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Sequential Notch activation regulates ventricular chamber development

Nature Cell Biology volume 18, pages 7–20 (2016)Cite this article

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Abstract

Ventricular chambers are essential for the rhythmic contraction and relaxation occurring in every heartbeat throughout life. Congenital abnormalities in ventricular chamber formation cause severe human heart defects. How the early trabecular meshwork of myocardial fibres forms and subsequently develops into mature chambers is poorly understood. We show that Notch signalling first connects chamber endocardium and myocardium to sustain trabeculation, and later coordinates ventricular patterning and compaction with coronary vessel development to generate the mature chamber, through a temporal sequence of ligand signalling determined by the glycosyltransferase manic fringe (MFng). Early endocardial expression of MFng promotes Dll4–Notch1 signalling, which induces trabeculation in the developing ventricle. Ventricular maturation and compaction require MFng and Dll4 downregulation in the endocardium, which allows myocardial Jag1 and Jag2 signalling to Notch1 in this tissue. Perturbation of this signalling equilibrium severely disrupts heart chamber formation. Our results open a new research avenue into the pathogenesis of cardiomyopathies.

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Figure 1: Dll4–Notch1 signalling abrogation disrupts trabeculation and chamber-gene expression.
Figure 2: Myocardial Jag1 inactivation disrupts chamber maturation and leads to cardiomyopathy and systolic dysfunction.
Figure 3: Jag1 gene profiling and validation.
Figure 4: Myocardial Jag2, together with Jag1, is required for ventricular maturation and compaction.
Figure 5: MFng modulates Notch selectivity towards its ligands and systemic Fng abrogation disrupts coronary vessel development.
Figure 6: Forced MFng expression in the endocardium disrupts chamber development.
Figure 7: Comparative expression profiling of Jag2flox;cTnT-Cre, Jag1flox;cTnT-Cre, Jag1flox;Jag2flox;cTnT-Cre, MFngGOF;Tie2-Cre (MFngtg;Tie2-Cre) and Mib1flox;cTnT-Cre mutants.
Figure 8: Sequential Notch ligand–receptor activation during ventricular chamber development.

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Acknowledgements

We thank Y. Fukushima (Osaka U., Japan) for help with the generation of the R26-MFng targeting vector, RIKEN CDB (Japan) for producing the chimaeric mice, the CNIC Genomics Unit for RNA-seq, the CNIC Advance Imaging Unit for CMRI analysis, B. Zhou (Albert Einstein College, NYC, USA) for the NFATc1-Cre driver line, A. Martín-Pendas (CSIC, Salamanca, Spain), P. Muñoz-Canoves (UPF, Barcelona, Spain) and J. M. Pérez-Pomares (Málaga U., Spain) for critical reading of the manuscript and S. Bartlett (CNIC) and K. McCreath for English editing. Funds were from grants SAF2013-45543-R, RD12/0042/0005 (RIC) and RD12/0019/0003 (TERCEL) from the Spanish Ministry of Economy and Competitiveness (MINECO), FP7-ITN 215761 (NotchIT) and 28600 (CardioNeT) from the EU and a grant from the BBVA Foundation for Research in Biomedicine (2014), all to J.L.d.l.P. G.D’A. holds a PhD fellowship linked to grant FP7-ITN 215761 (NotchIT). The MINECO and the Pro-CNIC Foundation support the CNIC.

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Authors and Affiliations

  1. Intercellular Signalling in Cardiovascular Development & Disease Laboratory, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029 Madrid, Spain

    Gaetano D’Amato, Guillermo Luxán, Gonzalo del Monte-Nieto, Beatriz Martínez-Poveda & José Luis de la Pompa

  2. Bioinformatics Unit, CNIC, Melchor Fernández Almagro 3, 28029 Madrid, Spain

    Carlos Torroja, Wencke Walter & Fernando Martinez

  3. Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210, USA

    Matthew S. Bochter & Susan Cole

  4. Molecular Genetics of Angiogenesis Laboratory, CNIC, Melchor Fernández Almagro 3, 28029 Madrid, Spain

    Rui Benedito

  5. Developmental Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA

    Anna-Katerina Hadjantonakis

  6. Department of Retinal Vascular Biology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan

    Akiyoshi Uemura

  7. Cardiovascular Imaging Laboratory, CNIC, Melchor Fernández Almagro 3, 28029 Madrid, Spain

    Luis J. Jiménez-Borreguero

  8. Instituto de Investigación Sanitaria Hospital La Princesa, 28006 Madrid, Spain

    Luis J. Jiménez-Borreguero

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  1. Gaetano D’Amato
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Contributions

G.D’A., G.L., G.d.M.-N., B.M.-P. and M.S.B. performed experiments. C.T., W.W. and F.M. analysed the RNA-seq data, A.-K.H., A.U. and S.C. provided the CBF:H2B-Venus reporter, the MFng transgenic line and the M−/−;L−/−;R−/− embryos. R.B. advised on the Fng experiments and L.J.J.-B. evaluated ultrasonography and CMRI. J.L.d.l.P. designed experiments, reviewed the data and wrote the manuscript. All authors reviewed the manuscript.

