Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Tension heterogeneity directs form and fate to pattern the myocardial wall

Abstract

How diverse cell fates and complex forms emerge and feed back to each other to sculpt functional organs remains unclear. In the developing heart, the myocardium transitions from a simple epithelium to an intricate tissue that consists of distinct layers: the outer compact and inner trabecular layers. Defects in this process, which is known as cardiac trabeculation, cause cardiomyopathies and embryonic lethality, yet how tissue symmetry is broken to specify trabecular cardiomyocytes is unknown. Here we show that local tension heterogeneity drives organ-scale patterning and cell-fate decisions during cardiac trabeculation in zebrafish. Proliferation-induced cellular crowding at the tissue scale triggers tension heterogeneity among cardiomyocytes of the compact layer and drives those with higher contractility to delaminate and seed the trabecular layer. Experimentally, increasing crowding within the compact layer cardiomyocytes augments delamination, whereas decreasing it abrogates delamination. Using genetic mosaics in trabeculation-deficient zebrafish models—that is, in the absence of critical upstream signals such as Nrg–Erbb2 or blood flow—we find that inducing actomyosin contractility rescues cardiomyocyte delamination and is sufficient to drive cardiomyocyte fate specification, as assessed by Notch reporter expression in compact layer cardiomyocytes. Furthermore, Notch signalling perturbs the actomyosin machinery in cardiomyocytes to restrict excessive delamination, thereby preserving the architecture of the myocardial wall. Thus, tissue-scale forces converge on local cellular mechanics to generate complex forms and modulate cell-fate choices, and these multiscale regulatory interactions ensure robust self-organized organ patterning.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Tension heterogeneity among cardiomyocytes at the onset of delamination.
Fig. 2: Crowding-induced tension heterogeneity triggers cardiomyocyte delamination.
Fig. 3: Differential contractility is sufficient to induce cardiomyocyte delamination in the absence of Nrg–Erbb2 signalling.
Fig. 4: Feedback interactions between mechanics and cell-fate cascades pattern the myocardial wall.

Similar content being viewed by others

Data availability

All of the data supporting Figs. 14 and Extended Data Figs. 110 are available within the manuscript and its Supplementary InformationSource data are provided with this paper.

Code availability

Custom script used for beating heart imaging is provided in the Supplementary Methods.

References

  1. Staudt, D. & Stainier, D. Uncovering the molecular and cellular mechanisms of heart development using the zebrafish. Annu. Rev. Genet. 46, 397–418 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Liu, J. et al. A dual role for ErbB2 signaling in cardiac trabeculation. Development 137, 3867–3875 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Staudt, D. W. et al. High-resolution imaging of cardiomyocyte behavior reveals two distinct steps in ventricular trabeculation. Development 141, 585–593 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Maître, J. L. et al. Adhesion functions in cell sorting by mechanically coupling the cortices of adhering cells. Science 338, 253–256 (2012).

    Article  ADS  PubMed  CAS  Google Scholar 

  5. Maître, J. L. et al. Asymmetric division of contractile domains couples cell positioning and fate specification. Nature 536, 344–348 (2016).

    ADS  PubMed  PubMed Central  Google Scholar 

  6. Miroshnikova, Y. A. et al. Adhesion forces and cortical tension couple cell proliferation and differentiation to drive epidermal stratification. Nat. Cell Biol. 20, 69–80 (2018).

    Article  CAS  PubMed  Google Scholar 

  7. Samarage, C. R. et al. Cortical tension allocates the first inner cells of the mammalian embryo. Dev. Cell 34, 435–447 (2015).

    Article  CAS  PubMed  Google Scholar 

  8. Cherian, A. V., Fukuda, R., Augustine, S. M., Maischein, H. M. & Stainier, D. Y. N-cadherin relocalization during cardiac trabeculation. Proc. Natl Acad. Sci. USA 113, 7569–7574 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Jiménez-Amilburu, V. & Stainier, D. Y. R. The transmembrane protein Crb2a regulates cardiomyocyte apicobasal polarity and adhesion in zebrafish. Development 146, dev171207 (2019).

    Article  PubMed  CAS  Google Scholar 

  10. Tinevez, J. Y. et al. Role of cortical tension in bleb growth. Proc. Natl Acad. Sci. USA 106, 18581–18586 (2009).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yonemura, S., Wada, Y., Watanabe, T., Nagafuchi, A. & Shibata, M. α-Catenin as a tension transducer that induces adherens junction development. Nat. Cell Biol. 12, 533–542 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Eisenhoffer, G. T. et al. Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia. Nature 484, 546–549 (2012).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  13. Eisenhoffer, G. T. & Rosenblatt, J. Bringing balance by force: live cell extrusion controls epithelial cell numbers. Trends Cell Biol. 23, 185–192 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Saw, T. B. et al. Topological defects in epithelia govern cell death and extrusion. Nature 544, 212–216 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. Marinari, E. et al. Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding. Nature 484, 542–545 (2012).

