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Coronary arterial development is regulated by a Dll4-Jag1-EphrinB2 signaling cascade

  1. Stanislao Igor Travisano
  2. Vera Lucia Oliveira
  3. Belén Prados
  4. Joaquim Grego-Bessa
  5. Rebeca Piñeiro-Sabarís
  6. Vanesa Bou
  7. Manuel J Gómez
  8. Fátima Sánchez-Cabo
  9. Donal MacGrogan  Is a corresponding author
  10. José Luis de la Pompa  Is a corresponding author
  1. Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Spain
  2. CIBER de Enfermedades Cardiovasculares, Spain
  3. Centro Nacional de Investigaciones Cardiovasculares, Spain
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Cite this article as: eLife 2019;8:e49977 doi: 10.7554/eLife.49977

Abstract

Coronaries are essential for myocardial growth and heart function. Notch is crucial for mouse embryonic angiogenesis, but its role in coronary development remains uncertain. We show Jag1, Dll4 and activated Notch1 receptor expression in sinus venosus (SV) endocardium. Endocardial Jag1 removal blocks SV capillary sprouting, while Dll4 inactivation stimulates excessive capillary growth, suggesting that ligand antagonism regulates coronary primary plexus formation. Later endothelial ligand removal, or forced expression of Dll4 or the glycosyltransferase Mfng, blocks coronary plexus remodeling, arterial differentiation, and perivascular cell maturation. Endocardial deletion of Efnb2 phenocopies the coronary arterial defects of Notch mutants. Angiogenic rescue experiments in ventricular explants, or in primary human endothelial cells, indicate that EphrinB2 is a critical effector of antagonistic Dll4 and Jag1 functions in arterial morphogenesis. Thus, coronary arterial precursors are specified in the SV prior to primary coronary plexus formation and subsequent arterial differentiation depends on a Dll4-Jag1-EphrinB2 signaling cascade.

Introduction

Coronary artery disease leading to cardiac muscle ischemia is the major cause of morbidity and death worldwide (Sanchis-Gomar et al., 2016). Deciphering the molecular pathways driving progenitor cell deployment during coronary angiogenesis could inspire cell-based solutions for revascularization following ischemic heart disease. The coronary endothelium in mouse derives from at least two complementary progenitor sources (Tian et al., 2015) that may share a common developmental origin (Zhang et al., 2016a; Zhang et al., 2016b). The sinus venosus (SV) commits progenitors to arteries and veins of the outer myocardial wall (Red-Horse et al., 2010; Tian et al., 2013), and the endocardium contributes to arteries of the inner myocardial wall and septum (Red-Horse et al., 2010; Wu et al., 2012; Tian et al., 2013). Regardless of origin, the endothelial precursors invest the myocardial wall along stereotyped routes and eventually interlink in a highly coordinated fashion. Subsequently, discrete components of the primitive plexus are remodeled into arteries and veins, and stabilized through mural cell investment and smooth muscle cell differentiation (Udan et al., 2013). Arterial-venous specification of endothelial progenitors is genetically pre-determined (Swift and Weinstein, 2009), whereas arterial differentiation and patterning depend on environmental cues, such as blood flow and hypoxia-dependent proangiogenic signals (le Noble et al., 2005; Jones et al., 2006; Fish and Wythe, 2015). Vascular endothelial growth factor (VEGF) binds endothelial receptors and drives the expansion of the blood vessel network as a response to hypoxia (Dor et al., 2001; Liao and Johnson, 2007; Krock et al., 2011).

The Notch signaling pathway is involved in angiogenesis in the mouse embryo and in the post-natal retina. In this processes, the ligand Dll4 is upregulated by VEGF, leading to Notch activation in adjacent endothelial cells (ECs), vessel growth attenuation, and maintenance of vascular integrity (Blanco and Gerhardt, 2013). In contrast, the ligand Jag1 has a proangiogenic Dll4-Notch–inhibitory function, suggesting that the overall response of ECs to VEGF is mediated by the opposing roles of Dll4 and Jag1 (Benedito et al., 2009). Dll4-Notch1 signaling is strengthened in the presence of the glycosyltransferase Mfng (Benedito et al., 2009; D’Amato et al., 2016).

Our understanding of how coronary vessels originate, are patterned, and integrate with the systemic circulation to become functional is still limited. Coronary arteries are distinct from peripheral arteries in that they originate from SV and ventricular endocardium, which is a specialized endothelium that lines the myocardium. Moreover, SV endothelium has a venous identity, as opposed to the retina vascular bed, which has no pre-determined venous identity. Given these differences of developmental context, it is essential to evaluate the role of Notch in coronary arterial development, and importantly, the implications for heart development and repair.

Several components of the Notch pathway have been examined in the context of coronary artery formation. Inactivation of the Notch modifier Pofut1 results in excessive coronary angiogenic cell proliferation and plexus formation (Wang et al., 2017), while endothelial inactivation of Adam10, required for Notch signaling activation, leads to defective coronary arterial differentiation (Farber et al., 2019). Transcriptomics has shown that pre-artery cells appear in the immature coronary vessel plexus before coronary blood flow onset, and express Notch genes, including Dll4 (Su et al., 2018). Here, we examine the early and late requirements of Notch ligands Jag1 and Dll4, and their downstream effector EphrinB2, for coronary arterial development.

Results

Jag1 and Dll4 are expressed in SV endocardium and coronary vessels endothelium

We examined SV and coronary vessels for the expression of Jag1 and Dll4. At embryonic day 11.5 (E11.5), Jag1 was detected in SV ECs and in ECs extending into the right atrium (Figure 1A). Dll4 was also expressed in ECs emanating from the SV and in the endocardium lining the right atrium (Figure 1A). These results suggest that either ligand could potentially activate Notch1 in the SV endocardium (Figure 1A). At E12.5, N1ICD was detected in endomucin (Emcn)-positive ECs in subepicardial capillaries emerging from the SV (Figure 1A). At E13.5, Jag1 and Dll4, were expressed in ECs of the developing coronary arteries (intramyocardial vessels; Figure 1—figure supplement 1A). Dll4 and Mfng were also expressed in prospective veins (subepicardial vessels; Figure 1—figure supplement 1A). At E15.5, Jag1, Dll4, and Mfng were all expressed in arterial ECs, whereas Mfng was still found in subepicardial vessels (Figure 1—figure supplement 1B). Thus, Jag1 and Dll4 expression is found in discrete ECs in SV endothelium, abates in sub-epicardial veins and becomes restricted to intramyocardial coronary arteries at later developmental stages.

Figure 1 with 4 supplements see all
Endocardial Jag1 or Dll4 inactivation disrupts coronary plexus formation.

(A) Jag1, Dll4, and N1ICD immunostaining (red) in E11.5 control hearts, sagittal views. Magnified views of boxed areas show details of sinus venosus (sv, arrowheads) and right atrium (ra, arrow). Whole-mount dorsal view of immunostainings for N1ICD (red) and Emcn (green) in E12.5 control heart. Magnified views show detail of sub-epicardial endothelium. Nuclei are counterstained with Dapi (blue). (B) Whole-mount dorsal view of immunostaining for Emcn (green) in E12.5 control, Jag1flox;Nfatc1-Cre, and Dll4flox;Nfatc1-Cre mutant hearts. Arrowheads indicate vascular malformations. Quantified data of average number of vascular malformations (VM) and average number of branching points in E12.5 control, Jag1flox;Nfatc1-Cre and Dll4flox;Nfatc1-Cre hearts. (C) Dorsal views of whole-mount E12.5 control, Jag1flox;Nfat-Cre, and Dll4flox;Nfatc1-Cre hearts stained for N1ICD (red) and VE-Caderin (white). Microscope: Leica SP5. Software: LAS-AF 2.7.3. build 9723. Objective: HCX PL APO CS 10 × 0.4 dry. HCX PL APO lambda blue 20 × 0.7 multi-immersion. (D) Dorsal views of whole-mount E12.5 control, Jag1flox;Nfat-Cre, and Dll4flox;Nfatc1-Cre hearts stained for EdU (green), ERG (red), and Emcn (blue). Scale bars, 100 μm. Microscope: Nikon A1-R. Software: NIS Elements AR 4.30.02. Build 1053 LO, 64 bits. Objectives: Plan Apo VC 20x/0.75 DIC N2 dry; Plan Fluor 40x/1.3 Oil DIC H N2 Oil. (E) Quantified data for vascular malformations (VM), average number (#) of branching points and EdU-ERG dual-positive nuclei as a percentage of all nuclei in sub-epicardial vessels. Data are mean ± s.d. (n = 7 control embryos and n = 4 Jag1flox;Nfat-Cre and n = 3 Dll4flox;Nfatc1-Cre mutant embryos for VM and average # of branching points. n = 3 control embryos and n = 3 mutant embryos for EdU-ERG). *p<0.05, ****p<0.0001 by one-way ANOVA with Tukey’s multiple comparison tests). Abbreviations: rv, right ventricle.

Nfatc1-positive progenitors give rise to the majority of subepicardial vessels

To inactivate Notch ligands in SV progenitors we used the Nfatc1-Cre driver line (Wu et al., 2012). To confirm the SV and endocardial specificity of this line, we crossed it with the Rosa26-LacZ reporter line (Soriano, 1999). X-gal-staining of heart sections of E11.5 embryos identified patchy LacZ expression in SV endothelium (Figure 1—figure supplement 2A). ß-gal staining was consistent with Nfatc1 protein nuclear localization in a subset of ECs lining the SV (Figure 1—figure supplement 2A). Uniform ß-gal staining was detected in ventricular endocardium and cushion mesenchyme derived from endocardial cells (Figure 1—figure supplement 2A). At E12.5, co-labelling with an anti-Pecam1 antibody revealed ß-gal-positive staining in 60% of Pecam1-positive subepicardial vessels in the right ventricle and about 50% in the left ventricle (Figure 1—figure supplement 2BSource data 1, sheet 1). Tracking the expression of Nfatc1-Cre-driven red fluorescent protein (RFP) and the endothelial-specific nuclear protein Erg at E12.5 indicated that about 80% of nuclei in the endothelial network were Nfatc1-positive and Erg-positive (Figure 1—figure supplement 2BSource data 1, sheet 1). Thus, SV-derived Nfatc1-positive progenitors give rise to 50–80% of subepicardial vessels in the ventricular wall, consistent with previous reports (Chen et al., 2014; Cavallero et al., 2015; Zhang et al., 2016a).

To trace the fate of Nfatc1-positive cells relative to Notch activity, we crossed Nfatc1-Cre;Rosa26-RFP mice with the Notch reporter line CBF:H2B-Venus. At E11.5, a subset of nuclear-stained RFP ECs extending from the SV into the right ventricle were co-labelled with CBF:H2B-Venus (Figure 1—figure supplement 2D). At E12.5, some RFP-labelled capillaries on the dorsal side of the heart were co-labeled with CBF:H2B-Venus while others were labelled with CBF:H2B-Venus alone (Figure 1—figure supplement 2E), indicating that Notch signaling activity is present in both Nfatc1-positive and Nfatc1-negative populations of SV-derived ECs.

Opposing roles of Jag1 and Dll4 in coronary plexus formation from SV

Jag1 inactivation with the Nfatc1-Cre driver line, specific of SV and endocardium, resulted in death at E13.5 (Supplementary file 1). Whole-mount Endomucin (Emcn) staining at E12.5 revealed a well-formed vascular network covering the dorsal aspect of control hearts (Figure 1B, E—figure supplement 3ASource data 1, sheet 2), whereas the vascular network in Jag1flox;Nfatc1-Cre mutants was poorly developed, and exhibited numerous capillary malformations (Figure 1B, E—figure supplement 3BSource data 1, sheet 2) with decreased endothelial branching (Figure 1B,ESource data 1, sheet 2). N1ICD expression was also more prominent (Figure 1C) and EC proliferation reduced 60% relative to control (Figure 1D,ESource data 1, sheet 2). Ventricular wall thickness in endocardial Jag1 mutants was reduced by 50–60% relative to controls (Figure 1—figure supplement 4ASource data 1, sheet 3). Thus, endocardial Jag1 deletion causes the growth arrest of the primitive coronary plexus.

Deletion of Dll4 with the Nfatc1-Cre driver resulted in the death of high proportion of embryos at E10.5 (Supplementary file 1). Nonetheless, about a third of mutant embryos survived until E12.5 (Supplementary file 1). Whole-mount Emcn immunostaining of E12.5 Dll4flox;Nfatc1-Cre hearts revealed a comparatively denser capillary network (Figure 1B,ESource data 1, sheet 2), characterized by numerous capillary malformations (Figure 1B and Figure 1—figure supplement 3A,CSource data 1, sheet 2), and increased endothelial branching (Figure 1B,ESource data 1, sheet 2). N1ICD expression was reduced (Figure 1C), as expected, and EC proliferation increased 30% relative to control (Figure 1D,ESource data 1, sheet 2). These defects were associated with 50–60% reduction in ventricular wall thickness (Figure 1—figure supplement 4ASource data 1, sheet 3). Thus, endocardial inactivation of Dll4 causes excessive growth of the primitive coronary plexus.

Endocardial Jag1 or Dll4 deletion results in a hypoxic and metabolic stress response

To determine the effect of early endocardial Jag1 or Dll4 inactivation on cardiac development we performed RNA-seq. This analysis yielded 211 differentially expressed genes (DEG) in the Jag1flox;Nfatc1-Cre transcriptome (130 upregulated, 81 downregulated; Figure 2ASupplementary file 2) and 274 DEGs in the Dll4flox;Nfatc1-Cre transcriptome (180 upregulated, 94 downregulated; Figure 2ASupplementary file 2).

Figure 2 with 1 supplement see all
Transcriptome profiling of endocardial Jag1 and Dll4 mutant hearts.

