/KO mice) and Dll4 (n = 7 control and 5 Flk1iΔEC/KO mice) in sorted Flk1 iΔEC/KO and control ECs. Data represent mean ± s.e.m. P values, two-tailed unpaired t-test. (g) Upregulation of VEGF-A (white) in P6 NICDiOEC/OEC ECs (white arrows) and avascular retina (yellow arrowheads). (h) Increased expression of ESM1 (white) in NICDiOEC/OEC sprouts (arrowheads) and plexus (arrows). (i) Increased expression of Dll4 (blue) in NICDiOEC/OEC capillaries, whereas CXCR4 (red) did not overtly change in mutant sprouts (arrowheads) and plexus (arrows). (j) Downregulation of VEGFR2 and Dll4 in Flk1iΔEC/KO vessels with some residual signal (arrowheads). k, Lost ESM1 (white) staining in Flk1iΔEC/KO sprouts (arrowheads) and ectopic plexus expression (arrows). Panels in centre and bottom rows show higher magnifications and ESM1 signal of insets. (l) Cleaved caspase 3 (CASP3; red) detects EC death (arrows) in Flk1iΔEC/KO retinal vessels. Panels in centre and bottom rows show higher magnifications and CASP3 signal of insets. (m) DAPT-induced ESM1 (white) expression in control (arrowheads) but not in Flk1iΔEC/KO vascular plexus. (n) Quantitation of VEGF-A in P6 total retina (n = 4 control and 4 NICDiOEC/OEC retinas) and ESM1 in IB4 + vessels sprouts (S) and plexus (P) (n = 5 control and 5NICDiOEC/OEC retinas).

Supplementary Figure 6 Genetic tracking and manipulation of tip cells in vivo.

(a) Confocal images of IB4 (blue) and ESM1 (green) stained retinas at the indicated postnatal stages. Note continuous presence of ESM1 in angiogenic sprouts. (b) Examples of clonal expansion of recombined ECs in Esm1-CreERT2T/+ R26R-ConfettiT/+ double transgenics. IB4 labels ECs (blue), while nuclear GFP and cytoplasmic YFP mark recombined cell clones in arteries or at the angiogenic front (encircled areas ECs). (c) Example of quantitation approach used to analyse the percentage of recombined GFP + sprout area (green; orange in bottom image) per total angiogenic front area (orange in top image) and to analyse the percentage of recombined GFP + cells in arteries (green; orange in right image) per total arterial EC area (orange in left image). (d) Quantitation of Cxcr4iΔTC/KO, Cxcr4iΔTC/+ and Cxcr4+/KO GFP + cells per total endothelial area (n = 10 Cxcr4+/KO, 10 Cxcr4iΔTC/+ and 8 Cxcr4iΔTC/KO retinas) and of GFP + sprouts per front area (n = 10 Cxcr4+/KO, 10 Cxcr4iΔTC/+ and 8 Cxcr4iΔTC/KO retinas) at 96 h after 4-OHT injection. Data represent mean ± s.e.m. P values, two-tailed unpaired t-test. (e) Confocal images of IB4 (blue), GFP (green) and ESM1 (white) staining in P6 Notch1iΔTC/ΔTC (Esm1-CreERT2T/+ Notch1loxlox R26-mTmGT/+) and control (Esm1-CreERT2T/+ R26-mTmGT/+) retinas at 96 h after 4-OHT injection. Note presence of ESM1 immunosignal in GFP + Notch1iΔTC/ΔTC tip cells (arrowheads) and capillaries (arrows). (f) IB4 (blue), GFP (green) and CXCR4 (red) staining of Esm1-CreERT2T/+ R26-mTmGT/+ retinas at 96 h after 4-OHT injection. Recombined GFP + cells found within the plexus area (arrowheads) are positive for CXCR4.

Supplementary Figure 7 Control of tip cell behaviour by Dll4 and Jag1.