    Article  ADS  CAS  PubMed  Google Scholar 

  16. Levayer, R., Dupont, C. & Moreno, E. Tissue crowding induces caspase-dependent competition for space. Curr. Biol. 26, 670–677 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Han, Y. et al. Vitamin D stimulates cardiomyocyte proliferation and controls organ size and regeneration in zebrafish. Dev. Cell 48, 853–863 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. D’Uva, G. et al. ERBB2 triggers mammalian heart regeneration by promoting cardiomyocyte dedifferentiation and proliferation. Nat. Cell Biol. 17, 627–638 (2015).

    Article  PubMed  CAS  Google Scholar 

  19. Uribe, V. et al. In vivo analysis of cardiomyocyte proliferation during trabeculation. Development 145, dev164194 (2018).

    Article  PubMed  CAS  Google Scholar 

  20. Chugh, P. et al. Actin cortex architecture regulates cell surface tension. Nat. Cell Biol. 19, 689–697 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Latorre, E. et al. Active superelasticity in three-dimensional epithelia of controlled shape. Nature 563, 203–208 (2018).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sehnert, A. J. et al. Cardiac troponin T is essential in sarcomere assembly and cardiac contractility. Nat. Genet. 31, 106–110 (2002).

    Article  CAS  PubMed  Google Scholar 

  23. Rasouli, S. J. & Stainier, D. Y. R. Regulation of cardiomyocyte behavior in zebrafish trabeculation by Neuregulin 2a signaling. Nat. Commun. 8, 15281 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  24. Jiménez-Amilburu, V. et al. In vivo visualization of cardiomyocyte apicobasal polarity reveals epithelial to mesenchymal-like transition during cardiac trabeculation. Cell Rep. 17, 2687–2699 (2016).

    Article  PubMed  CAS  Google Scholar 

  25. Peshkovsky, C., Totong, R. & Yelon, D. Dependence of cardiac trabeculation on neuregulin signaling and blood flow in zebrafish. Dev. Dyn. 240, 446–456 (2011).

    Article  PubMed  Google Scholar 

  26. Westcot, S. E. et al. Protein-trap insertional mutagenesis uncovers new genes involved in zebrafish skin development, including a Neuregulin 2a-based ErbB signaling pathway required during median fin fold morphogenesis. PLoS One 10, e0130688 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Auman, H. J. et al. Functional modulation of cardiac form through regionally confined cell shape changes. PLoS Biol. 5, e53 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Han, P. et al. Coordinating cardiomyocyte interactions to direct ventricular chamber morphogenesis. Nature 534, 700–704 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ninov, N., Borius, M. & Stainier, D. Y. Different levels of Notch signaling regulate quiescence, renewal and differentiation in pancreatic endocrine progenitors. Development 139, 1557–1567 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Shaya, O. et al. Cell–cell contact area affects Notch signaling and Notch-dependent patterning. Dev. Cell 40, 505–511 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bray, S. J. Notch signalling in context. Nat. Rev. Mol. Cell Biol. 17, 722–735 (2016).

    Article  CAS  PubMed  Google Scholar 

  32. Shaya, O. & Sprinzak, D. From Notch signaling to fine-grained patterning: modeling meets experiments. Curr. Opin. Genet. Dev. 21, 732–739 (2011).

    Article  CAS  PubMed  Google Scholar 

  33. del Monte-Nieto, G. et al. Control of cardiac jelly dynamics by NOTCH1 and NRG1 defines the building plan for trabeculation. Nature 557, 439–445 (2018).

    Article  ADS  PubMed  CAS  Google Scholar 

  34. Beach, J. R., Licate, L. S., Crish, J. F. & Egelhoff, T. T. Analysis of the role of Ser1/Ser2/Thr9 phosphorylation on myosin II assembly and function in live cells. BMC Cell Biol. 12, 52 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Bindels, D. S. et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat. Methods 14, 53–56 (2017).

    Article  CAS  PubMed  Google Scholar 

  36. Subauste, M. C. et al. Rho family proteins modulate rapid apoptosis induced by cytotoxic T lymphocytes and Fas. J. Biol. Chem. 275, 9725–9733 (2000).