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Correspondence to José Luis de la Pompa.

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Integrated supplementary information

Supplementary Figure 1

CBF:H2B-Venus activity in chamber endocardium (a) and Dll4 and Jag1 activate Notch during chamber development (b–h ′). (a) Two-photon whole-mount images of the left ventricle of E9.5 CBF:H2B-Venus, CBF:H2B-Venus;Notch1KO and CBF:H2B-Venus;RbpjKO embryos. Arrowheads indicate endocardial Venus expression in WT mice and abrogated expression in Notch1KO and RbpjKO embryos. (b) Images showing rear views of Amira 3D reconstructions of Dll4, Jag1 and N1ICD expression in the E9.5 WT heart. Atrioventricular canal region (avc) mesenchyme (yellow) does not express N1ICD. lv, left ventricle; rv right ventricle. (c,c ′) Dll4 (green), SMA (red), and Venus (grey) immunostaining in E12.5 WT CBF:H2B-Venus ventricles. Venus+ cells (c ′, arrowheads) distribute throughout the endocardium, similarly to Dll4 (arrows). (d,d ′) Jag1 (green), SMA (red), and Venus (grey) immunostaining in E12.5 WT CBF:H2B-Venus ventricles. In c–d ′ nuclei are counterstained with DAPI. Scale bar, 50 μm. (e–h ′) E15.5 WT ventricles. (e,e ′) ISH. Dll4 is transcribed in coronary vessel endothelium (e ′, white arrowheads) and some endocardial cells (black arrowhead). (f–h ′) Immunostainings. (f,f ′) Jag1 is expressed in trabecular myocardium (f ′, arrow) and coronary vessels (f ′, arrowhead). (g,g ′) N1ICD is expressed in trabecular endocardium (g ′, white arrowheads) and coronary vessels endothelium (g ′, red arrowhead). (h,h ′) Jag1 (green) and Venus (grey) expression in a CBF:H2B-Venus reporter embryo. Endocardial cells (yellow arrowheads) and coronary vessel endothelium (red arrowheads) are Venus+. Scale bar, 100 μm. Source data available in Supplementary Table 4.

Supplementary Figure 2

Dll4 abrogation disrupts Notch activity and trabecular marker expression (a–c) and Gpr126 expression responds to Notch activation (d-h). (a) Top, N1ICD immunostaining in E9.5 WT and Dll4flox;Nfat-Cre hearts. Middle, N1ICD expression in aortic endothelial cells. Bottom, N1ICD staining in E9.5 WT and Dll4flox;Tie2-Cre hearts. (b) Ratios of N1ICD-positive to total endocardial nuclei. Data are mean ± S.D. (n = 3 WT embryos, n = 3 Dll4flox;Nfat-Cre embryos and n = 3 Dll4flox;Tie2-Cre embryos, ∗∗P < 0.01, ∗∗∗P < 0.001, by Student’s t test). (c) Hey2, EfnB2 Nrg1 and Bmp10 ISH in E9.5 WT and Dll4flox;Tie2-Cre embryos. Scale bar, 50 μm. (d–f ′) Reduced gpr126 expression in zebrafish larvae with impaired Notch signalling. Panels show lateral and ventral views. Lateral and ventral views of the ISH of gpr126 in WT (d,d ′ 27 out of 29; 93%), mib1ta52b mutant (e,e ′, 35 out of 38; 92%) and RO-treated WT (f,f ′, 19 out of 22; 86%), 48-h.p.f. zebrafish embryos showing gpr126 transcripts in the heart tube (arrowhead) and ear region (arrow) in d, and reduced expression in e,f. Scale bar, 50 μm. (g) qRT-PCR analysis showing the effect of Dll4- or Dll4 + RO-stimulation on the transcription of Gpr126, Dll4 and the Notch targets Hey1, Hey2, HeyL and Nrarp in HUVEC. Data are mean ± S.D. (n = 4 independent biological replicates for each condition; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 by Student’s t test). (h) Gpr126 reporter activity measured by luciferase assay in BAEC. Data are mean ± S.D. (n = 4 independent biological replicates for each condition; ∗∗P < 0.01, ∗∗∗P < 0.001 by Student’s t test). Source data available in Supplementary Table 4.