(A) Left, Total number of differentially expressed genes identified by RNA-seq (Benjamini-Hochberg (B–H) adjusted p<0.05) in the indicated genotypes. Numbers indicate upregulated genes (red) and downregulated genes (blue). Center, circular plot of representative differentially expressed genes, presenting a detailed view of the relationships between expression changes (left semicircle perimeter) and IPA functions belonging to the Cardiovascular System Development and Function category (right semicircle perimeter). For both circular plots, in the left semicircle perimeter, the inner ring represents Jag1flox;Nfatc1-Cre data and the outer ring Dll4flox;Nfatc1-Cre data. Activation z-score scale: green, repression; orange, activation; white, unchanged. LogFC scale: red, upregulated; blue, downregulated; white, unchanged. Right, circular plot showing representative differentially expressed genes depending of selected upstream regulators. Details in Table supplement 2 and Table supplement 3. (B) In situ hybridization (ISH) of HeyL, Efnb2, Fabp4, Vegfa, Cx40 and Ldha on E12.5 control, Jag1flox;Nfatc1-Cre, and Dll4flox;Nfatc1-Cre heart sections. Arrowheads indicate Fabp4 expression in capillary vessels. (C) Immunohistochemistry of p27 (red) and IsoB4 (white) on E12.5 control, Jag1flox;Nfatc1-Cre, and Dll4flox;Nfatc1-Cre mutant heart sections. Dapi counterstain (blue). Microscope: Nikon A1-R. Software: NIS Elements AR 4.30.02. Build 1053 LO, 64 bits. Objectives: Plan Apo VC 20x/0,75 DIC N2 dry; Plan Fluor 40x/1,3 Oil DIC H N2 Oil. Quantified data for p27-positive nuclei as a % of total CM nuclei. Data are mean ± s.d. (n = 3 sections from 6 control embryos and n = 3 sections from 3 mutant embryos). **p<0.01 by one-way ANOVA with Tukey’s multiple comparison tests). Abbreviations: lv, left ventricle; rv, right ventricle. Scale bars, 100 μm.

Ingenuity Pathway Analysis (IPA) identified enrichment of EC functions (Figure 2A, left plot, Supplementary file 3). The main terms overrepresented in both genotypes were angiogenesis and EC development, and capillary vessel density, possibly reflecting the lack of a normal-sized capillary network (Figure 2ASupplementary file 3). EC proliferation and heart contraction were predicted to be upregulated in Jag1flox;Nfatc1-Cre embryos, while EC migration was upregulated in Dll4flox;Nfatc1-Cre mice (Figure 2ASupplementary file 3). Analysis of upstream regulators revealed activation of hypoxia (Hif1α), acute inflammatory response (Nf-κb1α), intracellular stress pathways (Atf4), and response to metabolic stress pathways (Foxo), while cell cycle and DNA repair pathways (Myc, Tp53) were negatively regulated (Figure 2A, right plot).

In situ hybridization (ISH) showed reduced expression of HeyL and the Notch target Efnb2 in subepicardial vessels (Figure 2B). Fabp4, a member of the fatty-acid-binding protein family, was upregulated (Figure 2A). Fabp4 is a DLL4-NOTCH target downstream of VEGF and FOXO1 in human EC (Harjes et al., 2014), required for EC growth and branching (Elmasri et al., 2009). Fapb4 expression was found exclusively in the atrio-ventricular groove in Jag1flox;Nfatc1-Cre hearts (Figure 2B), but extended sub-epicardially into the base of the heart in Dll4flox;Nfatc1-Cre hearts (Figure 2B). Vegfa expression was not globally affected in mutant hearts (Figure 2B), despite being upregulated in the RNA-seq (Figure 2A). Cell cycle-associated genes such as Cdkn1b/p27, a negative regulator of cell proliferation, were also upregulated in both genotypes (Figure 2A), and p27 nuclear staining was increased twofold in compact myocardium (Figure 2C,DSource data 1, sheet 4), indicating decreased cellular proliferation. Connexin 40 (Cx40) and Hey2, which label trabecular and compact myocardium respectively, showed no alteration in their expression domains by ISH (Figure 2BFigure 1—figure supplement 4B), suggesting that chamber patterning was normal in these mutants. Likewise, the glycolytic marker genes Ldha and Pdk1 (Menendez-Montes et al., 2016) were confined, as normal, to the compact myocardium (Figure 2B; Figure 1—figure supplement 4B), indicating maintenance of ventricular chamber metabolic identity.

We examined E12.5 Jag1flox;Nfatc1-Cre and Dll4flox;Nfatc1-Cre mutants for evidence of hypoxia given that the gene signatures in the RNA-seq analysis suggested an ongoing hypoxic/metabolic stress response. The hypoxic response might also explain the defect of ventricular wall growth. We performed immunohistochemical detection of pimonidazole on E12.5 Jag1flox;Nfatc1-Cre and Dll4flox;Nfatc1-Cre heart sections (Figure 2—figure supplement 1). Pimonidazole hydrochloride (Hypoxyprobe) immunostaining for hypoxic tissues was detected most intensely in cells lying within the ventricular septum and atrio-ventricular (AV) groove in the subepicardial area where the primitive coronary endothelium emerges to cover the myocardium (Figure 2—figure supplement 1Ai, arrowheads). Immunostaining for Glut1 (Slc2a1), a direct HIF1 target, showed myocardial expression partially overlapping with hypoxyprobe distribution (Figure 2—figure supplement 1Aii). There was no Glut1 immunostaining in the subepicardial mesenchyme zone (Figure 2—figure supplement 1Aii). We found no difference in the intensities of hydroxyprobe or Glut1 staining in either the ventricular free wall, or in the ventricular septum in Jag1flox;Nfatc1-Cre and Dll4flox;Nfatc1-Cre hearts (Figure 2—figure supplement 1). There was no obvious change in hydroxyprobe immunostaining of endothelial cells emerging at the AV groove either (Figure 2—figure supplement 1Ai-Ci). These results indicate that Jag1flox;Nfatc1-Cre and Dll4flox;Nfatc1-Cre mutant hearts are not overtly hypoxic at E12.5, suggesting that the hypoxic/metabolic stress gene signatures may be due to a cell autonomous defect of endocardial/endothelial cells.

Defective coronary remodeling and maturation in endothelial Jag1 or Dll4 mutants

We next examined the requirements of endothelial Jag1 and Dll4 for coronary vessel remodeling and maturation. To circumvent the early lethality of Jag1flox- or Dll4flox;Nfatc1-Cre mutants, we crossed Jag1flox and Dll4flox mice with the vascular endothelium-specific Pdgfb-iCreERT2 transgenic mice (Wang et al., 2010) to obtain the corresponding tamoxifen-inducible lines. Tamoxifen-induced Jag1 deletion at E12.5 resulted in 68% reduction in Jag1 expression (Figure 3—figure supplement 1A,CSource data 1, sheet 5) and a complete absence of arteries at E15.5, whereas the veins appeared unaffected (Figure 3A,CSource data 1, sheet 6). We measured NOTCH pathway activity by carrying out a N1ICD staining that showed a 50% increase in endothelial N1ICD (Figure 3B,CSource data 1, sheet 6), consistent with Jag1 acting as an inhibitory Notch ligand. Next, we examined perivascular coverage of the Jag1flox;Pdgfb-iCreERT2 endothelial coronary tree. We used α-smooth muscle actin (αSMA) and Notch3 to measure the extent of coronary vessel smooth muscle cell differentiation and pericyte coverage, respectively (Volz et al., 2015). We found that the proportion of αSMA- and Notch3-positive cells was significantly reduced in E15.5 Jag1flox;Pdgfb-iCreERT2 coronary arteries (Figure 3B,CSource data 1, sheet 6) reflecting the lack of differentiation of perivascular cells. Although αSMA is a commonly used marker of vascular smooth muscle cells, it is expressed more broadly in mesenchymal cells and cardiomyocytes throughout the embryonic heart prior to E16.5. To evaluate smooth muscle differentiation more specifically in E15.5 hearts, we used SM22a (Figure 3—figure supplement 2A,B). After co-staining with Notch3, we found that the proportion of SM22a and Notch3-positive cells in the Jag1flox;Pdgfb-iCreERT2 mutants was significantly reduced (Figure 3—figure supplement 2A,B,ESource data 1, sheet 7), confirming that pericytes fail to properly differentiate into smooth muscle.

Figure 3 with 4 supplements see all
Late endothelial Jag1 or Dll4 inactivation disrupts coronary plexus remodeling.

(A) Dorsal view of whole-mount immunochemistry for IsoB4 (red), labelling arteries and Emcn (green), labeling veins and capillaries, in E15.5 control and Jag1flox;Pdgfb-iCreERT2 mutant hearts, tamoxifen (Tx)-induced at E12.5. Scale bars, 100 μm. (B) E15.5 control and Jag1flox;Pdgfb-iCreERT2 mutant heart sections. Left, Immunostaining for N1ICD (red) and IsoB4 (green). Right, α-smooth-muscle actin (SMA, red) and Notch3 (green). Dapi counterstain (blue). Arrowheads point to N1ICD-positive nuclei. Arrows point to αSMA-Notch3 co-immunostaining. Scale bars, 100 μm. (C) Quantified data from E15.5 control and Jag1flox;Pdgfb-iCreERT2 hearts: area of coverage by coronary arteries (IsoB4-positive vessels) and veins (Emcn-positive vessels); Data are mean ± s.d. (n = 3 control embryos and 4 mutant embryos. N1ICD-positive nuclei as a percentage of total endothelial nuclei; and SMA-Notch3 co-immunostaining in coronary arteries. Data are mean ± s.d. (n = 3 sections from 4 control embryos and from 4 mutant embryos. (D) Whole-mount dorsal view of immunohistochemistry for Emcn (green) and IsoB4 (red) in E15.5 control and Dll4flox;Pdgfb-iCreERT2 mutant hearts. Tx-induced at E14.5. (E) Left, immunohistochemistry for N1ICD (red) and IsoB4 (green); right, immunohistochemistry for SMA (red) and Notch3 (green) on control E15.5 WT and Dll4flox;Pdgfb-iCreERT2 mutant heart sections. Dapi counterstain (blue). Arrowheads indicate N1ICD-positive nuclei. Arrows point to SMA-Notch3 co-immunostaining. Scale bars, 100 μm. Microscopes: Nikon A1-R, Leica SP5. Softwares: NIS Elements AR 4.30.02. Build 1053 LO, 64 bits (Nikon); LAS-AF 2.7.3. build 9723 (Leica). Objectives: Plan Apo VC 20x/0.75 DIC N2 dry; Plan Fluor 40x/1.3 Oil DIC H N2 Oil (Nikon); HCX PL APO CS 10 × 0.4 dry. HCX PL APO lambda blue 20 × 0.7 multi-immersion (Leica). (F) Quantified data from E15.5 control and Dll4flox;Pdgfb-iCreERT2 hearts: area covered by coronary arteries (IsoB4-positive vessels); area covered by veins (Emcn-positive vessels); percentage of N1ICD-positive nuclei in endothelium as a percentage (%) of total nuclei; and SMA-Notch3 co-immunostaining in coronary arteries. Data are mean ± s.d. (n = 3 sections from 3 control embryos and from three mutant embryos). *p<0.05, **p<0.01, by Student’s t-test; n.s., not significant. Scale bars, 100 μm.

To examine Dll4 function in coronary artery formation, we crossed Dll4fllox mice with Pdgfb-iCreERT2 to obtain Dll4flox;Pdgfb-iCreERT2 mice. Tamoxifen-induced Dll4 deletion from E12.5 or E13.5, resulted in embryonic lethality at E15.5, confirming that embryo survival is highly sensitive to reduction in endothelial Dll4 expression. However, induction at E14.5 resulted in a 82% reduction in Dll4 expression (Figure 3—figure supplement 1B,CSource data 1, sheet 5) and a complete absence of arteries at E15.5, whereas veins were unaffected (Figure 3D,FSource data 1, sheet 6). Endothelial N1ICD staining was decreased by 60% compared with controls (Figure 3E,FSource data 1, sheet 6), consistent with Dll4 activating Notch1 during angiogenesis. Furthermore, Dll4flox;Pdgfb-iCreERT2 mutants had deficient perivascular cell coverage as indicated by decreased αSMA- and Notch3-positive cells (Figure 3E,FSource data 1, sheet 6). This was confirmed by co-immunostaining with SM22a and Notch3 demonstrating near complete absence of smooth muscle differentiation in Dll4flox;Pdgfb-iCreERT2 mutants (Figure 3—figure supplement 2C,D,ESource data 1, sheet 7).

Endocardium/endothelial Notch ligand inactivation impairs coronary artery formation and ventricular growth

To confirm endothelial Jag1 requirement for coronary arterial formation, we crossed Jag1flox mice with Cdh5-CreERT2 driver line (Wang et al., 2010) to obtain homozygous Jag1flox;Cdh5CreERT2 mice. Tamoxifen-induced Jag1 inactivation from E9.5 onwards (Figure 3—figure supplement 3A) resulted in reduced coronary artery coverage and marginally increased vein coverage (Figure 3—figure supplement 3A,CSource data 1, sheet 8). N1ICD staining in the endothelium was increased (Figure 3—figure supplement 3B,CSource data 1, sheet 8), reflecting Jag1 inhibitory Notch function during angiogenesis. Furthermore, Jag1flox;Cdh5CreERT2 mutant heart coronaries had decreased perivascular cell coverage, as indicated by the reduced proportion of αSMA- and Notch3-positive cells (Figure 3—figure supplement 3B,CSource data 1, sheet 8).