    Article  CAS  PubMed  Google Scholar 

  37. Clark, B. S. et al. Loss of Llgl1 in retinal neuroepithelia reveals links between apical domain size, Notch activity and neurogenesis. Development 139, 1599–1610 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Lin, Y. F., Swinburne, I. & Yelon, D. Multiple influences of blood flow on cardiomyocyte hypertrophy in the embryonic zebrafish heart. Dev. Biol. 362, 242–253 (2012).

    Article  CAS  PubMed  Google Scholar 

  39. Mickoleit, M. et al. High-resolution reconstruction of the beating zebrafish heart. Nat. Methods 11, 919–922 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. D’Amico, L., Scott, I. C., Jungblut, B. & Stainier, D. Y. A mutation in zebrafish hmgcr1b reveals a role for isoprenoids in vertebrate heart-tube formation. Curr. Biol. 17, 252–259 (2007).

    Article  PubMed  CAS  Google Scholar 

  41. Reischauer, S., Arnaout, R., Ramadass, R. & Stainier, D. Y. R. Actin binding GFP allows 4D in vivo imaging of myofilament dynamics in the zebrafish heart and the identification of Erbb2 signaling as a remodeling factor of myofibril architecture. Circ. Res. 115, 845–856 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Fukuda, R. et al. Proteolysis regulates cardiomyocyte maturation and tissue integration. Nat.Commun. 8, 14495 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  43. Guerra, A. et al. Distinct myocardial lineages break atrial symmetry during cardiogenesis in zebrafish. eLife 7, e32833 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Revenu, C. et al. Quantitative cell polarity imaging defines leader-to-follower transitions during collective migration and the key role of microtubule-dependent adherens junction formation. Development 141, 1282–1291 (2014).

    Article  CAS  PubMed  Google Scholar 

  45. Lyons, D. A. et al. erbb3 and erbb2 are essential for Schwann cell migration and myelination in zebrafish. Curr. Biol. 15, 513–524 (2005).

    Article  CAS  PubMed  Google Scholar 

  46. Asakawa, K. & Kawakami, K. The Tol2-mediated Gal4-UAS method for gene and enhancer trapping in zebrafish. Methods 49, 275–281 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Berdougo, E., Coleman, H., Lee, D. H., Stainier, D. Y. & Yelon, D. Mutation of weak atrium/atrial myosin heavy chain disrupts atrial function and influences ventricular morphogenesis in zebrafish. Development 130, 6121–6129 (2003).

    Article  CAS  PubMed  Google Scholar 

  48. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    CAS  PubMed  Google Scholar 

  49. Bolte, S. & Cordelières, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J Microsc. 224, 213–232 (2006).

    Article  MathSciNet  CAS  PubMed  Google Scholar 

  50. Bornhorst, D. et al. Biomechanical signaling within the developing zebrafish heart attunes endocardial growth to myocardial chamber dimensions. Nat. Commun. 10, 4113 (2019).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  51. Priya, R. & Gomez, G. A. Measurement of junctional protein dynamics using fluorescence recovery after photobleaching (FRAP). Bio Protoc. 3, e937 (2013).

    Article  Google Scholar 

  52. Liang, X., Michael, M. & Gomez, G. A. Measurement of mechanical tension at cell–cell junctions using two-photon laser ablation. Bio Protoc. 6, e2068 (2016).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to A. S. Yap, C. J. Chan, P. Panza, C. C. Wu, F. Gunawan, M. Collins, G. Boezio, A. Munjal, T. Tsai and E. H. Barriga for discussions and critical reading of the manuscript. We thank our laboratory colleagues for their support, S. Howard for technical assistance with injections, R. Ramadass for imaging support and our colleagues elsewhere for their gifts of reagents. R.P. acknowledges support by postdoctoral fellowships from EMBO (LTF 1569) and the Alexander von Humboldt Foundation, as well as a start-up grant from the Cardio-Pulmonary Institute (CPI) (EXC 2026, project ID 390649896). S.A. is supported by the DFG-CRC1213 project B01. Research in the Stainier laboratory is supported by the Max Planck Society, EU (ERC), DFG and Leducq Foundation.

Author information

Authors and Affiliations

Authors

Contributions

R.P. conceived the project, designed and performed most of the experiments, analysed the data and wrote the manuscript with input from all authors. S.A. performed adult heart immunostaining and contributed to time-lapse imaging of beating hearts. A.G. performed some immunostaining and chemical treatment experiments. S.M. performed some immunostaining and chemical treatment experiments and contributed to data analysis. V.U. performed some mosaic experiments. H.-M.M. performed blastomere transplant experiments. D.Y.R.S. supervised the study and wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Rashmi Priya or Didier Y. R. Stainier.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Jeroen Bakkers, Carl-Philipp Heisenberg and Sara Wickström for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Cardiomyocytes delaminate in a stochastic fashion.