Supplementary Figure 3 Dll4-Notch1 activity is required for coronary vessel formation.

(a, c) Images of whole-mount E15.5 WT and Dll4flo/floxx;Cdh5-Cre/ + (Dll4flox;Cdh5-Cre) mutant embryos. Note the dorsal oedema (arrowhead) in the mutant embryos. Scale bar, 2mm. Dotted lines indicate the plane of the H&E stained sections shown in (b,d). The heart in the Dll4flox;Cdh5-Cre mutant has thinner ventricular walls (d, yellow bars) than its WT littermate (b). (e–f ′) ISH in E15.5 hearts. Dll4 is transcribed in coronary vessel endothelium (e ′, black arrowhead) and weakly expressed in endocardial cells (e ′, white arrowhead). Expression is severely impaired in the mutant heart (f,f ′). (g–h ′) N1ICD immunostaining. N1ICD staining is strong throughout the endocardium and in coronary vessels of WT ventricle (g ′, arrowhead), and weak in coronary vessels of mutant embryos (h ′, arrowhead). (i–p ′) ISH analysis. (i–j ′) Hey2 expression is similar in the compact myocardium of WT embryos (i,i ′) and mutants (j,j ′). Note the slightly thinner compact myocardium in the Dll4flox;Cdh5-Cre mutant heart (j ′, bracket). (k–p ′) Expression of the coronary artery endothelial cell markers HeyL (k–l ′), EfnB2 (m–n ′) and Cx40 (o–p ′) is markedly lower in mutant embryos. The arrowheads mark expression in coronary vessels which appear smaller in Dll4flox;Cdh5-Cre mutants. Scale bar in (b,d,e–p ′) = 100 μm.

Supplementary Figure 4

Summary of the comparative expression profiling of Dll4flox;Tie2-Cre, Dll4flox;Nfat-Cre, and Notch1flox;Nfat-Cre mutants (a,b) and attenuated gpr126 expression after Notch abrogation in zebrafish (c–e ′). (a) Log fold change (logFC) hierarchical clustering of the 858 genes differentially expressed at E9.5 in the hearts of at least two genotypes among E9.5 Dll4flox;Tie2-Cre, Dll4flox;Nfat-Cre and Notch1flox;Nfat-Cre mutants. The colour scheme represents the logFC in any of the genotypes compared with its control, with upregulated genes indicated in yellow, downregulated in blue, and black for no significant change. (b) Subsets of genes clustered into functional categories (right panels). The total number of deregulated genes for each genotype and the genes represented in the heat map can be found in Supplementary Table 1.

Supplementary Figure 5

Myocardial Jag1 is dispensable for Notch signalling activation during trabeculation (a–c) but is required for Notch activation during chamber maturation and compaction and ventricular maturation (d–f ′). (a) E10.5 WT and Jag1flox/flox;cTnT-Cre/ + (Jag1flox;cTnT-Cre) mutant hearts. (a) H&E staining. Trabeculae and compact myocardium thickness are similar in WT and mutant hearts. Jag1 (red) is expressed in the trabeculae of the WT ventricle (arrows) but is absent from the mutant ventricle. N1ICD immunostaining (red, arrowheads) in the endomucin-delineated endocardium (green) of WT and Jag1flox;cTnT-Cre mutant embryos. (b) Quantification of N1ICD-positive nuclei as the mean percentage of total nuclei in WT and Jag1flox;cTnT-Cre mutant embryos. Data are mean ± S.D. (n = 3 WT and n = 3 mutant embryos, n.s., not significant, by Student’s t test). (c) ISH. Hey2, EfnB2, Nrg1 and Bmp10 are normally expressed in E10.5 mutant hearts. (d) E13.5. Jag1 and N1ICD immunostainings in WT (d, left) and Jag1flox;cTnT-Cre mutant heart sections (d, right). Jag1 is strongly expressed in trabecular myocardium (arrowhead) and weakly in compact myocardium (arrow). N1ICD is expressed throughout the endocardium (arrowheads). Myocardial deletion of Jag1 attenuates Notch1 activity. (e–f ′) Anf ISH. E16.5 WT heart showing normal expression in trabeculae and septum (e,e ′, arrowheads) and reduced expression in Jag1flox;cTnT-Cre hearts (f,f ′, arrowheads). Abbreviations as in previous figures. Scale bar, 100 μm. Source data available in Supplementary Table 4.