(a) Representative overview and high magnification confocal images of GFP-labelled (green) Dll4iΔTC/ΔTC (Esm1-CreERT2T/+ Dll4loxlox R26-mTmGT/+) and control (Esm1-CreERT2T/+ R26-mTmGT/+) ECs in the retinal vasculature (IB4, blue) at 48 h after 4-OHT administration. Arrowheads indicate Dll4-deficient GFP + tip cells displaying increased extension of protrusions and filopodia. (b) Confocal images of IB4 (blue), GFP (green) and Dll4 (white) staining in P6 Dll4iΔTC/ΔTC and control retinas at 96 h after 4-OHT injection. Note absence of Dll4 immunosignal in GFP + Dll4iΔTC/ΔTC tip cells ECs (white arrowheads) and reduced expression of Dll4 in non-recombined ECs (yellow arrowheads). (c) IB4 (blue), GFP (green) and CXCR4 (red) staining in P6 Dll4iΔTC/ΔTC and control retinas at 96 h after 4-OHT injection. Note increased CXCR4 immunosignal in GFP + Dll4iΔTC/ΔTC cells in plexus area (arrows) but not in sprouts (arrowheads) as compared to control GFP + cells. (d) IB4 (blue), GFP (green) and Jag1 (white) staining in P6 Dll4iΔTC/ΔTC and control retinas at 96 h after 4-OHT injection. Note reduced Jag1 expression in GFP + Dll4iΔTC/ΔTC cells plexus area (arrowheads) as compared to control GFP + cells. (e) Confocal images of IB4 (blue), GFP (green) and CXCR4 (red) staining in P6 Jag1iΔTC/ΔTC (Esm1-CreERT2T/+ Jag1loxlox R26-mTmGT/+) and control (Esm1-CreERT2T/+ R26-mTmGT/+) retinas at 96 h after 4-OHT injection. Note no significant difference for CXCR4 immunosignal between Jag1iΔTC/ΔTC recombined sprouts (white arrowheads) and non-recombined tip cells (yellow arrowheads). (f) IB4 (blue), GFP (green) and Dll4 (white) staining in P6 Jag1iΔTC/ΔTC and control retinas at 96 h after 4-OHT injection. Note no obvious difference for Dll4 immunosignal between Jag1iΔTC/ΔTC recombined sprouts (white arrowheads) and non-recombined tip cells (yellow arrowheads). Images in a–f are representative of three mice analysed.

Supplementary Figure 8 Notch activation directs tip cell progeny into arteries.

(a) Maximum intensity projection of IB4 (red), Dll4 (white) and NICD (GFP, green) staining in NICDiOTC/OTC retinas at 96 h after 4-OHT administration. Arrowheads mark recombined nuclear GFP + cells; arteries (A) are indicated. Arrowheads indicate GFP + ECs with Dll4 immunostaining in and around arteries (top panels). Images are representative of two mice analysed. (b,c) Confocal images showing IB4 (red), NICD (GFP, green) and Dll4 (white) (b) or CXCR4 (white) (c) staining in NICDiOTC/OTC retinas at 24 h after 4-OHT administration. Arrowheads mark recombined GFP + ECs with Dll4 (b) and CXCR4 (c) immunosignal in capillaries at or near the angiogenic front. Whereas all NICDiOTC/OTC-expressing cells from three mice analysed express Dll4, only 72% of NICDiOTC/OTC-expressing cells from three mice analysed show CXCR4 immunostaining. (d) Representative images showing arterial incorporation of recombined GFP + cells in Dll4iΔTC/ΔTC, Jag1iΔTC/ΔTC, Notch1iΔTC/ΔTC, RbpjiΔTC/ΔTC and Cxcr4iΔTC/KO mice and their respective controls. Note reduced arterial contribution of GFP + ECs in Dll4iΔTC/ΔTC, Jag1iΔTC/ΔTC and Notch1iΔTC/ΔTC at 96 h after 4-OHT injection. This phenotype was also seen in the RbpjiΔTC/ΔTC at 72 h but not in the Cxcr4iΔTC/KO vasculature at 96 h after 4-OHT injection. Arrowheads point to GFP + ECs incorporated into arteries. (e) Whole-mount retina immunostaining of IB4 (green) and active NICD (NICD Val1744, red). Confocal images show the angiogenic growth front on top of the artery and central vascular plexus. NICD signal is detected in ECs (white arrowheads) and perivascular cells (yellow arrowheads). High NICD signal decorates arteries and ECs in the peri-arterial plexus (bottom). Bottom row panels show examples of sprouting ECs without (yellow arrows) or with high active NICD (white arrows). (f) Additional combinations of maximum intensity projections (same regions as in Figure 8d) for CXCR4 (blue), ERG (purple), RFP (red, ESM1 recombined cells) and active endogenous Notch (YFP/Venus, green) immunostaining in CBF:H2B Venus Notch reporter (Esm1-CreERT2T/+ CBF:H2B VenusT/+ ROSA26-tdRFPT/+). White arrows point to endothelial cells with high Notch activity, while white arrowheads indicate ERG + cells with no Notch activity. Yellow arrows indicate ERG + endothelial cells from capillary front, with detectable Notch activity and reversed CXCR4 polarization.

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Pitulescu, M., Schmidt, I., Giaimo, B. et al. Dll4 and Notch signalling couples sprouting angiogenesis and artery formation. Nat Cell Biol 19, 915–927 (2017). https://doi.org/10.1038/ncb3555

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