a, Representative frames from time-lapse beating heart imaging to visualize cardiomyocyte (CM) delamination starting at 60 hpf (n = 8/8 hearts). bc’, Representative mid-sagittal confocal images of 65 hpf (b), and 80 hpf (c) hearts, and distribution of delaminating (de) or TL cardiomyocytes along the compact layer at 65 hpf (b’, n = 31) and 80 hpf (c’, n = 20); red dashed line: position of first de or TL cardiomyocyte; blue dashed lines: relative distance between de or TL cardiomyocytes; white asterisks: TL cardiomyocytes; magenta asterisks: delaminating cardiomyocytes; arrowheads: CL cardiomyocytes. d, d’, 60 hpf heart immunostained for N-cadherin and GFP (membrane), and counterstained with DAPI; representative mid-sagittal confocal image (d), and fluorescence intensity (d’, n = 49). e, e’, Representative mid-sagittal confocal image of 67 hpf heart (e), and fluorescence intensity (e’, n = 36). f, f’, 60 hpf heart immunostained for ZO-1 and GFP (membrane), and counterstained with DAPI; representative mid-sagittal confocal image (f), and fluorescence intensity (f’, n = 33). g, g’, Representative mid-sagittal confocal image of 65 hpf heart (g), and fluorescence intensity (g’, n = 27). hh”, 60 hpf heart immunostained for Crb2a and GFP (membrane), and counterstained with DAPI; representative mid-sagittal confocal image (h), and fluorescence intensity (h’, n = 31), and number of delaminating cardiomyocytes with apical (yellow asterisk) or junctional (white asterisk) Crb2a localization (h”, n = 49). Data are mean ± s.d. Two-tailed Wilcoxon test. n refers to the number of cardiomyocytes. Asterisks, delaminating cardiomyocytes; arrowheads, CL cardiomyocytes. Scale bars, 50 μm (b, c, d, e, f, g, h); 5 μm (a). For more details on statistics and reproducibility, see Methods.

Source data

Extended Data Fig. 2 Proliferation-induced crowding triggers tension heterogeneity among cardiomyocytes.

a, a’, Representative mid-sagittal confocal image of 62 hpf heart (a), and fluorescence intensity (a’, n = 53). b, b’, Representative mid-sagittal confocal image of 65 hpf heart (b), and fluorescence intensity (b’, n = 71). c, c’, 62 hpf heart immunostained for GFP (membrane), and counterstained with phalloidin and DAPI; representative mid-sagittal confocal image (c), and fluorescence intensity (c’, n = 60). d, Representative mid-sagittal confocal image of 62 hpf heart immunostained for α−18 and GFP (membrane), and counterstained with DAPI, and fluorescence intensity profiles (d’, CL, n = 76; de, n = 88). e, f, Representative recovery profiles from FRAP of myosin (e, CL, n = 18; de, n = 16) and actin (f, CL, n = 16; de, n = 12) in CL and de cardiomyocytes. gg’, Recoil velocity (g) and rate constant (k) (g’) of CL (n = 44) and de (n = 47) cardiomyocytes. h, Maximum intensity projection (MIP) of 48 and 62 hpf hearts corresponding to 3D object maps in Fig. 2a. i, Mid-sagittal confocal and corresponding binary images of 48- and 62 hpf hearts corresponding to NND calculations in Fig. 2a. jj”, Representative MIP of 48- and 62 hpf hearts (j), apical cell surface area (j’, 48 hpf, n = 175; 62 hpf, n = 200) (j’), and aspect ratio (j”, 48 hpf, n = 167; 62 hpf, n = 193). k, k’, Representative maximum intensity projection (MIP) of 48 hpf heart (k), and number of mVenus-Gmnn+ (proliferating) cardiomyocytes (k’, n = 37). l, l’, 60 hpf hearts of DMSO-treated (n = 28), alfacalcidol-treated (Alfa, n = 21) or calcitriol-treated (Calci, n = 20) zebrafish embryos; representative MIP (l), and quantification (l’). m, m’, 60 hpf hearts of DMSO-treated (n = 30) or MEK inhibitor-treated (n = 29) zebrafish embryos; representative MIP (m), and quantification (m’). n, n’, 60 hpf hearts of DMSO-treated (n = 25) or Erbb2 inhibitor-treated (n = 24) zebrafish embryos; representative MIP (n), and quantification (n’). o, MIP of 60 hpf hearts of DMSO- or Alfa-treated zebrafish embryos corresponding to 3D object maps in Fig. 2b. p, MIP of 60 hpf hearts of DMSO-, MEK inhibitor- or Erbb2 inhibitor-treated zebrafish embryos corresponding to 3D object maps in Fig. 2d. q, q’, Representative mid-sagittal confocal images of 65 hpf hearts of DMSO-treated (n = 22) or MEK inhibitor-treated (n = 23) zebrafish embryos (q) and quantification (q’). Data are mean ± s.d. except for d’, g, g’ (mean ± s.e.m.). Two-tailed Wilcoxon test (a’, b’, c’); Kruskal–Wallis test (l’); two-tailed Student’s t-test (g’, m’, n’, q’). n refers to the number of hearts (e, f, g, k’, l’, m’, n’, q’) or number of cardiomyocytes (a’, b’, c’, d’, j’, j”). All box-and-whisker plots show median, 25th and 75th percentiles, and all data points extending from minimum to maximum. Asterisks, delaminating or TL cardiomyocytes; arrowheads, CL cardiomyocytes. Scale bars, 50 μm. For more details on statistics and reproducibility, see Methods.

