Regulation of cardiomyocyte behavior in zebrafish trabeculation by Neuregulin 2a signaling

Abstract

Trabeculation is crucial for cardiac muscle growth in vertebrates. This process requires the Erbb2/4 ligand Neuregulin (Nrg), secreted by the endocardium, as well as blood flow/cardiac contractility. Here, we address two fundamental, yet unresolved, questions about cardiac trabeculation: why does it initially occur in the ventricle and not the atrium, and how is it modulated by blood flow/contractility. Using loss-of-function approaches, we first show that zebrafish Nrg2a is required for trabeculation, and using a protein-trap line, find that it is expressed in both cardiac chambers albeit with different spatiotemporal patterns. Through gain-of-function experiments, we show that atrial cardiomyocytes can also respond to Nrg2a signalling, suggesting that the cardiac jelly, which remains prominent in the atrium, represents a barrier to Erbb2/4 activation. Furthermore, we find that blood flow/contractility is required for Nrg2a expression, and that while non-contractile hearts fail to trabeculate, non-contractile cardiomyocytes are also competent to respond to Nrg2a/Erbb2 signalling.

Figure 1. Nrg2a is required for cardiac trabeculation in zebrafish.

(a,b) Zebrafish larvae from nrg2a^+/− incrosses were imaged at 120 hpf; lateral view, anterior to the left; scale bars, 0.5 mm. nrg2a^−/− larvae can be recognized by their defective jaws (asterisk in b). (c–k) nrg2a mutants lack cardiac trabeculae. Confocal images (mid-sagittal sections) of larval hearts from Tg(myl7:LIFEACT-GFP);nrg2a^+/− incrosses at 75 (c–e), 120 (f–h) and 168 hpf (i–k); ventricular outer curvature (dashed boxes) magnified in upper right corners; arrows and asterisks indicate trabeculated and non-trabeculated walls, respectively; scale bars, 50 μm.

Figure 2. Nrg2a-mRFP expression during embryonic and larval cardiac development.

(a–i) Two-dimensional (2D) confocal images (mid-sagittal sections) of zebrafish hearts from Tg(kdrl:NLS-EGFP);nrg2a^+/− outcrosses at 52 (a–c), 78 (d–f) and 120 hpf (g–i) showing that Nrg2a-mRFP expression is clearly visible in the ventricular endocardium by 52 hpf, mainly in the outer curvature (a–c), and that it becomes stronger in both ventricular and atrial chambers at 78 and 120 hpf (d–i); however, it is weak in the atrioventricular canal; arrowheads point to the superior valve leaflet (a–i); AV, atrioventricular canal, At, atrium; V, ventricle; scale bars, 50 μm. (j) Cell-based mRFP intensity, measured with the ZEN Imaging Software and plotted as a graph, showing that Nrg2a-mRFP is more highly expressed in the ventricle compared to the AV canal and atrium; dots in this graph represent individual Nrg2a-mRFP expressing endocardial cells. (k) Nrg2a-mRFP positive endocardial cells counted in each chamber at 78 hpf, showing that there are more Nrg2a-mRFP positive endocardial cells in the ventricle than in the atrium at 78 hpf; dots in this graph represent individual hearts; N=5 hearts; values represent means±s.e.m.; **P≤0.01, ***P≤0.001 by Student's t-test.

Figure 3. Reduction of cardiac jelly thickness in developing zebrafish.

(a–h) Mid-sagittal confocal sections of late embryonic and early larval zebrafish hearts. Animals from Tg(kdrl:Hsa.HRAS-mCherry);Tg(myl7:EGFP-Hsa.HRAS) incrosses were synchronized at the tail bud stage (10 hpf) and imaged at 48 (a), 54 (b), 60 (c), 72 (d), 80 (e), 96 (f), 120 (g) and 144 hpf (h); endocardial and myocardial membranes are labelled in red and green, respectively; AV, atrioventricular canal, At, atrium; V, ventricle; scale bars, 50 μm. Higher magnification images of outer curvature area of ventricular (white dashed boxes) and atrial (yellow dashed boxes) walls are shown beneath each time point, respectively. The cardiac jelly is initially thicker in the atrium than in the ventricle at 48 hpf (a). It is greatly reduced in the ventricle by 72 hpf, (b–d) and almost fully gone by 96 hpf, while it is still detectable at 120 hpf in the atrium (e–h); arrowheads point to trabecular cardiomyocytes; arrows and asterisks indicate the presence and absence of cardiac jelly, respectively.

Figure 4. Myocardial nrg2a overexpression induces cardiomyocyte multilayering in nrg2a mutants.

(a) Cartoon of myocardial specific nrg2a construct. (b) Schematic representation of Nrg2a protein tagged by tdTomato. Due to the presence of the P2A peptide, cleavage occurs right after protein translation to separate the Nrg2a from the tdTomato fluorescent protein. (c–e) 2D confocal images (mid-sagittal sections) of Tg(myl7:LIFEACT-GFP);nrg2a^−/− hearts injected with myocardial specific nrg2a construct (myl7:nrg2a-p2a-tdTomato) at the one-cell stage. Mosaic overexpression of nrg2a in nrg2a^−/− cardiomyocytes led to the formation of a multilayered myocardial wall which is outlined by a white dashed box and magnified (c–e); arrows point to nrg2a overexpressing cardiomyocytes. (f–i) Confocal images (mid-sagittal sections) of 120 hpf Tg(myl7:EGFP-Hsa.HRAS) hearts from erbb2^+/− incrosses injected with the myl7:nrg2a-p2a-tdTomato construct (h–i). Magnified images of dashed boxes are shown below c–i; arrows point to nrg2a overexpressing cardiomyocytes, arrowheads point to trabeculae and asterisks indicate lack of trabeculae. At, atrium; V, ventricle; scale bars, 50 μm.

Figure 5. Myocardial specific nrg2a overexpression can lead atrial cardiomyocytes to form a multilayered wall.

(a) Illustration of cardiomyocyte specific nrg2a overexpression in zebrafish. (b–i) 2D confocal images (mid-sagittal views) of Tg(myl7:EGFP-Hsa.HRAS) and Tg(myl7:EGFP-Hsa.HRAS);Tg(myl7:nrg2a-p2a-tdTomato) hearts at 50 (b,c), 78 (d,e), 120 (f,g) and 144  hpf (h,i); magnified images of dashed boxes are shown below each time point; arrows point to multilayered walls; At, atrium; V, ventricle; scale bars, 50 μm.

Figure 6. Contractility/blood flow is required for endocardial expression of nrg2a but not the ability of cardiomyocytes to respond to nrg2a.

(a–i) Maximum intensity z-projections (25–30 z-stacks, mid-sagittal sections) of Tg(kdrl:NLS-EGFP);nrg2a^+/− hearts from non-treated (a–c), tnnt2a MO injected (d–f) and BDM treated (g–i) 78 hpf larvae; scale bars, 50 μm. (j–l) Graphs showing cell-based Nrg2a-mRFP intensity in outer curvature (yellow box) and inner curvature (green box) of hearts from non-treated (j), tnnt2a MO (k) and BDM treated (l) 78 hpf larvae; dots represent individual Nrg2a-mRFP expressing endocardial cells. Values represent means±s.e.m.; **P≤0.01, ***P≤0.001, NS (not significant), by Student's t-test. (m,n) 2D confocal images (mid-sagittal sections) of 78 hpf Tg(myl7:EGFP-Hsa.HRAS) (m) or Tg(myl7:EGFP-Hsa.HRAS);Tg(myl7:nrg2a-p2a-tdTomato) hearts (n) showing that myocardial overexpression of nrg2a can induce cardiomyocyte multilayering in tnnt2a morphants; asterisks and arrows indicate single-layered and multilayered ventricular walls, respectively. (o–r) Maximum intensity z-projections of TgBAC(cdh2:cdh2-EGFP) hearts from non-injected (o), injected with tnnt2a MO alone (p) or injected with tnnt2a MO and myl7:nrg2a-p2a-tdTomato plasmid (q,r) 96 hpf larvae. (r) 2D confocal image (sagittal section) of heart shown in q. Magnified images of dashed boxes are shown below (o–r); arrowheads and lozenges indicate presence and absence of Cdh2-EGFP proteins on the basal side of cardiomyocytes, respectively; At: atrium; V, ventricle; scale bars, 50 μm. (s) Schematic diagram of modulation of Nrg2a/Erbb2 signaling by cardiac contractility/blood flow.