Supplementary Figure 6

Quantification of regional compact myocardium thickness and trabecular area in E16.5 Jag1flox;Jag2flox;cTnT-Cre M-Fngtg;Tie2-Cre and Mib1flox;cTnT-Cre mutants (a), compact myocardium thickness and complexity of trabecular myocardium in E16.5 Mib1flox;cTnT-Cre mutants (a) and cellular proliferation in E13.5 Mib1flox;cTnT-Cre mice (b,c–d ′). (a) For quantification of the morphological parameters the following number of samples were analysed per genotype: Jag1flox;Jag2flox;cTnT-Cre (n = 3 WT and n = 3 mutant embryos), M-Fngtg;Tie2-Cre (n = 3 WT and n = 4 mutant embryos) and Mib1flox;cTnT-Cre (n = 3 WT and n = 3 mutant embryos). (b,c–d ′) Cellular proliferation measured by BrdU immunostaining. BrdU (green), SMA (red) and DAPI (blue) staining of E13.5 WT (c,c ′) and Mib1flox;cTnT-Cre heart (d,d ′). The arrows point to BrdU-positive endocardial nuclei and the arrowheads to BrdU-positive cardiomyocytes. Scale bar, 100 μm. Data are means ± S.D. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, by Student’s t test. n.s., not significant). Source data available in Supplementary Table 4.

Supplementary Figure 7 Notch signalling abrogation disrupts compaction.

E16.5 heart sections stained with endomucin (green) and cTnT antibodies (red) to delineate chamber endocardium and myocardium. The WT heart (a–a ′′) has a thick, cTnT-positive compact myocardium in both ventricles, with compacting trabeculae covered by endomucin-positive endocardium. The Jag1flox;Jag2flox;cTnT-Cre (b–b ′′), M-Fngtg;Tie2-Cre (c–c ′′) and Mib1flox;cTnT-Cre hearts (d–d ′) show very thin compact myocardium, uncompacted trabeculae and disrupted ventricular septum. The white bars in (a ′ d ′′) indicate compact myocardium thickness. The red and yellow brackets in (a) indicate the basal and apical regions measured to determine the compact myocardium thickness shown in Fig. 2,4,5 and 6. The yellow bar indicates the thickness of compact myocardium. Scale bar, 100 μm.

Supplementary Figure 8

Analysis of Lunatic Fringe and Radical Fringe expression in the embryonic heart (a,b) and characterization of gene expression in GFP-MEVEC and MFng-MEVEC (c). (a) ISH analysis of LFng and RFng in WT hearts. LFng expression is detected in proepicardial cells of E9.5 embryos (arrowhead) and is restricted to the epicardium of E10.5 and E11.5 hearts (arrowheads). RFng is not detected in the heart at these stages. (b) Relative mRNA expression of LFng and R-Fng in E.8.5E11.5 ventricles. Data are means ± S.D. (n = 3 pools of 5 WT hearts at E8.5, and n = 3 pools of 3 ventricles per pool at E9.5-E11.5 (c) MEVEC. qRT-PCR analysis of Notch pathway genes and endocardial markers, indicating similar expression levels of these genes in GFP-MEVEC and MFng-MEVEC. Note the very high MFng transcript expression after lentiviral transduction in MEVEC. Data are means ± S.D. (n = 2 independent biological replicates for each condition). Scale bar, 100 μm. Source data available in Supplementary Table 4.

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Imaris 3D reconstruction of whole-mount stained E9.5 CBF1:2HB-Venus heart.

The myocardial surface in red was built from the SMA staining. Venus (white nuclei), revealing Notch activity in CD31/Pecam1-positive cells (green), can be observed in the ventricular endocardium. Nuclei are counterstained with DAPI. (MOV 6211 kb)

Representative Z-stack of CMRI short axis views of hearts from 6-month-old WT and Jag1flox;cTnT-Cre mice.

The mutant heart exhibits dilated ventricles and a thinner septum than the WT heart. (MOV 21291 kb)

Representative M-mode echocardiography analysis of the hearts of 6-month-old WT and Jag1flox;cTnT-Cre mice.

The mutant heart shows contraction defects. (MOV 13239 kb)

Representative Z-stack of CMRI short axis views of hearts from 6-month-old WT and Jag1flox;cTnT-Cre mice.

The mutant heart exhibits a remarked segmental dyskinesia in the RV free wall. (MOV 8548 kb)

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D’Amato, G., Luxán, G., del Monte-Nieto, G. et al. Sequential Notch activation regulates ventricular chamber development. Nat Cell Biol 18, 7–20 (2016). https://doi.org/10.1038/ncb3280

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