Source data

Extended Data Fig. 3 Inducing or abrogating the proliferation of CL cardiomyocytes increases or decreases cell morphology and tension heterogeneity.

a, b, Representative mid-sagittal confocal images of 60 hpf hearts of zebrafish embryos treated with DMSO, alfacalcidol (Alfa), MEK inhibitor or Erbb2 inhibitor, immunostained for p-myo and GFP (membrane), and counterstained with DAPI, and quantification (b, DMSO, n = 42; Alfa, n = 39; MEK inhibitor, n = 28; Erbb2 inhibitor, n = 33). Boxed area indicates high-p-myo cardiomyocytes in DMSO- and Alfa-treated zebrafish embryos. cc”, Representative MIP of 62 hpf hearts (c), apical cell surface area (c’, Erbb2 inhibitor, n = 217; Alfa, n = 229) and aspect ratio (c”, Erbb2 inhibitor, n = 199; Alfa, n = 205). d, d’, Representative MIP of 48- and 60 hpf hearts immunostained for p-myo and GFP (membrane), and counterstained with DAPI (d), and fluorescence intensity (d’, 48 hpf, n = 91; 60 hpf, n = 89). e, e’, Representative MIP of 60 hpf hearts of DMSO-, Alfa- and MEK inhibitor-treated zebrafish embryos immunostained for p-myo, GFP (membrane), and counterstained with DAPI (e), and fluorescence intensity (e’, DMSO, n = 125; Alfa, n = 114; MEK inhibitor, n = 120). Data are mean ± s.d. One-way ANOVA (b), two-tailed Mann–Whitney U-test (d’); Kruskal–Wallis test (e’). n refers to the number of hearts (b) or number of cardiomyocytes (c’, c”, d’, e’). All box-and-whisker plots show median, 25th and 75th percentiles, and all data points extending from minimum to maximum. Asterisks, delaminating cardiomyocytes; arrowheads, CL cardiomyocytes. Scale bars, 50 μm. For more details on statistics and reproducibility, see Methods.

Source data

Extended Data Fig. 4 Differential cellular contractility is necessary for cardiomyocyte delamination.

aa”, Representative mid-sagittal confocal images of hearts of WT-MYL9-GFP- (n = 25), DN-MYL9-GFP- (n = 37) or CA-MYL9-GFP- (n = 34) injected 96 hpf zebrafish larvae. bc, Hearts of mScarlet-WT-RHOA- (WT, n = 57), mScarlet-DN-RHOA- (DN, n = 37) or mScarlet-CA-RHOA- (CA, n = 31) injected 98 hpf zebrafish larvae; representative mid-sagittal confocal images (bb”), and quantification (c). d, d’, Hearts of mCherry-CAAX- or DN-shroom3-P2A-tdtomato-injected 96 hpf zebrafish larvae; representative mid-sagittal confocal images (d), and quantification (d’, n = 27). e, Representative frames from time-lapse imaging of a beating heart (50 hpf) of a CA-MYL9-mScarlet-injected animal (n = 5/5 hearts). f, f’, Hearts of mScarlet-DN-RHOA- and CA-MYL9-GFP-injected 100 hpf zebrafish larvae; representative mid-sagittal confocal images (f), and quantification (f’, CA-MYL9, n = 32; DN-RHOA, n = 18; DN-RHOA + CA-MYL9, n = 36). gg”, Heart of 98 hpf zebrafish larvae transplanted with control (ctrl) or tnnt2a morpholino (MO) -injected blastomeres; experimental plan (g), representative mid-sagittal confocal images (g’), and quantification (g”, n = 17). hh”, Hearts of ctrl (n = 12) or tnnt2a (n = 27) MO-injected 75 hpf zebrafish larvae transplanted with myl7: mKate-CAAXblastomeres; experimental plan (h), representative mid-sagittal confocal images (h’), and quantification (h”). Data are mean ± s.d. Kruskal–Wallis test (c, f’), two-tailed Student’s t-test (d’, h”). n refers to the number of hearts. Asterisks, delaminating or TL cardiomyocytes; arrowheads, CL cardiomyocytes. Scale bars, 50 μm (aa”, bb”, d, f, g’, h’); 5 μm (e). For more details on statistics and reproducibility, see Methods.