To confirm Dll4 requirement for coronary arterial formation and circumvent the lethality resulting from E12.5 tamoxifen administration in Dll4flox;Pdgfb-iCreERT2 mice, we crossed Dll4flox mice with Cdh5-CreERT2 driver line (Wang et al., 2010) to obtain homozygous Dll4flox;Cdh5-CreERT2 mice (Figure 3—figure supplement 3D). Tamoxifen induction at E12.5 resulted in the reduction of coronary arterial coverage, unchanged vein coverage but reduced vein caliber (Figure 3—figure supplement 3D,FSource data 1, sheet 8). N1ICD staining in the endothelium was decreased by 60% compared with controls (Figure 3—figure supplement 3E,FSource data 1, sheet 8), likely due to reduced EC contribution to the arteries. Furthermore, Dll4flox;Cdh5-CreERT2 mutant heart coronaries had decreased perivascular cell coverage as indicated by the reduced proportion of αSMA- and Notch3-positive cells (Figure 3—figure supplement 3E,FSource data 1, sheet 8). The impact of impaired coronary vessel formation in endothelial Jag1- or Dll4- mutants was also indicated by their diminished ventricular wall thickness (Figure 3—figure supplement 4ASource data 1, sheet 9).

To examine the effect of endothelial Dll4 deletion on cardiac gene expression, we performed RNA-seq on E15.5 Dll4flox;Cdh5-CreERT2 mutant ventricles. Therefore of 163 DEGs, 138 were upregulated, while 25 were downregulated (Figure 3—figure supplement 4B and Supplementary file 2, sheet 3). As expected, Dll4 was downregulated, while Vegfa was upregulated, suggesting cardiac hypoxia (Figure 3—figure supplement 4BSupplementary file 2, sheet 3). Cdkn1a/p21 was upregulated, indicating impaired cell proliferation (Figure 3—figure supplement 4BSupplementary file 2, sheet 3). IPA identified strong upregulation of endothelial cell functions (Figure 3—figure supplement 4B and Supplementary file 3), whereas analysis of upstream regulators identified an enrichment for transcriptional activators associated with innate inflammatory responses (Irfs and Stats), suggesting defective vascular integrity. Other upstream regulators were associated with cardiovascular disease, including hypertrophy (Nfatc2) and oxidative stress responses (Nfe212; Figure 3—figure supplement 4B right and Supplementary file 3). Moreover, ISH showed reduced endothelial expression of Efnb2 and Dll4 in smaller caliber vessels (Figure 3—figure supplement 4C), suggesting a loss of arterial identity, whereas Fapb4 was upregulated (Figure 3—figure supplement 4C) consistent with increased coronary vessel density of the un-remodeled coronary plexus. Thus, endothelial Jag1 or Dll4 signaling promotes remodeling and maturation of the coronary vascular tree, and in the absence of adequate vascular remodeling, cardiac growth is impaired.

Forced endothelial Dll4 expression disrupts coronary vascular remodeling

To further characterize the role of Notch in coronary development, we generated a transgenic line (Dll4GOF) bearing a Rosa26-CAG-floxNeoSTOPflox-Dll4-6xMycTag expression cassette (see Materials and methods). Tie2-Cre-mediated removal of the floxed NeoSTOP sequences resulted in a mild 1.2-fold endothelial Dll4 overexpression that permitted survival of transgenic embryos bearing a single copy of the Dll4GOF allele (not shown). At E14.5, forced Dll4 expression lead to marginally increased, sub-epicardial vessel (Emcn-positive) coverage (Figure 4A,FSource data 1, sheet 10) and vascular malformations similar to those found in Dll4flox;Pdgfb-iCreERT2 and Dll4flox;Cdh5-CreERT2 mutants (Figure 1—figure supplement 3E). However, by E16.5 IsoB4-positive (arteries) intramyocardial vessels were substantially decreased (Figure 4B,FSource data 1, sheet 10). Accordingly, Dll4 and Efnb2 expression was restricted to smaller caliber vessels in Dll4GOF transgenics (Figure 4C). In contrast, superficial Emcn-positive vessels (veins) were more numerous and dense (Figure 4B,FSource data 1, sheet 10). N1ICD expression was increased and extended to the prospective veins (Figure 4D,GSource data 1, sheet 10), consistent with Notch1 gain-of-function in endothelium. Arterial smooth muscle cell coverage was also substantially reduced (Figure 4E,GSource data 1, sheet 10), suggesting defective pericyte and/or smooth muscle differentiation. These coronary vascular defects were associated with below-normal cardiomyocyte proliferation and reduced myocardial thickness (not shown). Therefore, endothelial Dll4 overexpression blocks coronary artery formation, vessel remodeling and maturation, and impairs cardiac growth. These results were surprising, as our loss-of-function data support a pro-arteriogenic role for Dll4.

Figure 4 with 1 supplement see all
Forced Dll4 expression in endothelium disrupts coronary arteriovenous differentiation and remodeling.

(A) Dorsal view of whole-mount immunochemistry for IsoB4 (red), labeling arteries, and Emcn (green), labeling veins and capillaries, in E14.5 control and Dll4GOF;Tie2-Cre heart. Scale bar, 100 μm. (B) Dorsal view of whole-mount immunochemistry for IsoB4 (red) and Emcn (green) in E16.5 control and Dll4GOF;Tie2-Cre mutant heart. Scale bar, 100 μm. (C) ISH of Dll4 and Efnb2 on E16.5 control and Dll4GOF;Tie2-Cre heart sections. Arrowheads indicate coronary arteries. Scale bar, 50 μm. (D) Immunohistochemistry for N1ICD (red) and IsoB4 (green) on E16.5 control and Dll4GOF;Tie2-Cre heart sections. Dapi counterstain (blue). Arrowheads indicate N1ICD-stained nuclei. Scale bar, 50 μm. (E) Immunohistochemistry for SMA (red) and Notch3 (green) on E16.5 control and Dll4GOF;Tie2-Cre heart sections. Dapi counterstain (blue). The arrow points to a coronary vessel stained by SMA and Notch3. Scale bar, 50 μm. Microscope: Nikon A1-R. Software: NIS Elements AR 4.30.02. Build 1053 LO, 64 bits. Objectives: Plan Apo VC 20x/0,75 DIC N2 dry; Plan Fluor 40x/1,3 Oil DIC H N2 Oil. (F) Quantified data for control and Dll4GOF;Tie2-Cre hearts: E14.5, area covered by veins (Emcn-positive vessels). Data are mean ± s.d, (n = 2 control embryos and n = 3 mutant embryos); E16.5, area covered by coronary arteries (IsoB4-positive vessels), and area covered by veins (Emcn-positive vessels). Data are mean ± s.d, (n = 3 control embryos and n = 2 mutant embryos). (G) Quantified data of the percentage of N1ICD-positive nuclei in E16.5 endothelium as a percentage (%) of total nuclei; and SMA-Notch3 co-immunostaining in coronary arteries. Data are mean ± s.d, (n = 3 sections from 3control embryos and n = 4 sections from 3 mutant embryos) *p<0.05, **p<0.01, by Student’s t-test; ns. not significant.

To support our Dll4GOF results, we used a transgenic line conditionally overexpressing Mfng (MfngGOF) (D’Amato et al., 2016) to test whether increased Mfng activity favored Dll4-mediated signaling, and thus impaired arteriogenesis. At E11.5, MfngGOF;Tie2-Cre embryos displayed reduced SV sprouting, increased Notch1 signaling and myocardial wall thinning (Figure 4—figure supplement 1A,B). However, by E14.5 MfngGOF;Tie2-Cre embryos displayed an extensive vascular network (Figure 4—figure supplement 1C), although the arteries appeared atrophied (Figure 4—figure supplement 1C) and prospective veins appeared more numerous and intricately branched (Figure 4—figure supplement 1C). MfngGOF;Tie2-Cre embryos displayed numerous capillary malformations at E14.5 (Figure 1—figure supplement 3F), suggesting defective arterial-venous differentiation. The decrease in arterial coverage and increase in venous coverage in MfngGOF;Tie2-Cre embryos became more obvious by E16.5 (Figure 4—figure supplement 1C,GSource data 1, sheet 11). N1ICD expression was markedly increased (Figure 4—figure supplement 1D,GSource data 1, sheet 11), as expected, while the expression of the Notch targets HeyL and Efnb2 was restricted to smaller caliber vessels (Figure 4—figure supplement 1E). Vegfa appeared upregulated throughout (Figure 4—figure supplement 1E), suggesting that MfngGOF;Tie2-Cre hearts are hypoxic. Pericyte coverage and smooth muscle differentiation, as shown by αSMA and Notch3 co-immunostaining, were significantly decreased at E16.5 (Figure 4—figure supplement 1F,GSource data 1, sheet 11), suggesting defective pericyte recruitment and/or differentiation. Ink injection confirmed that MfngGOF;Tie2-Cre coronaries were ‘leaky’ (Figure 4—figure supplement 1H), and therefore functionally deficient.

EphrinB2 is required for coronary arteriogenesis and vessel branching

Efnb2 is necessary for arterial-venous differentiation and vascular maturation (Kania and Klein, 2016) and is a Notch target during ventricular chamber development (Grego-Bessa et al., 2007). We find Efnb2 expression in ventricular endocardium at E10.5 (Figure 5—figure supplement 1A) but not in SV endothelium (not shown). At E13.5, Efnb2 was expressed in emerging arteries and prospective veins (Figure 5—figure supplement 1A), and at E16.5 was confined to arteries (Figure 5—figure supplement 1A). Therefore, Efnb2 is expressed dynamically in a pattern similar to Dll4 and Mfng.

To examine EphrinB2 function in coronary angiogenesis, we crossed mice bearing a conditional Efnb2flox allele with the Nfatc1-Cre line. Whole-mount IsoB4 immunostaining revealed reduced artery coverage in E15.5 Efnb2flox;Nfatc1-Cre hearts (Figure 5A,FSource data 1, sheet 12), while vein coverage (Emcn-positive) was unchanged (Figure 5A,FSource data 1, sheet 12). Histological examination showed that the compact myocardium in the ventricles was 30% thinner at E16.5 (Figure 5—figure supplement 1B,CSource data 1, sheet 13), which could be attributed to the reduced proliferation observed at E14.5 (Figure 5—figure supplement 1C,DSource data 1, sheet 13). The presence of smaller caliber arteries coincided with higher Notch1 activity (Figure 5B,FSource data 1, sheet 12) and increased Hey1 expression (Figure 5C), suggesting negative feedback between EphrinB2 and Notch signaling. Although the patterning of prospective veins was unchanged, we found occasional malformations (Figure 1—figure supplement 3G,H) and transmural communications or shunts between the endocardium and epicardium, which we interpret to be arteriovenous fistulae (Figure 5—figure supplement 1E, F).

Accordingly, the venous marker EphB4 was expressed ectopically in a subset of intramyocardial vessels at E16.5 (Figure 5—figure supplement 1E), indicative of abnormal venous identity. Vegfa was upregulated throughout the heart (Figure 5—figure supplement 1F), suggesting that Efnb2flox;Nfatc1-Cre hearts are hypoxic. Arterial smooth muscle coverage was reduced substantially (Figure 5E,FSource data 1, sheet 12), suggesting defective vessel maturation. Dye perfusion in Efnb2flox;Nfatc1-Cre hearts revealed absence of left anterior descending coronary artery at E16.5 (Figure 5—figure supplement 1G) and ink perfusion revealed vascular hemorrhaging in the most distal section of the coronary arterial tree at E18.5 (Figure 5—figure supplement 1H), consistent with vessel leakiness, while aortic connections were normal (Figure 5—figure supplement 1H). Thus, endocardial EphrinB2 is required for coronary arterial remodeling, vessel structural integrity, and cardiac growth.

Figure 5 with 1 supplement see all
Endocardial Efnb2 inactivation disrupts coronary artery differentiation and remodeling.

(A) Dorsal view of whole-mount immunochemistry for IsoB4 (red), labelling arteries and Emcn (green), labeling veins and capillaries, in E15.5 control and Efnb2flox;Nfatc1-Cre hearts. Scale bars,100 μm. (B) Immunohistochemistry of N1ICD (red) and Emcn (green) on E16.5 control and Efnb2flox;Nfatc1-Cre heart sections. Dapi-counterstain (blue). Arrowheads indicate N1ICD-stained nuclei. Scale bars,100 μm. (C) ISH of Hey1 on E16.5 control and Efnb2flox;Nfatc1-Cre heart sections. Arrowheads indicate Hey1-expressing coronaries. Scale bars, 100 μm. (D) Immunohistochemistry for EphB4 (red) and IsoB4 (green) on E16.5 control and Efnb2flox;Nfatc1-Cre heart sections. Dapi-counterstain (blue). Yellow arrowheads indicate EphB4-stained vessels. Scale bar, 50 μm. (E) Immunohistochemistry for SMA (red) and Notch3 (green) on E16.5 control and Efnb2flox;Nfatc1-Cre heart sections. Dapi-counterstain (blue). Arrow points to a coronary artery stained by SMA and Notch3. Microscope: Nikon A1-R. Software: NIS Elements AR 4.30.02. Build 1053 LO, 64 bits. Objectives: Plan Apo VC 20x/0,75 DIC N2 dry; Plan Fluor 40x/1,3 Oil DIC H N2 Oil. (F) Quantified data for control and E16.5 WT and Efnb2flox;Nfatc1-Cre hearts: E15.5, area covered by coronary arteries (IsoB4-positive vessels), area covered by veins (Emcn-positive vessels). Data are mean ± s.d, (n = 3 control embryos and n = 3 mutant embryos), and percentage of N1ICD-stained nuclei in endothelium as percentage (%) of total nuclei Data are mean ± s.d. (n = 3 sections from 3 control embryos and n = 3 sections from 4 mutant embryos); E16.5, SMA-Notch3 co-immunostaining in coronary arteries. Data are mean ± s.d. (n = 3 sections from three control embryos and n = 3 sections from 3 mutant embryos). *p<0.05, **p<0.01 by Student’s t-test; n.s., not significant.

EphrinB2 mediates Jag1 and Dll4 signaling in arterial branching morphogenesis

To model coronary angiogenesis ex vivo, we developed a ventricular explant assay (Figure 6A; (Zhang and Zhou, 2013). Compared with controls, Jag1flox;Nfatc1-Cre explants had a lower angiogenic potential, manifested as smaller caliber vessels, longer distances between branching points and a lower number of transmural endothelial branches (Figure 6B,C,KSource data 1, sheet 14). Conversely, Dll4flox;Nfatc1-Cre or Notch1flox;Nfatc1-Cre explants yielded more densely interconnected vascular networks of larger vessel caliber, longer distances between vessel branching points, and more transmural endothelial branches (Figure 6B,D,E,KSource data 1, sheet 14). Notch1flox;Nfatc1-Cre explants displayed a phenotype similar to Dll4flox;Nfatc1-Cre explants, although the differences in vessel caliber and endothelial branch number did not reach significance (Figure 6B,E,KSource data 1, sheet 14). MfngGOF;Tie2-Cre and Dll4GOF;Tie2-Cre explants showed a broadly similar phenotype, with substantial lengthening of branching point distance and a below-normal number of endothelial branches (Figure 6B,I,J,KSource data 1, sheet 14). In contrast, MfngGOF;Tie2-Cre explants had smaller caliber vessels than Dll4GOF;Tie2-Cre explants (Figure 6I,J,KSource data 1, sheet 14). Efnb2flox;Nfatc1-Cre explants formed sparsely interconnected networks with decreased vessel caliber, reduced number of transmural endothelial branches, and increased distances between branching points, similar to those seen in Jag1flox;Nfatc1-Cre explants (Figure 6B,F,KSource data 1, sheet 14).

EPHRINB2 rescues disrupted arterial branching in ventricular explants from Jag1 and Dll4 mutant hearts.

(A) Ventricular explant assay procedure. (B–J) Representative images of E11.5 cultured ventricular explants from the following embryos: (B) control (n = 8–10), (C) Jag1flox;Nfatc1-Cre (n = 4), (D) Dll4flox;Nfatc1-Cre (n = 5), (E) Notch1flox;Nfatc1-Cre (n = 4), (F) MfngGOF;Tie2-Cre (n = 3), (G) Dll4GOF;Tie2-Cre (n = 3), (H) Efnb2flox;Nfatc1-Cre (n = 5–7), (I) Dll4flox;Nfatc1-Cre infected with Efnb2-overexpressing lentivirus (n = 5–7). Microscope: Nikon A1-R. Software: NIS Elements AR 4.30.02. Build 1053 LO, 64 bits. Objectives: Plan Apo VC 20x/0,75 DIC N2 dry; Plan Fluor 40x/1,3 Oil DIC H N2 Oil. (J) Quantification of vessel caliber (arrowheads), branching point distance (double ended arrowhead), and mean endothelial branch (asterisk) number. Data are means ± s.d. ***p<0.001; **p<0.01; *p<0.05, by Student’s t-test; Benjamini-Hochberg adjusted p-value. n.s not significant. Scale bars, 100 μm.

Based on the congruence of endocardial/endothelial Jag1, Dll4 and Efnb2 loss-of-function phenotypes, we tested whether EphrinB2 mediates Notch function in coronary angiogenesis. Thus, delivery of a lentivirus expressing Efnb2 to Jag1flox;Nfatc1-Cre ventricular explants normalized the reduced endothelial branch number to the number seen in control explants (Figure 6B,G,KSource data 1, sheet 14). Likewise, Efnb2 expression restored the elevated number of endothelial branches in Dll4flox;Nfatc1-Cre explants to control levels (Figure 6B,H,KSource data 1, sheet 14), showing that the branching defect could be normalized in both mutants.

EphrinB2 functions downstream of Jag1 and Dll4 in capillary tube formation

The ventricular explant assay assesses the collective behaviors of endocardium and coronary endothelial outgrowth. In order to determine endothelial behavior exclusively, we performed capillary tube formation assays with primary human endothelial cells (HUVEC). shRNA directed against JAG1, DLL4 or EFNB2 were lentivirally-transduced into HUVEC. This resulted in reduction of mRNA levels of 30% and 60% for JAG1 and DLL4, respectively (Figure 7ASource data 1, sheet 15), and of 50% for EFNB2 (Figure 7—figure supplement 1ASource data 1, sheet 16). The NOTCH target HEY1 was unchanged in shJAG1-infected cells and 50% decreased in shDLL4-infected cells (Figure 7ASource data 1, sheet 15). The activity of EFNB2 lentivirus was verified by rescuing shRNA-mediated EFNB2 knockdown (Figure 7—figure supplement 1B,CSource data 1, sheet 16). EFNB2 knockdown resulted in decreased capillary network complexity (Figure 7—figure supplement 1B,CSource data 1, sheet 16). These parameters were restored to control levels by lentiviral-mediated EFNB2 overexpression (Figure 7—figure supplement 1B,CSource data 1, sheet 16).

Figure 7 with 1 supplement see all
EPHRINB2 rescues defective capillary network formation resulting from shRNA-mediated silencing of JAG1 and DLL4 in HUVEC.

(A) qRT-PCR of JAG1, DLL4 and HEY1 after transduction of shRNA. (B) Representative phase contrast images (1 of 2 experiments) of the HUVEC network, after transduction of indicated shRNA and rescue by EFNB2 analyzed by the Angiogenesis Analyzer from ImageJ. (C) Significantly changed measurements in the analyzed area: nodes surrounded by junctions (red dot surrounded by dark blue circle). Isolated elements (dark blue line). Segments (yellow). Number of branches (green) and meshes (light blue) were not significantly altered (not shown). Total segments length: sum of length of the segments. Number of pieces: sum of number of segments, isolated elements and branches detected. Total isolated branches length: sum of the length of the isolated elements. Red asterisks refer to comparisons between experimental and control situations. Black asterisks refer to comparisons between shRNA-mediated inhibition and LV-mediated rescue. Data are means ± s.d. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05, by ANOVA.

We next tested whether EPHRINB2 acts downstream of JAG1 and DLL4 in capillary tube formation. JAG1 knockdown inhibited capillary tube formation and decreased network complexity as determined by measuring EC junctions, nodes, segments and pieces that were below control, while the ‘total isolated branches length’ was above control (Figure 7B,CSource data 1, sheet 15). However, these parameters were restored to control levels in presence of the EFNB2 transgene (Figure 7B,CSource data 1, sheet 15), suggesting that EPHRINB2 compensates for JAG1 knockdown in this assay. In contrast, knockdown of DLL4 increased capillary tube formation and network complexity (junctions, nodes, segments and pieces; Figure 7B,CSource data 1, sheet 15), while measurements were restored and even went beyond control by overexpressing EFNB2 (Figure 7B,CSource data 1, sheet 15). Thus, EFNB2 overexpression not only compensates for the absence of DLL4 in this assay, but has an added effect as well.

Taken together, our observations with ventricular explants and HUVEC indicate that JAG1 and EPHRINB2 promote coronary vessel sprouting and branching, whereas DLL4, MFNG and NOTCH1 inhibit these processes. Moreover, EPHRINB2 mediates signaling from both JAG1 and DLL4 to regulate endothelial branching during coronary angiogenesis.

Discussion

The origin of the coronary endothelium from a venous source has prompted the suggestion that arteries need to be ‘reprogrammed’ from veins (Red-Horse et al., 2010). We found Jag1, Dll4, Mfng, and N1ICD expression in SV endothelium and sub-epicardial capillaries, with Dll4 and Mfng expression maintained subsequently in prospective veins. These Notch pathway elements are co-expressed with the venous marker endomucin, which is also expressed in the endocardium and SV (Cavallero et al., 2015). This implies that nascent subepicardial vessels have a mixed arterial/venous identity. During embryonic angiogenesis, Notch is necessary for artery-vein specification—the reversible commitment of ECs to arterial or venous fate—before the onset of blood flow (Swift and Weinstein, 2009). Our finding of SV endothelial cell heterogeneity in relation to arterial-venous identity is consistent with the notion that early vascular beds are phenotypically plastic during embryonic development (Moyon et al., 2001; Chong et al., 2011; Fish and Wythe, 2015). Our results are consistent with recently published data showing that pre-arterial cells expressing Notch pathway elements are present in coronary endothelium (Su et al., 2018), prior to blood flow onset.

We show that Notch ligands Jag1 and Dll4 are required for SV sprouting angiogenesis, further supporting the notion that coronary arteries are specified by Notch prior to blood flow (Su et al., 2018) (Figure 8A). Dll4 is the key Notch ligand activator regulating embryonic (Duarte, 2004; Gale et al., 2004; Krebs et al., 2004; Benedito et al., 2008) and postnatal retinal angiogenesis (Hellström et al., 2007; Lobov et al., 2007), whereas Jag1 antagonizes Dll4-Notch1 signaling (Benedito et al., 2009) and acts downstream of Dll4-Notch1 to promote smooth muscle differentiation (Pedrosa et al., 2015). Jag1 or Dll4 inactivation in the SV results in arrested and excessive angiogenesis, respectively, resembling the situation in the retina (Benedito et al., 2009) (Figure 8B). Moreover capillary ‘entanglements’ suggestive of vascular malformations were present in the early plexus of Jag1flox;Nfatc1-Cre and Dll4flox;Nfatc1-Cre mutants. Vascular malformations in Notch mutants have been attributed to unresolved intermingling of arteries and veins during differentiation, or to failed maintenance of arterial and venous identities in the vascular bed (Gale et al., 2004; Krebs et al., 2004). The presence of these malformations in Jag1flox;Nfatc1-Cre and Dll4flox;Nfatc1-Cre mutants could therefore indicate that endothelial progenitors begin to differentiate into arteries and veins very soon after exiting the SV.

Coronary arterial development is regulated by a Dll4-Jag1-EphrinB2 signaling cascade.

(A,B) Embryonic stages E11.5-E12.5. Development of the primitive coronary plexus. (A) Left: sinus venous (SV)-derived vessels covering the dorsal side of a wild-type heart. Zoomed view showing a subepicardial vessel with a subset of ECs expressing N1ICD (red). These N1ICD-expressing cells are equivalent to pre-artery cells defined by Su et al. (2018). Center: cross-section through the ventricular wall. SV-derived ECs invade the sub-epicardial space over the myocardium to cover the heart dorsally. Some pre-artery ECs invade the myocardium and begin to differentiate into arteries. Right: during EC sprouting and proliferation, a regulatory balance between Dll4/Mfng and Jag1 modulates Notch signaling output downstream of Vegf. (B) Left: E12.5 Jag1flox;Nfatc1-Cre (Jag1LOF; top) exhibit arrested coronary angiogenesis, Dll4flox;Nfatc1-Cre mutants (Dll4LOF; bottom) display increased angiogenesis, and both mutants display vascular malformations. The zoomed views show details of a sub-epicardial vessels with increased N1ICD expression in the Jag1LOF mutants (red), and decreased expression in the Dll4LOF mutants (blue). Center: the sub-epicardial capillary plexus is either poorly developed in Jag1LOF mutants or over-developed in Dll4LOF mutants, and with vascular malformations. Mutant hearts have a thin myocardial wall, but maintain myocardial patterning (grey-brown boundary). Right: Endocardial Jag1 deletion disrupts the regulatory balance with Dll4/Mfng, leading to increased N1ICD and arrested migration and proliferation. In contrast, endocardial Dll4 deletion results in decreased N1ICD signaling, and increased network complexity and proliferation. (C,D) Embryonic stages E14.5-E16.5. Arterial differentiation and plexus remodeling. (C) Left: coronary vasculature at the dorsal aspect of the wild-type heart. Zoomed view showing a coronary artery with an inner layer of ECs expressing N1ICD (red) and an outer layer of smooth muscle cells (yellow). Center: capillary differentiation and patterning give rise to large veins sub-epicardially and large arteries intra-myocardially. Right: systemic blood flow activates Dll4/Jag1/Mfng/N1ICD/EphrinB2 signaling to drive terminal arterial differentiation and remodeling. (D) Left: in coronary endothelial-Dll4flox;Pdgfb-iCreERT2 or -Jag1flox;Pdgfb-iCreERT2 mutants, the coronary vasculature is mis-patterned. Zoomed view detailing near absence of arteries in Dll4 mutants, and comparatively smaller caliber coronary artery in Jag1 mutants. The arteries in both mutants have an inner layer of a ‘leaky’ endothelium and an outer layer of poorly differentiated perivascular cells (white). Center: Dll4 mutants are characterized by the near-absence of coronary arteries while arteries in Jag1 mutants are decreased. In either case, veins are unaffected. Mutant hearts have a thin myocardial wall but maintain myocardial patterning (grey-brown boundary). Right: Coronary endothelial-Jag1 or Dll4LOF leads to increased or decreased N1ICD respectively, causing arrested arterial differentiation and remodeling. Inactivation of Efnb2 leads to a similar phenotype. Endothelial Notch gain-of-function (GOF), resulting from Dll4 or Mfng overexpression, also leads to increased N1ICD and disrupted arterial differentiation and remodeling.

Consistent with the known roles of the Notch ligands in angiogenesis, the hierarchical organization of the coronary vascular tree was profoundly altered in the late-induced Jag1 and Dll4 mutants, implying that both Notch ligands are required for high-order complexity of the coronary vessels (Figure 8C). Jag1 or Dll4 inactivation during coronary arterial remodeling results in smaller diameter arteries, consistent with Notch promoting vascular remodeling and arterial fate commitment, as described in other developmental settings (Figure 8D; (Duarte, 2004; Gale et al., 2004; Krebs et al., 2004). However, gain of Notch function also led to smaller caliber arteries (Figure 8D), when the opposite outcome was anticipated (Uyttendaele et al., 2001; Trindade et al., 2008; Krebs et al., 2010). This result implies that non-physiological variations of Notch activity lead to the arrest of coronary artery differentiation. Dll4 mutants also showed coronary vessel hemorrhaging reminiscent of that found in vascular disorders (Park-Windhol and D'Amore, 2016) or tumors (Goel et al., 2011) and indicative of disrupted endothelial integrity.

We reasoned that the coronary maturation defects observed in Jag1 or Dll4 mutants are due, at least in part, to the loss of EphrinB2 function given that Efnb2 is a direct Dll4-Notch signaling target in ECs (Iso et al., 2006) and endocardium during ventricular development (Grego-Bessa et al., 2007). Moreover, EphrinB2 and VEGF are functionally linked during angio- and lymphangiogenesis; EphrinB2 is a direct activator of VEGFR2 and VEGFR3, and therefore cooperates in the mechanism leading to tip cell extension and vessel sprouting (Sawamiphak et al., 2010; Wang et al., 2010). Thus, Efnb2 inactivation leads to coronary artery remodeling defects, similar to those resulting from Jag1 or Dll4 inactivation, suggesting that EphrinB2 functions downstream of Notch to promote coronary arterial remodeling (Figure 8D). This notion is supported by the angiogenic ventricular explant and EC capillary tube assays, in which opposite effects of Jag1 or Dll4 deficiency on vessel branching are rescued by transduction of an EFNB2-expressing lentivirus, identifying EphrinB2 as a Notch effector during coronary artery development.

A common feature among the endocardial or endothelial Notch loss- and gain-of-function models analyzed in our study is the thin ventricular wall (Figure 8B,D). Of interest is that myocardial inactivation of Jag1, or combined inactivation of Jag1 and Jag2, or Mib1, leads to thinner ventricular walls, accompanied by reduced cardiomyocyte proliferation, disrupted ventricular chamber patterning, and cardiomyopathy (Luxán et al., 2013; D’Amato et al., 2016). In contrast, chamber patterning is maintained in endocardial Jag1, Dll4, and Efnb2 mutants and endothelial Dll4 and Jag1 mutants. Our results suggest that disturbed ventricular wall growth in the earlier E11.5-E12.5 Jag1flox;Nfatc1-Cre is caused by altered endocardial-myocardial signaling, as suggested for Dll4flox;Nfatc1-Cre mutants (D’Amato et al., 2016). In the later E14.5-E16.5 Jag1flox;Pdgfb-iCreERT2, Dll4flox;Pdgfb-iCreERT2, Jag1flox;Cdh5-CreERT2 and Dll4flox;Cdh5-CreERT2 mutant embryos, a thinner ventricular wall would be due to the lack of a well-formed coronary plexus (Figure 8D). At E12.5-E13-5, myocardial growth may also depend on ‘angiocrine signals’ from the un-perfused primitive coronary plexus (Rafii et al., 2016), as the diffusion limit of oxygen and nutrients from the endocardium is reached during the transition from endocardial to coronary myocardial perfusion. Therefore, ventricular compaction relies on two interconnected Notch-dependent processes: patterning and maturation of the chamber myocardium, and timely development of a functional coronary vessel network, as previously suggested (D’Amato et al., 2016). This may be clinically relevant to the study and treatment of cardiomyopathies.

Materials and methods

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagentMus Musculus (Mouse strain)(Nowotschin et al., 2013)CBF1:H2B-Venus
Genetic reagentMus Musculus (Mouse strain)(Kisanuki et al., 2001)Tie2-Cre
Genetic reagentMus Musculus (Mouse strain)(Wu et al., 2012)Nfatc1-Cre
Genetic reagentMus Musculus (Mouse strain)(Wang et al., 2010)Pdgfb-iCreERT2
Genetic reagentMus Musculus (Mouse strain)(Wang et al., 2010)Cdh5-CreERT2
Genetic reagentMus Musculus (Mouse strain)(Koch et al., 2008)Dll4flox
Genetic reagentMus Musculus (Mouse strain)(Mancini et al., 2005)Jag1flox
Genetic reagentMus Musculus (Mouse strain)(Grunwald et al., 2004)Efnb2flox
Genetic reagentMus Musculus (Mouse strain)(Radtke et al., 1999)Notch1flox
Genetic reagentMus Musculus (Mouse strain)(D’Amato et al., 2016)MfngGOF
Sequenced-based reagentFH1_DLL45’-GTTACACAGTGAAAAGCCAG-3’KiCqStart_
SIGMA qPCR primer
Sequenced-based reagentRH1_DLL45’-CTCTCCTCTGATATCAAACAC-3’KiCqStart_
SIGMA qPCR primer
Sequenced-based reagentFH1_JAG15’-ACTACTACTATGGCTTTGGC-3’KiCqStart_
SIGMA qPCR primer
Sequenced-based reagentRH1_JAG15’-ATAGCTCTGTTACATTCGGG-3’KiCqStart_
SIGMA qPCR primer
Squenced-based reagentFH1_HEY15’-CCGGATCAATAACAGTTTGTC -3’KiCqStart_
SIGMA qPCR primer
Sequenced-based reagentRH1_HEY15’-CTTTTTCTAGCTTAGCAGATCC-3’KiCqStart_
SIGMA qPCR primer
Sequenced-based reagentFH1_EFNB25’-AAAGTTGGACAAGATGCAAG-3’KiCqStart_
SIGMA qPCR primer
Sequenced-based reagentRH1_EFNB25’-TGTACCAGCTTCTAGTTCTG-3’KiCqStart_
SIGMA qPCR primer
Transfected
construct (human)
VSV-GViral Vectors Unit, CNIC, SpainLentiviral construct to
transfect
shRNA
Cell line (include species here) HUVEC Lonza  
Transfected construct (mouse)pRLL-IRESeGFPAddgene
Lentiviral construct to
transfect eGFP or
Full-length murine EphrinB2
Transfected construct (human)shRNA to JAG1 SIGMASHCLNG-NM_000214 Clone ID:NM_000214.2-3357s21c1;Clone ID:NM_000214.2-1686s21c1transfected construct (human)
Transfected construct (human)shRNA to DLL4SIGMASHCLNV-NM_019074 Clone ID:NM_019074.2-2149s21c1;
Clone:NM_019074.2-2276s21c1
transfected construct (human)
AntibodyAnti-CD31/Pecam1 (Monoclonal Rat)
BD Biosciences Pharmingen
 550274 MEC13.3IF=1:100
AntibodyDll4
(Polyclonal Rabbit)
Santa Cruz BiotechnologySc-28915IF=1:100
AntibodyEndomucin
(Polyclonal Rat)
Santa Cruz
Biotechnology
sc-65495 V.7C7IF=1:200
AntibodyJag1
(Monoclonal Rabbit)
Cell Signaling
Technology
2620
28H8
IF=1:100
AntibodyNFATc1
(Monoclonal Mouse)
Enzo Life
Sciences
ALX-
804-022-R100
7A6
IF=1:100
AntibodyCleaved
Notch1 (Val1744)
(Monoclonal Rabbit)
Cell Signaling Technology4147 D3B8IF=1:100
Antibodyp27
(Polyclonal Mouse)
Medical and
Biological
Laboratories
K0082-3 p27 Kip1IF=1:100
AntibodyNotch 3
(Polyclonal Rabbit)
Abcamab23426 G00041IF=1:100
AntibodyERG
(Monoclonal Rabbit)
Abcamab110639 EPR3863
IF=1:100
AntibodyGlut1
(Polyclonal Rabbit)
MERCK
Millipore
07-1401 C00222IF=1:100
AntibodyBrdU
(Monoclonal Mouse)
BD Biosciences347580
B-44
IF=1:50
AntibodyIsolectin IB4-
Alexa Fluor 647
Molecular
Probes
I32450IF=1:300
AntibodyBiotin
Anti-rat
Vector
laboratories
BA4001IF=1:200
AntibodyBiotin
Anti-rabbit
Jackson111-066-003IF=1:200
AntibodyAnti-mouse
Alexa Fluor 647
Jackson115-606-003IF=1:200
AntibodyAnti-mouse
Alexa Fluor
488
InvitrogenA-11029IF=1:200
AntibodyAnti-ratInvitrogenA-11006IF=1:200
Peptide, recombinant proteinHuman-Vegf165Human
Peprotech
100-20 10ng/ml
Commercial assay or kitClick-iT EdU Imaging KitThermo Fisher ScientificC10340 
Commercial assay or kitHydroxyprobeTM-1 Plus Kit
Hydroxyprobe, Inc (HPI).(Pimonidazole Hydrochloride CAS#70132-50-3)
OtherMatrigelCorning354234
SoftwareCutadapt v1.6(Martin, 2011)
SoftwareRSEM v1.2.20(Li and Dewey, 2011)
SoftwareLimma(Ritchie et al., 2015)Bioconductor package
SoftwareGOplot(Walter et al., 2015)
SoftwareIPAhttp://www.ingenuity.com

Mouse strains and genotyping

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Animal studies were approved by the CNIC Animal Experimentation Ethics Committee and by the Madrid regional government (Ref. PROEX 118/15). All animal procedures conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 1201/2005. Mouse strains were CBF1:H2B-Venus (Nowotschin et al., 2013), Tie2-Cre (Kisanuki et al., 2001), Nfatc1-Cre (Wu et al., 2012), Pdgfb-iCreERT2 (Wang et al., 2010), Cdh5-CreERT2 (Wang et al., 2010), Dll4flox (Koch et al., 2008), Jag1flox (Mancini et al., 2005), Efnb2flox (Grunwald et al., 2004), Notch1flox (Radtke et al., 1999), MfngGOF (D’Amato et al., 2016). To generate the R26CAGDll4GOF transgenic line, a full-length mouse Dll4 cDNA was obtained from clone IMAGE 6825525. The sequence was PCR amplified with Phusion High-Fidelity DNA Polymerase (NEB) and primers containing BamHI and ClaI sites and was cloned in-frame with 6 Myc Tag epitopes into the BamHI and ClaI sites of a pCS2-MT plamid. The Dll4-MT fragment was subcloned into pCDNA3.1 with BamHI and EcoRI and excised with BamHI and NotI. We then modified pCCALL2 (Lobe et al., 1999) by cloning new XbaI sites before and after a CAG cassette, which includes a CMV enhancer/β-actin promoter and a rabbit β-globin polyA signal. The KpnI and NotI PCR Dll4-MT was cloned into the BglII-NotI sites of the modified pCCALL2. The XbaI-cassette containing CAG-loxP-β-Geo-loxP-Dll4-IRESeGFP was obtained by digestion and cloned into the XbaI site of the pROSA26-1 plasmid (Soriano, 1999). The final construct was linearized with XhoI and electroporated into R1 mESCs derived from a cross of 129/Sv x 129/Sv-CP mice (Nagy et al., 1993). After G418 (200 μg/ml) selection for 7 days, 231 clones were picked. Homologous recombination was identified by Southern blot of EcoRV-digested DNA and hybridized with 5’ and 3’ probes. About 25% of the clones were positive, and we selected three clones to confirm karyotype. One positive clone was injected into C57/BL6C blastocysts to generate chimaeras that transmitted the transgene to their offspring. The resulting founders were genotyped by PCR of tail genomic DNA using primers targeting the R26 locus before and after the cloning site and the transgene polyA signal.

Throughout the MS, Jag1flox/flox;Nfatc1-Cre mice are called Jag1flox;Nfatc1-Cre for simplicity; this also applies to the Dll4flox;Nfatc1-Cre mice, Jag1flox;Pdgfb-iCreERT2 mice, Dll4flox;Pdgfb-iCreERT2, Dll4flox;Cdh5-CreERTT2, Jag1flox;Cdh5-CreERT2 mice, Dll4flox;Cdh5-CreERT2, Dll4flox;Cdh5-CreERTT2 MfngGOF;Tie2-Cre mice (which carry two extra copies of Mfng), and Efnb2flox;Nfatc1-Cre mice, but not to Dll4GOF;Tie2-Cre mice, which carry only one extra copy of Dll4.

Tamoxifen induction

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Double heterozygous Cdh5-CreERTT2/+;Jag1flox/+ females were crossed with homozygous Jag1flox/flox males, and pregnant females were administered by oral gavage 200 µl of tamoxifen solution (Sigma, T5648; 5 mg/ ml; generated by diluting 50 mg of tamoxifen in ml of 95% ethanol plus 9 ml of Corn Oil). To obtain Jag1flox/flox;Cdh5-CreERTT2 embryos, pregnant females were induced at E9.5 and opened at E16.5. To obtain Dll4flox/flox;Cdh5-CreERTT2 embryos, Dll4flox/flox males were crossed with double heterozygous Cdh5-CreERTT2/+;Dll4flox /+ females, and pregnant females were induced at E12.5 and opened at E15.5. With Pdgfb-iCreERT2, double heterozygous Pdgfb-iCreERT2/+;Jag1flox/+(or Dll4flox/+) males were crossed with homozygous Jag1flox/flox or Dll4flox/flox females. Pregnant females were induced at E12.5 and dissected at E15.5 (for Jag1flox), or induced at E14.5 and dissected at E15.5 for Dll4flox.

Histology and in situ hybridization

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Hematoxylin and eosin (H & E) staining and in situ hybridization (ISH) on sections were performed as described (Kanzler et al., 1998). Details of probes will be provided on request.

Whole-mount immunofluorescence

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Embryos were dissected and fixed for 2 hr in 4% paraformaldehyde (PFA) in PBS at 4°C, then permeabilized for 1 hr with 0.5% Triton X‐100/PBS and subsequently blocked for 1 hr in Histoblock solution (5% goat serum, 3% BSA, 0.3% Tween‐20 in PBS). After several washes in PBS‐T (PBS containing 0.1% Tween‐20), embryos were mounted in 1% agar in a 60 mm petri dish. For whole-mount immunofluorescence on E11.5-E12.5 CBF:H2B-Venus embryos endogenous GFP, was imaged with a NIKON A1R confocal microscope. Z-stacks were captured every 5 μm.

For EdU immunofluorescence, pregnant females at E12.5 were injected intraperitoneally with 100 μl EdU nucleotides (2 mg/ml in PBS). The mice were euthanized 1 hr later and fixed in 4% PFA. EdU incorporation was detected with the Click-iT EdU Imaging Kit (Thermo Fisher Scientific, C10340) according to manufacturer's instructions. For IsoB4 and Endomucin immunostaining on E11-4-E16.5 embryos, imaging by confocal z-projection of the deeper section of the myocardium captures the arteries (IsoB4-stained) with the outer section corresponding to the veins (IsoB4+Endomucin-stained). N1ICD embryos were fixed for 5 hr at 4°C in frozen Methanol, washed and incubated 20 min at 98°C in DAKO target retrieval solution pH6 (Agilent). After washing in H2O, hearts were fixed again in acetone at −20°C for 10 min. After washing in PBT, the hearts were blocked in 10% FBS, 5%BSA, 0.4% TritonX100 for 3 hr at RT under gentle rocking. Hearts were incubated with primary N1ICD-antibody at 1:500 for 2 days at 4°C and allowed to settle at RT for 1 hr. This was followed by washing 5 hr in PBS/0.4% TritonX100 at RT. Hearts were then incubated in presence of anti-rabbit HRP (1:500), DAPI (1:1500) and anti-VE cadherin (1:500) overnight at 4°C. After resting for 1 hr at RT, hearts were washed in PBT, and 30 min in presence TSA (tyramides) diluted 1:200 in PBS. Quantifications were carried out using ImageJ software. The main coronary trees were selected with the tool ‘Freehand selection’ manually and measured directly from the autoscaled images obtained by Z-projection. The selected area was quantified in μm2. Confocal microscopy analysis was carried out on a Nikon A1R or Leica LAS-AF 2.7.3.

Immunofluorescence on sections

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Paraffin sections (7 μm) were incubated overnight with primary antibodies, followed by 1 hr incubation with a fluorescent-dye–conjugated secondary antibody. N1ICD, Dll4, Jag1, and p27 were immunostained using tyramide signal amplification (TSA) (Del Monte et al., 2007); see Supplementary file 4 for antibodies. All immunostainings were performed in the same way except for co-immunostaining of SM22a (+ secondary chicken anti-goat-594) and Notch3 (+secondary donakey anti-rabbit-488), which required continued permeabilization in PBS 1X + 0.3% TritonX100. Images were processed using ImageJ software. For p21 and N1ICD quantification, the number of positive nuclei was divided by the total number of nuclei counted on sections (≥3). For αSMA and Notch3 quantification, the number of mural cells surrounding intramyocardial vessels expressing both SMA+ and Notch3+ (double positives) was counted and divided by the total number of intramyocardial vessels defined by Iso B4 staining in the same section. For SM22a and Notch3 quantification, the number of double- SM22 and Notch3 positive cells was divided by the total number Notch3-positive cells counted on sections (≥3).

Hypoxia analysis

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Pimonidazole hydrochloride (Hypoxyprobe) was injected intraperitoneally at 60 mg/kg to E12.5 pregnant females. After 2.5 hr, embryos were dissected in cold PBS and fixed O/N in 4% PFA. Embryos were paraffin embedded. Sections at 7 µm were deparaffinized and boiled in 10 mM, pH = 6 Na Citrate solution. For immunofluoresence, sections were permeabilized with 0.4% Triton in PBS, blocked with 5% FBS and incubated anti-Glut1 (1:100) and anti-Pymonidazole (1:200) antibodies O/N at 4°C. Secondary antibodies (anti-Rabbit Alexa568 for Glut1 and anti-mouse biotinylated for Pymonidazole 1:400, respectively) were incubated for 1 hr at RT. Pymonidazole signal was amplified using Fluorescein Tyramides (1:100). Counterstaining: IB4 (endocardium/endothelium) 1:200; DAPI (nuclei) 1:3000.

Mouse ventricular explant culture and immunofluorescence

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Heart ventricular explants were performed as previously described with minor modifications (Wu et al., 2012). Ventricles were dissected from E10.5 or E11.5 embryos (with removal of the outflow tract and atria), rinsed with PBS to remove circulating cells, and placed in Nunc four-well plates. Matrigel (Corning Matrigel Basement Membrane Matrix, *LDEV-Free, 10 mL growth factor reduced, BD Biosciences 354234) was diluted 1:1 with DMEM plus 10% FBS and 10 ng/ml Vegf (Human-Vegf 165 Peprotech). Each well had a total volume of 400 µl, and 3–4 hearts were cultured for 6 days and then fixed in 4% PFA4% 10 min and washed twice with PBS1X for 15 min. Explants were permeabilized with 0.5% Triton X-100 for 1 hr, blocked with Histoblock solution (FBS Histoblock) for 2–3 hr at RT, and incubated with anti-CD31 1:100 (Purified Rat Anti-Mouse CD31 BD Biosciences Pharmingen) overnight at 4°C. Anti-rat biotinylated was used as secondary antibody and diluted in BSA 1:150. Staining was amplified with the ABC kit and 3 min of TSA (tyramides) diluted 1:100 in PBS.

Quantification of compact myocardium thickness

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The method used was a modification of that described by Chen et al. (2009); Yang et al. (2012). Briefly, 7 μm paraffin sections from E12.5, E15.5 and E16.5 wild type (WT) and mutant hearts were stained with anti-CD31 or Endomucin (Emcn) and anti-MF20 antibodies to visualize ventricular structures. Confocal images were obtained with a NIKON A1R confocal microscope. Measurements were made using ImageJ software. In E15.5 and E16.5 heart sections, endocardial cells were stained with anti-Emcn and myocardium with anti-cTnT. Left and right ventricles were analyzed separately. For each measurement, settings were kept constant for all images, using the scale bar recorded in each image as the reference distance. The thickness of the compact myocardium was measured by dividing the ventricle into left and right parts. Several measurements were taken in each region and the mean was expressed in μm.

Quantification of Jag1 and Dll4 expression in sections

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Quantification of ISH signal were carried out using ImageJ software. Compact myocardium area was selected manually with the tool ‘Polygon selection’. Coronary area was identified in this selection by color intensity using ‘Color Threshold plugin’ with the settings: Hue: Min = 145 Max = 199, Saturation: Min = 60 Max = 255, Brightness: Min = 0 Max = 255. Positive coronary area for Dll4 or Jag1 expression was calculated dividing the coronary area positive for each gene by the total compact myocardium area. Statistical analysis was assessed by Student’s t-test.

India ink perfusion

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Embryos were collected from E15.5 to E18.5 in PBS containing 2 μg/ml heparin. The thoracic cavities were immediately cut and transferred to DMEM containing 10%FBS and heparin on ice. Hearts were carefully dissected from the surrounding tissue and then placed on an inverted Petri dish and gently dried to avoid movement during perfusion. India ink or red tempera was diluted in PSB/heparin and injected from the ascending aorta with a borosilicate glass tube thinned to the appropriate diameter and attached to a mouth pipette. India ink was slowly injected by minute puffs of breath during diastolic intervals. Hearts were fixed in 4% PFA, dehydrated, cleared in BABB (benzyl alcohol: benzyl benzoate, 1:1) and imaged with a stereomicroscope.

Lentiviral production

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Bacterial glycerol stocks for JAG1 and DLL4 MISSION shRNA were purchased from SIGMA. MISSION shRNAs: shRNA JAGGED1_2: clone ID: NM_000214.2–3357 s21c1; shRNA JAGGED1_3: clone ID: NM_000214.2–1686 s21c1; shRNA DLL4_1: clone ID_ NM_019074.2–2149 s21c1; shRNA DLL4_1: clone ID_ NM_019074.2–821 s21c1. Concentrated lentiviral particles were produced by triple transfection in HEK293T cells. Briefly, HEK293T cells were cultured in 150 mm plates for transfection of the lentiviral vectors. The psPAX2 packing plasmid, the pMD.G envelope plasmid coding for the VSV-G glycoprotein (Viral Vectors Unit, CNIC, Spain) and the LTR-bearing shuttle lentiviral plasmids were cotransfected using the calcium phosphate method. The transfection medium was replaced with fresh medium 16 hr post-transfection. Viral supernatants were harvested at 72 hr post-transfection, filtered through a 0.45 µm filter (Steriflip-HV, Millipore, MA) and concentrated by ultracentrifugation. Precipitated viruses were resuspended in pre-chilled 1X PBS, aliquoted and stored at −80°C for further use. Viral titer was measured on viral genomes by qPCR using the standard curve method with primers against the LTRs. Forward primer: 5’-AGCTTGCCTTGAGTGCTTCAA-3’; Reverse primer: 5’-AGGGTCTGAGGGATCTCTAGTTA-3’. Full-length murine EphrinB2 (Efnb2), kindly provided by Ralf Adams (MPI for Molecular Biomedicine, University of Münster, Germany), was subcloned into the lentiviral vector pRLL-IRES-eGFP (Addgene). Concentrated lentiviruses expressing pRLL-IRES-eGFP or pRLL-Mfng-IRES-eGFP were obtained as described (Esteban et al., 2011). Viruses were titrated in Jurkat cells, and infection efficiency (GFP-expressing cells) and cell death (propidium iodide staining) were monitored by flow cytometry.

Culture and infection on HUVEC

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Human umbilical vein endothelial cells (HUVECs) were maintained in supplemented EGM2 medium (EGM2 Bulletkit, K3CC-3162 Lonza). Cells were transduced on suspension at a multiplicity of infection (m.o.i.) of 80 with combinations of 2 shRNAs against JAG1 or DLL4 together with GFP or EFNB2 overexpressing lentivirus and seeded onto 24-well plates or 12-well plates at a density of 3 × 104 or 6 × 104 cells/well respectively. Infection efficiency was near 100%. HUVECs were harvested after 48 hr. Gene expression analysis was performed using KiCqStart SYBR Green predesigned primers (Sigma-Aldrich).

Angiogenesis assays on matrigel

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Angiogenesis in vitro was investigated using endothelial cell tube formation assay on Matrigel (#354234 Corning) as previously described (Grant et al., 1991). Briefly 50 μl of ice cold Matrigel was coated on a 96 micro-well plate as a base for tube formation. After allowing the gel to settle in the incubator for 30 min at 37°C, 5% CO2, HUVECs in basic EGM medium (EBM2 H3CC-3156, Lonza) were seeded in triplicate at a density of 12.5 × 104 on Matrigel and incubated at 37°C, 5% CO2. After 5 hr, tube formation was monitored by phase-contrast microscope. The tube networks were quantified using NIH Image J with Angiogenesis plugin software (http://image.bio.methods.free.fr/ImageJ/?Angiogenesis-Analyzer-for-ImageJ). Excel data were represented on Graphpad Prism seven as mean with SD. For statistical analysis a one-way ANOVA and Tukey post-hoc tests were performed.

Statistical analysis

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Statistical analysis was carried out using Prism 7 (GraphPad). All statistical tests were performed using two-sided, unpaired Student’s t-tests, except for Figures 1, 2 and 7 and figures Supplements 4, 9 and 12 where we performed one-way ANOVA to assess statistical significance with a 95% confidence interval, where numerical data are presented as mean ± SD; results are marked with one asterisk (*) if p<0.05, two (**) if p<0.01, and three (***) if p<0.001. Sample size was chosen empirically according to previous experience in the calculation of experimental variability. No statistical method was used to predetermine sample size. All experiments were carried out with at least three biological replicates. The numbers of animals used are described in the corresponding figure legends. Animals were genotyped before the experiment and were caged together and treated in the same way. Variance was comparable between groups throughout the manuscript. We chose the appropriate tests according to the data distributions. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

RNA-sequencing

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RNA was isolated at E12.5, from whole hearts of Jag1flox;Nfatc1-Cre and Dll4flox;Nfatc1-Cre embryos, as well as their WT counterparts (four replicate samples for each condition, pooling four embryos per sample, in all cases). At E15.5, RNA was isolated from ventricles of Dll4flox;Cdh5-CreERT2 (three replicate samples, pooling three embryos per sample), as well as their WT counterparts (four replicate samples, pooling three embryos per sample). RNA-Seq data for Jag1flox;Nfatc1-Cre, Dll4flox;Nfatc1-Cre, and Dll4flox;Cdh5-CreERT2 experiments was generated by CNIC's Genomics Unit. RNA-Seq sequencing reads were pre-processed by means of a pipeline that used FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to assess read quality and Cutadapt v1.6 (Martin, 2011) to trim sequencing reads, thus eliminating Illumina adaptor remains, and to discard reads shorter than 30 bp. Resulting reads were mapped against the mouse transcriptome (GRCm38 assembly, Ensembl release 76) and quantified using RSEM v1.2.20 (Li and Dewey, 2011). Around 80–90% of the reads participated in at least one reported alignment. Expected expression counts calculated with RSEM were then processed with an analysis pipeline that used Bioconductor package Limma (Ritchie et al., 2015) for normalization and differential expression testing in six pairwise contrasts involving mutant versus WT comparisons. We discarded two of the Dll4flox;Nfatc1-Cre samples based on preliminary analyses of Dll4 expression levels and clustering patterns in diagnostic principal component analysis plots. Changes in gene expression were considered significant if associated with a Benjamini-Hochberg (BH) adjusted p-value<0.05. The number of differentially expressed genes detected in comparisons between Jag1flox;Nfatc1-Cre, Dll4flox;Nfatc1-Cre and Dll4flox;Cdh5-CreERT2 and their control counterparts was 205, 257 and 156, respectively. Enrichment analyzes were performed with IPA (Ingenuity Pathway Analysis, Qiagen, http://www.ingenuity.com). IPA was used to identify collections of genes associated with canonical pathways, common upstream regulators, or functional terms significantly overrepresented in the sets of differentially expressed genes; statistical significance was defined by Benjamini-Hochberg adjusted p-value<0.05. Circular plots summarizing the association between genes and enriched pathways, upstream regulators, and functional terms were generated with GOplot (Walter et al., 2015).

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Decision letter

  1. Didier Y Stainier
    Senior Editor; Max Planck Institute for Heart and Lung Research, Germany
  2. Bin Zhou
    Reviewing Editor; Chinese Academy of Sciences, China
  3. Bin Zhou
    Reviewer; Chinese Academy of Sciences, China
  4. Nicola Smart
    Reviewer

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Thank you for submitting your article "Coronary arterial development is regulated by a Dll4-Jag1-EphrinB2 signaling cascade" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Bin Zhou as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Nicola Smart (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

Unraveling the molecular mechanism regulating coronary vessel formation provides important information to neovascularization in coronary artery diseases. In this study, Travasiano et al. used mouse genetic Cre-loxP system as well as in vitro culture experiments to determine whether Notch ligands Dll4 and Jag1 promote or attenuate coronary angiogenesis. Their data showed that sinus venosus endocardial knockout of Jag1 inhibits coronary sprouting from sinus venosus, while Dll4 removal promotes excessive coronary sprouting, indicating the ligands antagonism in regulating coronary angiogenesis. Gain of function by Dll4 or glycosyltransferase MFng over expression inhibit coronary artery formation. Authors then linked Notch signaling to downstream target EphrinB2, which mediates arterial differentiation. While Notch signaling in angiogenesis regulation and artery formation has been reported in other vascular beds (e.g. retina vascular plexus), the role in coronary angiogenesis and artery maturation has not been reported and remains incompletely understood currently. This study, therefore, offers a sufficient advance to the field of coronary vascular development and may provide new insight to coronary neovascularization in diseases such as myocardial infarction. Overall, this finding is interesting and important, and the major conclusion is supported by the data presented.

Essential revisions:

1) Deletion efficiency of genes by in situ or immunostaining detection. The authors claimed that Late endothelial Jag1 or Dll4 inactivation disrupts coronary plexus remodeling. The authors should show the relative target gene Dll4, Jag1 deletion efficiency in inducible mice, by immunostaining or in-situ?

2) A schematic model need to be provided. Since Jag1 and Dll4 have opposing effects on Notch signaling, based on the current data, how do the authors propose these ligands and Notch signaling shape the pre-artery fate and coronary vessel development temporally and spatially? Since phenotypes were analyzed at different stages during coronary plexus formation, arterial differentiation and myocardial growth, the authors could consider to provide a schematic model to help with the readability.

3) More detailed in situ hypoxia measurement need to be included. It is difficult to understand why hypoxia should manifest as early as E12.5 in the Dll4/ Jag1 mutants, if, as inferred, this is due to malformed coronary plexus and perfusion defects. At this stage, SV and endocardial sprouting is not very advanced (in control hearts) and the mutant hearts are less compacted, thus less reliant on vessels for perfusion. Perhaps this could be reconciled if the authors assessed hypoxia in situ e.g. with hypoxyprobe (or at the very least, Glut1 across the whole heart) to correlate regional hypoxia with coronary vessel sprouting. If, instead, this is a stress response to the loss of angiocrine stimuli, as alluded, the authors should validate some putative factors from the RNA-Seq data to support this conclusion.

4) Other more specific markers for smooth muscle should be used. αSMA is used to label smooth muscle cells, however, it is expressed more broadly in mesenchymal cells and cardiomyocytes throughout the embryonic heart prior to ~E16.5 and this is reflected in the images. The authors should attempt to stain for SM22a which, although similarly non-specific, shows better enrichment in VSMCs, relative to the other cell types. Improved visualization may provide further insight into the nature of the defect e.g. are epicardium-derived precursors trapped or poorly differentiated?

Reviewer #1:

Understanding how coronary arteries are formed is important for developmental biology and also postnatal coronary neovascularization after cardiac injuries. In this study, the authors reported that coronary arterial precursors are specified in the SV prior to the formation of primary coronary plexus, while Dll4-Jag1-EphrinB2 signaling mediated subsequent arterial differentiation. Inactivation of Dll4 increase capillary growth while Jag1 removal blocks sprouting. Deletion of arterial differentiation target Efnb2 causes coronary arterial defects. This finding is interesting and important to the field. Overall, the conclusion is supported by the data presented. I have a few points for author to consider and clarify.

The ligand antagonism mechanism proposed by author for coronary angiogenesis regulation is interesting. Is this Jag1/Dll4 antagonism restricted to coronary development or has already been known in other organotypic vessel development?

Deletion of Dll4 causes excessive expansion of the primitive coronary vessels and at the same time has thin (half) myocardium in the mutant. This is an interesting mutant phenotype as it separates myocardial growth and regulation of coronary angiogenesis. Could author elaborate more on this and also study how the angiogenesis factors such as VEGF in thin myocardium regulates coronary vessels?

The authors claimed that Late endothelial Jag1 or Dll4 inactivation disrupts coronary plexus remodeling. The authors should show the relative target gene Dll4, Jag1 deletion efficiency in inducible mice, by immunostaining or in-situ?

Does Pdgfb-iCreERT2 also target endocardial cells in the heart in addition to coronary vascular endothelial cells?

There is reduced artery coverage in Efnb2 knockout hearts. How about the pre-artery cells (Su et al., 2018) in the mutant heart? How about pericyte/smooth muscle cells recruitment in the mutant?

The authors described in the manuscript that p27 nuclear staining was increased 2-fold which indicate decreased cell proliferation, so in which cell p27 increased expression level, cardiomyocytes or endothelial?

The authors should pay more attention to the details in the manuscript and figures. Such as how the EDU inject in the experiments? please describe it. More detailed information for experiments should be provided in the Materials and methods section.

Reviewer #2:

Travisano and colleagues performed a comprehensive analysis of the roles of Notch ligands in coronary vessel development by manipulating Jag-1 and Dll4 using combinations of various Cre-floxed lines. Using both gain and loss of function analysis of Notch signaling pathway components and both in vivo and in vivo (including a novel ventricular explant) assays, the authors showed that Notch signaling is essential for sinus venosus sprouting angiogenesis for coronary plexus formation, coronary vessel maturation and artery formation. The authors also linked Notch functions to a known downstream target, EphrinB2, which mediates angiogenesis and arterial differentiation. Overall, the authors have done extensive phenotypic analyses and provided data of excellent quality. The manuscript is also very well written. The manuscript can be improved by providing more specific mechanistic insights of certain phenotypes. However, considering that the work covered in this manuscript has filled in a missing gap in our knowledge in Notch signaling in coronary vessel development and provided entry points for more future investigations, it warrants publication in eLife.

1) The overall finding here is consistent with the known roles of Notch signaling. Since Jag1 and Dll4 have opposing effects on Notch signaling, based on the current data, how do the authors propose these ligands and Notch signaling shape the pre-artery fate and coronary vessel development temporally and spatially? Since phenotypes were analyzed at different stages during coronary plexus formation, arterial differentiation and myocardial growth, the authors could consider to provide a schematic model to help with the readability.

2) Figure 6 legend, the authors should clarify the labels for the vessel caliber, branch point distance and the endothelial number (i.e. what are labeled by arrowheads, asterisks and arrows?). Since these features seem to vary in different areas of the explants, the authors should label these in all magnified panels.

Reviewer #3:

The study by Travasiano et al. examined the requirement of the Notch pathway in development of the coronary vasculature, specifically to determine whether the ligands Jag1 and Dll4 promote and attenuate angiogenesis, respectively, from the main embryonic sources (sinus venosus and endocardium, mirroring their roles in the systemic vasculature. Essentially, these hypothesised roles are confirmed, and roles in plexus remodelling, arterial differentiation and perivascular cell recruitment are additionally demonstrated. Extrapolating from the systemic vasculature, such roles are neither unexpected nor novel, however, they have not been previously demonstrated in the coronary vasculature, only indirectly inferred e.g. in recent scRNA-Seq analyses. The work, therefore, offers a sufficient advance to the field and the analysis has been carefully and rigorously performed. Substantive concerns are few:

1) Conceptually, it is difficult to understand why hypoxia should manifest as early as E12.5 in the Dll4/ Jag1 mutants, if, as inferred, this is due to malformed coronary plexus and perfusion defects. At this stage, SV and endocardial sprouting is not very advanced (in control hearts) and the mutant hearts are less compacted, thus less reliant on vessels for perfusion. Perhaps this could be reconciled if the authors assessed hypoxia in situ e.g. with hypoxyprobe (or at the very least, Glut1 across the whole heart) to correlate regional hypoxia with coronary vessel sprouting. If, instead, this is a stress response to the loss of angiocrine stimuli, as alluded, the authors should validate some putative factors from the RNA-Seq data to support this conclusion.

2) αSMA is used to label smooth muscle cells, however, it is expressed more broadly in mesenchymal cells and cardiomyocytes throughout the embryonic heart prior to ~E16.5 and this is reflected in the images. The authors should attempt to stain for SM22a which, although similarly non-specific, shows better enrichment in VSMCs, relative to the other cell types. Improved visualisation may provide further insight into the nature of the defect e.g. are epicardium-derived precursors trapped or poorly differentiated?

3) How do the authors explain the differences in lethality between the PdgfbiCrERT2 vs. Cdh5CreERT2 loss of Dll4, induced at E12.5?

https://doi.org/10.7554/eLife.49977.sa1

Author response

Essential revisions:

1) Deletion efficiency of genes by in situ or immunostaining detection. The authors claimed that Late endothelial Jag1 or Dll4 inactivation disrupts coronary plexus remodeling. The authors should show the relative target gene Dll4, Jag1 deletion efficiency in inducible mice, by immunostaining or in-situ?

We agree with the reviewer that the efficiency of Jag1 and Dll4 deletion in the inducible loss of function models needs to be demonstrated. We now show in the revised Figure 3—figure supplement 1 that Jag1 and Dll4 are efficiently deleted after tamoxifen induction of Pdgfb-iCreERT2. We have performed ISH for Jag1 and Dll4 on Jag1flox;Pdgfb-CreERTand Dll4flox;Pdgfb-CreERT2 hearts that were tamoxifen-induced at E12.5 and E14.5, respectively. As shown in Figure 3—figure supplement 1, Jag1 or Dll4 mRNA are severely decreased in coronaries of E15.5 Jag1flox (or Dll4flox);Pdgfb-creERT2 mutants. Moreover, we previously showed that Dll4 mRNA was markedly decreased in E15.5 Dll4flox;Cdh5-CreERT2 coronaries tamoxifen-induced at E12.5 (see Figure 3—figure supplement 4C).

The revised text says (Results): “Defective coronary remodeling and maturation in endothelial Jag1 or Dll4 mutantsWe next examined the requirements of endothelial Jag1 and Dll4 for coronary vessel remodeling and maturation. […] This was confirmed by co-immunostaining with SM22a and Notch3 (Figure 3—figure supplement 7C, D) demonstrating near complete absence of smooth muscle differentiation in Dll4flox;Pdgfb-iCreERT2mutants (Supplementary file 1, sheet 7).”

2) A schematic model need to be provided. Since Jag1 and Dll4 have opposing effects on Notch signaling, based on the current data, how do the authors propose these ligands and Notch signaling shape the pre-artery fate and coronary vessel development temporally and spatially? Since phenotypes were analyzed at different stages during coronary plexus formation, arterial differentiation and myocardial growth, the authors could consider to provide a schematic model to help with the readability.

We thank the reviewers for this suggestion. We have now included a schematic model summarizing our findings on the opposing roles of Jag1 and Dll4 during coronary vessel sprouting and their later roles in vessel remodeling. This model is referred to as Figure 8 (new) in the Discussion.

3) More detailed in situ hypoxia measurement need to be included. It is difficult to understand why hypoxia should manifest as early as E12.5 in the Dll4/ Jag1 mutants, if, as inferred, this is due to malformed coronary plexus and perfusion defects. At this stage, SV and endocardial sprouting is not very advanced (in control hearts) and the mutant hearts are less compacted, thus less reliant on vessels for perfusion. Perhaps this could be reconciled if the authors assessed hypoxia in situ e.g. with hypoxyprobe (or at the very least, Glut1 across the whole heart) to correlate regional hypoxia with coronary vessel sprouting. If, instead, this is a stress response to the loss of angiocrine stimuli, as alluded, the authors should validate some putative factors from the RNA-Seq data to support this conclusion.

Following the reviewers suggestion, we have performed immunohistochemical detection of pimonidazole and Glut1 on E12.5 Jag1flox;Nfatc1Creand Dll4flox;Nfatc1Cre heart sections.

The revised text says (Results): “We examined E12.5 Jag1flox;Nfatc1-Cre and Dll4flox;Nfatc1-Cre mutants for evidence of hypoxia given that the gene signatures in the RNA-seq analysis suggested an ongoing hypoxic/metabolic stress response. […] These results indicate that Jag1flox;Nfatc1-Cre and Dll4flox;Nfatc1-Cre mutant hearts are not overtly hypoxic at E12.5, suggesting that the hypoxic/metabolic stress gene signatures may be due to a cell autonomous defect of endocardial/endothelial cells”.

Thus, the hydroxyprobe and Glut-1 immunostainings suggests that contrary to our initial interpretation, E12.5 Jag1flox;Nfatc1Creand Dll4flox;Nfatc1Cre mutants are not more hypoxic than the WT. This would imply that ventricular wall thinning in the mutants is not due to improper coronary plexus development. One possibility is that impaired ventricular wall growth in E12.5 Jag1flox;Nfatc-Cre and Dll4flox;Nfatc1-Cre hearts may is due to defective endocardial-myocardial signaling, as we have previously suggested for E10.5 Dll4flox;Nfatc1Cre(D'Amato et al., 2016).

The revised manuscript now says in Discussion: “A common feature among the endocardial or endothelial Notch loss- and gain-of-function models analyzed in our study is the thin ventricular wall (Figure 8B, D). […] This may be clinically relevant to the study and treatment of cardiomyopathies”

Angiocrine factors. Following the reviewer’s suggestion, we also examined the possibility that coronaries might be providing angiocrine factors to sustain myocardial growth and proliferation in absence of coronary perfusion. Tissue-specific instructive functions of ECs have been demonstrated in organ repair without compromising blood supply (Rafii et al., 2016). We have used GSEA (http://software.broadinstitute.org/gsea/index.jsp) to test for the enrichment of a gene set collection of 52 angiocrine factors assembled from Nolan et al. (Nolan et al., 2013) in E12.5 Jag1flox;Nfatc1-Cre and Dll4flox;Nfatc1-Cre mutants, relative to controls (see Author response image 1 and Author response table 1). The gene set was enriched with positive normalized enrichment score (NES) in both analyses, indicating that angiocrine factor expression was increased in both mutant conditions (see GSEA profiles). However, enrichment was statistically significant only for the Jag1flox;Nfatc1-Cre mutant (FDR = 0.04). The leading-edge subset of genes (those contributing mostly to the enrichment score) consisted of 11 genes in both analyses. Seven genes of the 11 genes were shared by both subsets: VEGFA, PDGFB, ANGPT2, EDN1, FGF9, CCL9, BMP6 (see heatmaps), suggesting the increased expression of these angiocrine factors occurs independently of the mechanism of angiogenesis in Jag1flox;Nfatc1-Cre and Dll4flox;Nfatc1-Cre mutants. We are currently refining our bioinformatics analysis by cross-validating our data with sc-RNA-seq made by (Su et al., 2018). We will validate our data by ISH and eventually, functional analysis, that unfortunately, are out of the scope (and time-limitations) of this manuscript.

Author response image 1
GSEA analysis of Jag1flox;Nfatc1Creand Dll4flox;Nfatc1Cre RNA-seq datasets compared against respective controls (Ctrl).

Figure 2. GSEA analysis of Jag1flox;Nfatc1Cre and Dll4flox;Nfatc1Cre expression profiles, compared against their respective WT controls. An angiocrine factor gene set consisting of 52 genes was obtained from a previous publication (see Author response table 1 and Nolan et al. Dev Cell. 2013 Jul 29; 26(2)). Of the 52 angiocrine factors, 38 were detected as expressed in the Dll4 contrast (14,302 genes), and 34 in the Jag1 contrast (13,982 genes), as represented by the Venn diagrams at the upper left corner (panel A). GSEA results indicated that angiocrine factors were enriched among the genes that are more expressed in both Jag1 and Dll4 mutant backgrounds, as indicated by the respective enrichment profiles (panels B and C). In these profiles, red to blue gradient stripes represent the complete collections of expressed genes, sorted by decreasing fold change value in mutant versus WT contrasts: red colours indicate higher expression in mutant background (positive fold change), and blue colours indicate higher expression in WT background (negative fold change). Angiocrine factors, represented by black segments perpendiculary oriented along the gradient stripes, tend to concentrate in positive regions, as indicated by the green enrichment curves. Normalized enrichment score (NES) was therefore positive for both contrasts (1.54 and 0.93, for the Jag1 and Dll4 contrasts, respectively), although enrichment was statistically significant only for the Jag1 analysis (FDR = 0.04). Black rectangles indicate the groups of genes that contribute mostly to calculated NES values, which casually consisted in 11 genes for both analyses. The two leading edge subsets shared 7 genes (VEGFA, PDGFB, ANGPT2, EDN1, FGF9, CCL9 and BMP6), as represented by the Venn diagrams at the lower left corner (panel F). The central scatterplot (panel D) compares the fold change rank of the set of 34 angiocrine factors that were detected in both contrasts, and indicates (with a colour code) which of them were detected as differentially expressed (with p-value < 0.05) in each of the contrasts. Six leading edge subset angiocrine factors were detected as differentially expressed in at least one contrast, and four of them (VEGFA, BMP6, EDN1 and PDGFB) were detected in both. Heatmaps represent relative expression values for 38 angiocrine factors in Dll4 KO samples and their respective WT controls (panel E), and 34 angiocrine factors in Jag1 KO samples and the respective WT controls (panel G). Red and blue colours represent expression values above and below average, respectively. The four genes with asterisks in panel E are those that were detected as expressed only in the DLL4 contrast.

Author response table 1
List of angiocrine gene from Nolan et al. (2013) compared with our RNA-seq data from Jag1flox;Nfatc1-Cre and Dll4flox;Nfatc1-Cre embryos (Figure 2A and Supplementary file 3).
Nolan (52 genes)Dll4 (38 genes)Jag1 (34 genes)Dll4 and Jag1
(34 genes)
Not common to Dll4 and Jag1
(14 genes)
Dll4 specific
(4 genes)
ANGPT2ANGPT2ANGPT2ANGPT2CCL3CCL6
BMP2BMP2BMP2BMP2CXCL2CXCL13
BMP5BMP5BMP5BMP5DKKL1FGF7
BMP6BMP6BMP6BMP6GJB2IL1F9
CCL2CCL2CCL2CCL2IL1A
CCL3CCL6CCL9CCL9IL6
CCL6CCL9COL14A1COL14A1LAMA-4
CCL9COL14A1COL4A1COL4A1LAMB1-1
COL14A1COL4A1CXCL12CXCL12MMP13
COL4A1CXCL12DKK2DKK2MMP27
CXCL12CXCL13EDN1EDN1MMP8
CXCL13DKK2EGFL7EGFL7MMP9
CXCL2EDN1ELNELNTNF
DKK2EGFL7ESM1ESM1WNT8A
DKKL1ELNFGF1FGF1
EDN1ESM1FGF9FGF9
EGFL7FGF1FN1FN1
ELNFGF7GJA1GJA1
ESM1FGF9GJA4GJA4
FGF1FN1GJC1GJC1
FGF7GJA1IGF1IGF1
FGF9GJA4IL33IL33
FN1GJC1JAG1JAG1
GJA1IGF1KITLKITL
GJA4IL1F9LAMB2LAMB2
GJB2IL33LAMC1LAMC1
GJC1JAG1PDGFBPDGFB
IGF1KITLPDGFCPDGFC
IL1ALAMB2PDGFDPDGFD
IL1F9LAMC1TGFB2TGFB2
IL33PDGFBVEGFAVEGFA
IL6PDGFCVEGFCVEGFC
JAG1PDGFDWNT2WNT2
KITLTGFB2WNT5AWNT5A
LAMA-4VEGFA
LAMB1-1VEGFC
LAMB2WNT2
LAMC1WNT5A
MMP13
MMP27
MMP8
MMP9
PDGFB
PDGFC
PDGFD
TGFB2
TNF
VEGFA
VEGFC
WNT2
WNT5A
WNT8A

4) Other more specific markers for smooth muscle should be used. αSMA is used to label smooth muscle cells, however, it is expressed more broadly in mesenchymal cells and cardiomyocytes throughout the embryonic heart prior to ~E16.5 and this is reflected in the images. The authors should attempt to stain for SM22a which, although similarly non-specific, shows better enrichment in VSMCs, relative to the other cell types. Improved visualization may provide further insight into the nature of the defect e.g. are epicardium-derived precursors trapped or poorly differentiated?

In our study we used double staining of pericytes and smooth muscle using Notch3 and αSMA respectively. This method was used previously by (Volz et al., 2015), to demonstrate that pericytes are precursors of smooth muscle cells in coronaries in E14.5-E16.5 hearts. We cite this paper (Volz et al., 2015). Moreover, we avoided αSMA-immunostained mesenchyme and cardiomyocytes by counting αSMA/Notch3 double-positive cells surrounding coronaries, thus we are sure that the cell type that we label with αSMA is vascular smooth muscle. Nevertheless, to address the reviewer´s concern we have performed Notch3/SM22a double immunostaining on Pdgfb-iCreERT2/+;Jag1flox/flox (and Dll4flox/flox) heart sections in triplicate. After quantifying the percentage of SM22a/Notch3+-positive staining in Notch3-positive cells, we obtained very similar results, namely that smooth muscle differentiation is significantly impaired in Pdgfb-iCreERT2/+;Jag1flox/flox(and Dll4flox/flox) mutants (new Figure 3—figure supplement 2A). These data indicate that Jag1 and Dll4 are required for coronary vascular smooth muscle cell differentiation.

The revised text says (Results): “Although αSMA is a commonly used marker of vascular smooth muscle cells, it is expressed more broadly in mesenchymal cells and cardiomyocytes throughout the embryonic heart prior to E16.5. […] After co-staining with Notch3, we found that the proportion of SM22a and Notch3-positive cells in the Jag1flox;Pdgfb-iCreERT2mutants was significantly reduced (Supplementary file 1, sheet 7), confirming that pericytes fail to properly differentiate into smooth muscle.”

“Furthermore, Dll4flox;Pdgfb-iCreERT2mutants had deficient perivascular cell coverage as indicated by decreased αSMA- and Notch3-positive cells (Supplementary file 1, sheet 6). This was confirmed by co-immunostaining with SM22a and Notch3 (Figure 3—figure supplement 7C, D) demonstrating near complete absence of smooth muscle differentiation in Dll4flox;Pdgfb-iCreERT2mutants (Supplementary file 1, sheet 7)”.

Reference:

Nolan DJ, Ginsberg M, Israely E, Palikuqi B, Poulos MG, James D, Ding BS, Schachterle W, Liu Y, Rosenwaks Z et al. 2013. Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration. Dev Cell26: 204-219.

https://doi.org/10.7554/eLife.49977.sa2

Article and author information

Author details

  1. Stanislao Igor Travisano

    1. Intercellular Signalling in Cardiovascular Development and Disease Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
    2. CIBER de Enfermedades Cardiovasculares, Madrid, Spain
    Contribution
    Investigation, Visualization
    Competing interests
    No competing interests declared
  2. Vera Lucia Oliveira

    1. Intercellular Signalling in Cardiovascular Development and Disease Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
    2. CIBER de Enfermedades Cardiovasculares, Madrid, Spain
    Contribution
    Investigation, Visualization, Methodology
    Competing interests
    No competing interests declared
  3. Belén Prados

    1. Intercellular Signalling in Cardiovascular Development and Disease Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
    2. CIBER de Enfermedades Cardiovasculares, Madrid, Spain
    Contribution
    Validation, Investigation
    Competing interests
    No competing interests declared
  4. Joaquim Grego-Bessa

    1. Intercellular Signalling in Cardiovascular Development and Disease Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
    2. CIBER de Enfermedades Cardiovasculares, Madrid, Spain
    Contribution
    Validation, Investigation, Visualization
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0938-2346
  5. Rebeca Piñeiro-Sabarís

    1. Intercellular Signalling in Cardiovascular Development and Disease Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
    2. CIBER de Enfermedades Cardiovasculares, Madrid, Spain
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  6. Vanesa Bou

    1. Intercellular Signalling in Cardiovascular Development and Disease Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
    2. CIBER de Enfermedades Cardiovasculares, Madrid, Spain
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  7. Manuel J Gómez

    Bioinformatics Unit, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain
    Contribution
    Software, Validation, Investigation
    Competing interests
    No competing interests declared
  8. Fátima Sánchez-Cabo

    Bioinformatics Unit, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain
    Contribution
    Software, Supervision, Validation, Investigation
    Competing interests
    No competing interests declared
  9. Donal MacGrogan

    1. Intercellular Signalling in Cardiovascular Development and Disease Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
    2. CIBER de Enfermedades Cardiovasculares, Madrid, Spain
    Contribution
    Data curation, Formal analysis, Supervision, Investigation
    For correspondence
    dmacgrogan@cnic.es
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2808-8422
  10. José Luis de la Pompa

    1. Intercellular Signalling in Cardiovascular Development and Disease Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
    2. CIBER de Enfermedades Cardiovasculares, Madrid, Spain
    Contribution
    Resources, Supervision, Funding acquisition, Visualization, Project administration
    For correspondence
    jlpompa@cnic.es
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6761-7265

Funding

Spanish Ministry of Science, Innovation and Universities (SAF2016-78370-R)

  • José Luis de la Pompa

Spanish Ministry of Science, Innovation and Universities (CB16/11/00399)

  • José Luis de la Pompa

Spanish Ministry of Science, Innovation and Universities (RD16/0011/0021)

  • José Luis de la Pompa

Fundación BBVA (BIO14_298)

  • José Luis de la Pompa

Fundació la Marató de TV3 (20153431)

  • José Luis de la Pompa

Comunidad de Madrid (Atracción de Talento Program)

  • Joaquim Grego-Bessa

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank the CNIC Genomics Unit for RNA-seq experiments and S Bartlett (CNIC) for English editing. This study was supported by grants SAF2016-78370-R, CB16/11/00399 (CIBER CV), and RD16/0011/0021 (TERCEL) from the Spanish Ministry of Science, Innovation and Universities (MCIU) and grants from the Fundación BBVA (Ref.: BIO14_298) and Fundación La Marató (Ref.: 20153431) to JLDLP. JG-B is funded by Atracción de Talento Program from the Comunidad de Madrid. The cost of this publication was supported in part with funds from the ERDF. The CNIC is supported by the MCIU and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015–0505).

Ethics

Animal experimentation: Animal studies were approved by the CNIC Animal Experimentation Ethics Committee and by the Madrid regional government (Ref. PROEX 118/15). All animal procedures conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 1201/2005.

Senior Editor

  1. Didier Y Stainier, Max Planck Institute for Heart and Lung Research, Germany

Reviewing Editor

  1. Bin Zhou, Chinese Academy of Sciences, China

Reviewers

  1. Bin Zhou, Chinese Academy of Sciences, China
  2. Nicola Smart

Publication history

  1. Received: July 5, 2019
  2. Accepted: December 1, 2019
  3. Accepted Manuscript published: December 2, 2019 (version 1)
  4. Accepted Manuscript updated: December 3, 2019 (version 2)
  5. Accepted Manuscript updated: December 4, 2019 (version 3)
  6. Version of Record published: December 17, 2019 (version 4)

Copyright

© 2019, Travisano et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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