Source data

Extended Data Fig. 5 Differential cellular contractility augments cardiomyocyte delamination.

a, a’, Heart of CA-MYL9-GFP-injected 58 hpf zebrafish embryos; representative mid-sagittal confocal images (a), and quantification of apical domain length (a’, n = 12). bc’, Representative mid-sagittal confocal images of 61 hpf (b) and 70 hpf (c) hearts of control or CA-MYL9-GFP-injected zebrafish embryos, and quantification (b’, n = 16; c’; n = 14). d, d’, Representative mid-sagittal confocal images of 60 hpf (d, n = 12/12) and 70 hpf (d’, n = 24/24) hearts of CA-MYL9-mScarlet-injected zebrafish embryos; asterisks: depolarized TL cardiomyocytes. e, e’, Representative MIP of 60 hpf hearts of H2B-BFP- (n = 19) or CA-MYL9-BFP- (n = 23) injected zebrafish embryos (e), and quantification of the percentage of BFP and mVenus-Gmnn double-positive cardiomyocytes (e’); asterisks, BFP and mVenus-Gmnn double-positive cardiomyocytes. f, Representative confocal images of adult heart expressing CA-MYL9-mScarlet, and immunostained for MF-20 (myocardium) and counterstained with DAPI (n = 5/5). gh, Representative MIP of 96 hpf hearts of control or CA-MYL9-BFP-injected zebrafish larvae treated with DMSO or Erbb2 inhibitor (g), and quantification (h; g, n = 13; g’, n = 18; g”, n = 24). Data are mean ± s.d. Two-tailed Student’s t-test (a’, b’, c’, e); Kruskal–Wallis test (h). n refers to the number of hearts (b’, c’, e’, h); n = number of cardiomyocytes (a’). Asterisks: delaminating or TL cardiomyocytes; arrowheads: CL cardiomyocytes. Scale bars, 50 μm (a, b, c, d, d’, e, f, gg”), 200 μm (f). For more details on statistics and reproducibility, please see Methods.

Source data

Extended Data Fig. 6 Differential cellular contractility is sufficient to drive cardiomyocyte delamination and apicobasal depolarization in the absence of Nrg–Erbb2 signalling.

ab, Hearts of control or CA-MYL9-mScarlet-injected 96 hpf zebrafish larvae treated with DMSO or Erbb2 inhibitor; representative mid-sagittal confocal images (aa”), and quantification (b; a’, n = 30; a”, n = 27). cc”, Hearts of nrg2a:mRFP;myl7:LIFEACT-GFP 72 hpf zebrafish larvae injected with CA-MYL9-BFP; representative confocal images and 3D surface-rendered images (n = 17). de, Hearts of nrg2a:mRFP; myl7:eGFP-Podxl 96 hpf zebrafish larvae injected with CA-MYL9-BFP; representative mid-sagittal confocal images (ee”), and quantification (d; e’, n = 12; e’’, n = 15). f, f’, Hearts of ctrl MO-, amhc MO- or amhc MO + CA-MYL9-mScarlet-injected 80 hpf zebrafish larvae; representative confocal images (f), and quantification (f’, n = 18). gg” Hearts of ctrl- or tnnt2a MO- (n = 26) injected 70 hpf zebrafish embryos, transplanted with myl7:MYL9-GFP (n = 22) or myl7:CA-MYL9-GFP (n = 32) blastomeres; experimental plan (g), representative mid-sagittal confocal images (g’), and quantification (g”). Data are mean ± s.d. Two-tailed Student’s t-test (f’); Two-tailed Mann–Whitney U-test (b, d); Kruskal–Wallis test (g”). n refers to the number of hearts. Asterisks, delaminating or TL cardiomyocytes. Scale bars, 50 μm. For more details on statistics and reproducibility, see Methods.

Source data

Extended Data Fig. 7 Contractility-induced spatial segregation of cardiomyocytes is necessary and sufficient to trigger Notch reporter expression in CL cardiomyocytes.

a, Representative MIP of 48 hpf heart; Notch reporter expression is observed only in the endocardium (n = 15/15 hearts). b, b’, Hearts of DMSO- (n = 20) or alfacalcidol- (Alfa, n = 27) treated 60 hpf zebrafish embryos; representative mid-sagittal confocal images (b), and quantification (b’). c, c’ Hearts of DMSO- (n = 16) or MEK inhibitor- (n = 20) treated 65 hpf zebrafish embryos; representative mid-sagittal confocal images (c), and quantification (c’). dd’, Hearts of nrg2a:mRFP;TP1:VenusPest 100 hpf zebrafish larvae injected with CA-MYL9-BFP (d), and quantification (d’, nrg2a−/−, n = 40; nrg2a−/− + CA-MYL9, n = 56). e, e’, Hearts of tnnt2a MO-injected 72 hpf zebrafish larvae (n = 22) transplanted with myl7:CA-MYL9-mScarlet blastomeres (n = 27) (e), and quantification (e’). f, f’, Representative mid-sagittal confocal image of 62 hpf hearts (f), and quantification of apical domain length in Notch+ CL and Notch delaminating cardiomyocytes (f’, n = 17). Data are mean ± s.d. Two-tailed Student’s t-test (b’, c’, d’, f’); two-tailed Mann–Whitney U-test (e’). n refers to the number of hearts (b’, c’, d’, e’) or number of cardiomyocytes (f’). Red arrowheads, Notch+ cardiomyocytes; yellow arrowheads, Notch+ endocardial cells; white arrowheads, CL cardiomyocytes; asterisks, delaminating or TL cardiomyocytes. Scale bars, 50 μm. For more details on statistics and reproducibility, see Methods.

Source data

Extended Data Fig. 8 Notch signalling suppresses the actomyosin network in CL cardiomyocytes.

a, a’, Representative mid-sagittal confocal and skeletonized images of 70 hpf hearts of DMSO- or Notch-inhibitor-treated zebrafish embryos (a), and quantification (a’; DMSO, n = 55; LY411575, n = 34; RO4929097; n = 35). Asterisks, TL cardiomyocytes; arrowheads, Notch+ cardiomyocytes. b, b’, Representative mid-sagittal confocal images of 94 hpf hearts of zebrafish larvae injected with NICD-P2A-tdTomato and/or CA-MYL9-GFP (b), and quantification (b’; NICD, n = 23; CA-MYL9, n = 41; NICD + CA-MYL9, n = 33). c, c’, Actin localization in Notch and Notch+ (blue dashed line) CL cardiomyocytes of 62 hpf hearts; representative confocal images (en face view) (c), and quantification (c’, n = 20). dd”, Phalloidin localization in Notch and Notch+ (blue dashed line) CL cardiomyocytes of 62 hpf hearts; representative confocal images (en face view) (d), and FI profiles (d’; Notch, n = 55; Notch+, n = 56), and quantification (d”, n = 21). ee”, p-myo localization in NICD and NICD+ cardiomyocytes; representative confocal images (en face view) (e), and FI profiles (n = 42) (e’), and quantification (e”; n = 21). ff”, Representative frames from FRAP of myosin in Notch and Notch+ CL cardiomyocytes (f), and representative recovery profiles (f’, Notch, n = 11; Notch+, n = 12), and mobile fraction values (f”, Notch; n = 43; Notch+ n = 47). g, g’, Representative recovery profile (g, Notch, n = 12; Notch+, n = 9) and mobile fraction values calculated from FRAP of actin in Notch (n = 44) and Notch+ (n = 39) CL cardiomyocytes (g’). Data are mean ± s.d., except for d’ and e’ (mean ± s.e.m.). Two-tailed Student’s t-test (d”, e”, f”, g’); two-tailed Mann–Whitney U-test (c’); Kruskal–Wallis test (a’, b’). n refers to the number of hearts (a’, b’, c’, d”, e”f’, f”g, g’) or number of cardiomyocytes (d’, e’). Asterisks, delaminating or TL cardiomyocytes; arrowheads, CL cardiomyocytes. Scale bars, 50 μm (a, b); 20 μm (c, d, e). For more details on statistics and reproducibility, see Methods.

Source data

Extended Data Fig. 9 Erbb2 signalling does not regulate actomyosin localization at the onset of trabeculation.

aa”, Hearts of 65 hpf erbb2+/− and erbb2−/− zebrafish embryos, immunostained for Alcam, and counterstained with phalloidin and DAPI; representative MIP and en face view of boxed area (a), and FI profiles (a’, erbb2+/−, n = 116; erbb2−/−, n = 111), and quantification (a”, n = 17). b, b’, Hearts of 65 hpf erbb2+/− and erbb2−/− zebrafish embryos, immunostained for p-myo and counterstained with DAPI; representative MIP and en face view of boxed area (b), and quantification (b’, erbb2+/−, n = 17; erbb2−/−, n = 18). cc”’, Hearts of DMSO- or Erbb2 inhibitor-treated 60 hpf zebrafish embryos, immunostained for p-myo and Alcam, and counterstained with phalloidin and DAPI; representative MIP and en face view of boxed area (c), and FI profiles (c’, DMSO, n = 237; Erbb2 inhibitor, n = 166), and quantification (c”, DMSO, n = 26; Erbb2 inhibitor, n = 22; c”’, DMSO, n = 19; Erbb2 inhibitor, n = 18). Data are mean ± s.d., except for a’ and c’ (mean ± s.e.m.). Two-tailed Student’s t-test. n refers to the number of hearts (a”, b’, c”, c”’) or number of cardiomyocytes (a’, c’). Scale bars, 50 μm. For more details on statistics and reproducibility, see Methods.

Source data

Extended Data Fig. 10 Erbb2 signalling does not regulate actomyosin localization and stability at the onset of trabeculation.

a, a’, Representative MIP and en face view of boxed area of 62 hpf hearts of DMSO or Erbb2 inhibitor-treated zebrafish embryos (a), and quantification (a’, DMSO, n = 25; Erbb2 inhibitor, n = 27). bb”, Representative MIP and en face view of boxed area of 62 hpf hearts of DMSO or Erbb2- inhibitor- treated zebrafish embryos (b), and FI profiles (b’, DMSO, n = 189; Erbb2 inhibitor, n = 183), and quantification (b”, DMSO, n = 22; Erbb2 inhibitor, n = 25). cc”, FRAP of actin in CL cardiomyocytes of DMSO or Erbb2 inhibitor-treated zebrafish embryos; representative frames (c), and representative recovery profiles (c’, DMSO, n = 16; Erbb2 inhibitor, n = 17), and mobile fraction values (c”, DMSO, n = 39; Erbb2 inhibitor, n = 45). dd”, FRAP of myosin in CL cardiomyocytes of DMSO- or Erbb2 inhibitor-treated animals; representative frames (d), and recovery profiles (d’, n = 43), and mobile fraction values (d”, n = 42). Data are mean ± s.d., except for b’ (mean ± s.e.m.). Two-tailed Mann–Whitney U-test (a’, c”, d”); two-tailed Student’s t-test (b”). n refers to the number of hearts (a’, b”c’, c”d’, d”) or number of cardiomyocytes (b’). Scale bars, 50 μm. For more details on statistics and reproducibility, see Methods.

Source data

Supplementary information

Supplementary Methods

This file contains Supplementary Method 1: Macro for time-lapse live imaging of beating hearts on a Zeiss CSU-X1 Yokogawa spinning disk; and Supplementary Method 2: Laser nano-ablation data analysis.

Reporting Summary

Supplementary Video 1

Delaminating cardiomyocytes constrict their apical domain and exhibit enhanced myosin recruitment. Time-lapse imaging of a delaminating cardiomyocyte in a 60 hpf myl7:HRAS-GFP (green); myl7:myl9-mScarlet (magenta) heart. Scale bar, 10 µm.

Supplementary Video 2

Cardiomyocytes expressing CA-MYL9 delaminate to seed the trabecular layer. Time-lapse imaging of a 50 hpf myl7:HRAS-GFP (green) heart injected with myl7:CA-MYL9-mScarlet (magenta). Scale bar, 10 µm.

Supplementary Video 3

Cardiomyocytes expressing CA-MYL9 form trabecular ridges. Representative 3-D surface rendered animation of 15 dpf hearts of myl7:GFP or myl7:CA-MYL9-GFP injected animals. Scale bar, 30 µm.

Supplementary Video 4

Cardiomyocytes expressing CA-MYL9 form trabecular ridges in the absence of Erbb2 signalling. Representative 3-D surface rendered animation of 96 hpf hearts of control or myl7:CA-MYL9-mScarlet injected animals treated with DMSO or Erbb2 inhibitor. Scale bar, 20 µm.

Supplementary Video 5

Cardiomyocytes expressing CA-MYL9 form trabecular ridges in the absence of Nrg2a signalling. Representative 3-D surface rendered animation of 72 hpf hearts of nrg2a:mRFP; myl7:LIFEACT-GFP animals injected with myl7:CA-MYL9-BFP. Scale Bar, 20 µm.

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Priya, R., Allanki, S., Gentile, A. et al. Tension heterogeneity directs form and fate to pattern the myocardial wall. Nature 588, 130–134 (2020). https://doi.org/10.1038/s41586-020-2946-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-020-2946-9

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing