pbabe neo gateway erbb2  (Addgene inc)

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 1. Variations in erbb2 expression in developing heart. (A) Schematic diagram illustrating the purification of embryonic hearts, isolation of cardiac cells, and sub- sequent single-cell RNA sequencing analysis. (B) Heatmap showing the expression of enriched markers in cell clusters of zebrafish hearts at 72 hours postfertilization (hpf). CM-V, ventricular cardiomyocytes; FB, fibroblasts; EP, epicardial cells; CM-A, atrial cardiomyocytes; RBC, erythrocytes; VMC, valve myocardial cell; EC, endocardial cells; VEC, valve endocardial cells; IC, immune cells; EPDC, epicardium-derived cells. Yellow, high expression; magenta, low expression. (C) UMAP plot showing the cardiac cell clusters in zebrafish hearts at 72 hpf. (D) UMAP plot showing the heterogeneous expression of erbb2 in CM-V subpopulations. (E) UMAP plot showing reclustering of CM-V. (F) erbb2 heterogeneity in CM-V. Red, high expression; gray, low expression. Three independent experiments were performed. (G to I) Confocal slices of cardiac ventricles expressing erbb2:GFP;myl7:mCherry at (G) 60 hpf, (H) 72 hpf, and (I) 96 hpf. (J) Confocal sections of erbb2:GFP;myl7:mCherry hearts at 45 days postfertilization (dpf). Enlarged views of boxed areas are shown in the right panels. White arrowheads, erbb2:GFP+ cardiomyocytes; magenta arrowheads, erbb2:GFP− cardiomyocytes. CL CMs, compact layer cardiomyocytes; TL CMs, trabecular layer cardiomyocytes. [(G) to (J)] n = 10 zebrafish for each stage. Scale bars, 20 μm [(G) to (I)] and 100 μm (J).

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Expressing, Purification, Isolation, RNA Sequencing, Derivative Assay

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 2. The erbb2 heterogeneity correlates with trabeculae formation. (A) erbb2:RFP;β-act2:BSR embryos were injected with photoactivatable green fluorescent pro- tein (PAGFP) mRNA at the one-cell stage. Time series images of the same embryonic heart at 60 hpf before ultraviolet (UV) photoactivation (top), immediately following photoactivation (middle), and at 80 hpf (bottom). Magenta arrowheads: erbb2:GFP+ cardiomyocytes; white arrowheads: erbb2:GFP− cardiomyocytes. (B) Schematic of photoactivation experiments to track the delamination of single cardiomyocytes in (A). (C) Quantification of the spatial distribution of erbb2+ and erbb2− cardiomyocytes in the trabecular and compact layers at 80 hpf in (A). n = 12 embryos. (D) Schematic diagram showing the dual-side illumination objectives coupled with an imaging module. (E) Time-lapse images of erbb2:GFP;myl7:H2A-mCherry heart between 60 and 80 hpf. Blue and white arrowheads indicate erbb2+ and erbb2− cardiomyocytes, re- spectively. (F) Curved lines in (E) represent the moving trajectories of erbb2+ (magenta) and erbb2− (blue) cardiomyocyte nuclei. Blue circles indicate the starting points, and yellow circles denote the end points. (G) The schematic depicts the distance along the apicobasal axis of cell delamination over a 20-hour time window. (H) Quantifi- cation of the movement distance of erbb2+ and erbb2− cardiomyocytes. n = 12 cardiomyocytes per group. For in toto live imaging, three independent experiments were performed using different hearts on different days. Data are presented as mean ± SEM. Two-tailed Mann-Whitney U tests were used for comparison. Scale bars, 25 μm.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Injection, Imaging, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 3. erbb2 directly controls tension heterogeneity to guide cardiomyocyte delamination. (A) Left, p-myo staining in erbb2:GFP hearts at 60 hpf. Dashed arrows, the posi- tions subjected to fluorescence intensity profiling; blue and magenta arrowhead, erbb2:GFP+ and erbb2:GFP− cardiomyocytes, respectively. Middle and right: fluorescence in- tensity profiles and average fluorescence intensities of p-myo. n = 12 cardiomyocytes for each group. (B) Apical view and schematics of F-actin maximum intensity projections in erbb2:GFP embryos at 96 hpf. White and magenta dashed lines, erbb2+ and erbb2− cells, respectively. (C) Confocal image of erbb2:GFP;myl7:WT-MYL9-mRuby embryo at 60 hpf. White and magenta arrowheads, erbb2:GFP+ and erbb2:GFP− cardiomyocytes. (B and C) n = 7 embryos. (D) Fluorescence recovery after photobleaching (FRAP) analysis of WT- MYL9-mRuby in erbb2:GFP;myl7:WT-MYL9-mRuby hearts. Right: quantifications of WT-MYL9-mRuby mobile fraction. n = 7 cardiomyocytes for each group. (E) F-actin staining in myl7:memGFP hearts treated with DMSO, PD168393, and AG1478. Arrowheads, the apical enrichment of F-actin. (F) Schematic of a dominant-negative human ERBB2 (DN-ERBB2). (G) myl7:DN-ERBB2-P2A-mCherry clones were confined to the compact layer at 96 hpf. (H) Quantifications of the spatial distributions of DN-ERBB2 clones at 72 and 96 hpf. CL CMs, compact layer cardiomyocytes; TL CMs, trabecular layer cardiomyocytes. [(E), (G), and (H)] n = 8 embryos for each stage. (I) Apical view and schematics of F-actin in WT and myl7:DN-ERBB2-P2A-GFP hearts. (J) Confocal image and statistics of trabeculae in myl7:WT-MYL9-mRuby (left), myl7:CA-MYL9-mRuby treated with DMSO (middle), and PD168393 (right) at 72 hpf. Arrowheads, trabeculae. (K) Confocal image showing DN-ERBB2-P2A-GFP+ clones in myl7:WT-MYL9-mRuby and myl7:CA-MYL9-mRuby hearts. (L) Quantifications of DN-ERBB2 clone distributions in (K). [(I) to (L)] n = 7 to 10 embryos for each group. Data are presented as mean ± SEM. Unpaired two-tailed Student’s t tests [(A) and (D)], one-way ANOVA (J), and two-tailed Mann-Whitney U tests [(H) and (L)] were applied to assess statistical significance. Scale bars, 20 μm.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Staining, Fluorescence, Dominant Negative Mutation, Clone Assay, Two Tailed Test, MANN-WHITNEY

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 4. erbb2 promotes trabeculation by triggering PI3K-mediated remodeling of the actomyosin network. (A) Confocal imaging of myl7:PH-AKT-GFP;erbb2:RFP hearts at 60 hpf. PH-AKT-GFP was enriched on the plasma membrane of erbb2:RFP+ cardiomyocytes, while its cytosolic distribution was observed in erbb2:RFP− cells. Insets are magnifications of boxed areas, and the dashed arrows indicate the position subjected to fluorescence intensity analysis. Right: fluorescence intensity profiles of PH- AKT-GFP. n = 10 embryos. (B) PD168393-treated myl7:PH-AKT-GFP;erbb2:RFP heart at 60 hpf. (C) Confocal image of myl7:PH-AKT-GFP;myl7:DN-ERBB2-P2A-mCherry heart at 60 hpf. (D) myl7:PH-AKT-GFP;erbb2:RFP hearts treated with LY294002 to inhibit PI3K signaling. [(B) to (D)] n = 8 embryos. (E) myl7:memGFP embryos, carrying myl7:actr2b- mRuby clones, were treated with DMSO (left) and PD168393 (right). Arrowheads denote the membrane localization of the actr2b-mRuby in delaminating cardiomyocytes. (F) Apical views of maximum intensity projections of F-actin in wild-type (WT) and myl7:arpin-mRuby hearts. F-actin is restricted to the lateral sides of cardiomyocytes in myl7:arpin-mRuby hearts. (G) Confocal images of myl7:memGFP (left) and myl7:arpin-mRuby;myl7:memGFP (right) hearts at 84 hpf. Arrowheads, trabeculae. (H) Apical views of F-actin network in DMSO and JLY-treated hearts. JLY decouples PI3K signaling from its ability to regulate actin dynamics. JLY, a combination of jasplakinolide (0.8 μM), latrunculin A (0.125 μM), and Y27632 (1 μM). [(E) to (H)] n = 6 to 10 embryos per group. Insets are enlarged views of boxed areas. [(F) and (H)] Dashed arrows indicate the intensity profile of F-actin. (I) Confocal images of 72 hpf myl7:WT-MYL9-mRuby;myl7:memGFP (left) and myl7:CA-MYL9-mRuby;myl7:memGFP (right) embryos treated with DMSO, LY294002, and CK666. Arrowheads indicate trabeculae. Quantifications of trabeculae are displayed on the right. n = 7 embryos per group. One-way ANOVA, followed by Tukey’s multiple comparisons test was used. Data are presented as mean ± SEM. Scale bars, 20 μm. (J) Schematic diagram showing erbb2 promotes actomyo- sin network remodeling through the PI3K-Arp2/3 axis.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Imaging, Clinical Proteomics, Membrane, Fluorescence, Clone Assay

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 5. Notch-mediated lateral inhibition suppresses trabeculation by reducing erbb2 expression. myl7:mCherry embryos were treated with 100 μM DAPT and DMSO from 84 to 96 hpf. Three independent replicates were performed, with each replicate containing approximately 1000 embryonic hearts. (A) RNA sequencing analysis re- vealed that erbb2 expression was significantly up-regulated following DAPT-treatment. (B) Gene set enrichment analysis (GSEA) revealed an increased expression level of key genes in the PI3K-AKT signaling pathway in Notch-inhibited hearts. Magenta, high expression; blue, low expression. (C) Confocal section (top) and maximum projec- tions (bottom) of Tp1:d2GFP;erbb2:RFP heart at 96 hpf. Note the mutually exclusive distributions of Tp1:d2GFP+ and erbb2:RFP+ cells. White arrowheads, Tp1:d2GFP+ cells; blue arrowheads, erbb2:RFP+ cells. (D) Maximum projections of Tp1:d2GFP;erbb2:RFP embryos treated with DMSO and DAPT. (E) erbb2:RFP embryos were injected with the myl7:NICD-P2A-GFP plasmid at the one-cell stage and imaged at 72 and 84 hpf. White arrowheads, NICD-P2A-GFP+ cells; blue arrowheads, erbb2:GFP+ cells. (F) Confocal images of myl7:PH-AKT-GFP hearts exposed to DMSO and DAPT. Insets are magnifications of boxed areas. (C to F) n = 10 embryos per group. (G) F-actin staining of erbb2:GFP hearts after treatment with either DMSO or DAPT. (H) Confocal images of myl7:WT-MYL9-mRuby (left) and myl7:DN-MYL9-mRuby (right) embryos treated with DMSO and DAPT. Notably, inhibiting Notch activity was unable to rescue the trabeculation defects in myl7:DN-MYL9-mRuby hearts. (I) Confocal images of myl7:WT-MYL9- mRuby (left), myl7:WT-MYL9-mRuby;myl7:NICD-P2A-GFP (middle), and myl7:CA-MYL9-mRuby;myl7:NICD-P2A-GFP (right) hearts. [(G) to (I)] n = 8 embryos per group. Scale bar, 20 μm. (J) Schematic illustration of the trabeculae formation paradigm: Initially, erbb2 is differentially expressed in the single-layer myocardium (left). Subsequently, erbb2 activates the PI3K-Arp2/3 axis, inducing cell tension heterogeneity and cardiomyocyte sprouting (middle). Last, the nascent trabeculae trigger Notch activity in adjacent cardiomyocytes, which then abolishes their erbb2 expression to confine these cardiomyocytes in the compact layer.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Inhibition, Expressing, RNA Sequencing, Injection, Plasmid Preparation, Staining, Activity Assay

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 1. Variations in erbb2 expression in developing heart. (A) Schematic diagram illustrating the purification of embryonic hearts, isolation of cardiac cells, and sub- sequent single-cell RNA sequencing analysis. (B) Heatmap showing the expression of enriched markers in cell clusters of zebrafish hearts at 72 hours postfertilization (hpf). CM-V, ventricular cardiomyocytes; FB, fibroblasts; EP, epicardial cells; CM-A, atrial cardiomyocytes; RBC, erythrocytes; VMC, valve myocardial cell; EC, endocardial cells; VEC, valve endocardial cells; IC, immune cells; EPDC, epicardium-derived cells. Yellow, high expression; magenta, low expression. (C) UMAP plot showing the cardiac cell clusters in zebrafish hearts at 72 hpf. (D) UMAP plot showing the heterogeneous expression of erbb2 in CM-V subpopulations. (E) UMAP plot showing reclustering of CM-V. (F) erbb2 heterogeneity in CM-V. Red, high expression; gray, low expression. Three independent experiments were performed. (G to I) Confocal slices of cardiac ventricles expressing erbb2:GFP;myl7:mCherry at (G) 60 hpf, (H) 72 hpf, and (I) 96 hpf. (J) Confocal sections of erbb2:GFP;myl7:mCherry hearts at 45 days postfertilization (dpf). Enlarged views of boxed areas are shown in the right panels. White arrowheads, erbb2:GFP+ cardiomyocytes; magenta arrowheads, erbb2:GFP− cardiomyocytes. CL CMs, compact layer cardiomyocytes; TL CMs, trabecular layer cardiomyocytes. [(G) to (J)] n = 10 zebrafish for each stage. Scale bars, 20 μm [(G) to (I)] and 100 μm (J).

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Expressing, Purification, Isolation, RNA Sequencing, Derivative Assay

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 2. The erbb2 heterogeneity correlates with trabeculae formation. (A) erbb2:RFP;β-act2:BSR embryos were injected with photoactivatable green fluorescent pro- tein (PAGFP) mRNA at the one-cell stage. Time series images of the same embryonic heart at 60 hpf before ultraviolet (UV) photoactivation (top), immediately following photoactivation (middle), and at 80 hpf (bottom). Magenta arrowheads: erbb2:GFP+ cardiomyocytes; white arrowheads: erbb2:GFP− cardiomyocytes. (B) Schematic of photoactivation experiments to track the delamination of single cardiomyocytes in (A). (C) Quantification of the spatial distribution of erbb2+ and erbb2− cardiomyocytes in the trabecular and compact layers at 80 hpf in (A). n = 12 embryos. (D) Schematic diagram showing the dual-side illumination objectives coupled with an imaging module. (E) Time-lapse images of erbb2:GFP;myl7:H2A-mCherry heart between 60 and 80 hpf. Blue and white arrowheads indicate erbb2+ and erbb2− cardiomyocytes, re- spectively. (F) Curved lines in (E) represent the moving trajectories of erbb2+ (magenta) and erbb2− (blue) cardiomyocyte nuclei. Blue circles indicate the starting points, and yellow circles denote the end points. (G) The schematic depicts the distance along the apicobasal axis of cell delamination over a 20-hour time window. (H) Quantifi- cation of the movement distance of erbb2+ and erbb2− cardiomyocytes. n = 12 cardiomyocytes per group. For in toto live imaging, three independent experiments were performed using different hearts on different days. Data are presented as mean ± SEM. Two-tailed Mann-Whitney U tests were used for comparison. Scale bars, 25 μm.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Injection, Imaging, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 3. erbb2 directly controls tension heterogeneity to guide cardiomyocyte delamination. (A) Left, p-myo staining in erbb2:GFP hearts at 60 hpf. Dashed arrows, the posi- tions subjected to fluorescence intensity profiling; blue and magenta arrowhead, erbb2:GFP+ and erbb2:GFP− cardiomyocytes, respectively. Middle and right: fluorescence in- tensity profiles and average fluorescence intensities of p-myo. n = 12 cardiomyocytes for each group. (B) Apical view and schematics of F-actin maximum intensity projections in erbb2:GFP embryos at 96 hpf. White and magenta dashed lines, erbb2+ and erbb2− cells, respectively. (C) Confocal image of erbb2:GFP;myl7:WT-MYL9-mRuby embryo at 60 hpf. White and magenta arrowheads, erbb2:GFP+ and erbb2:GFP− cardiomyocytes. (B and C) n = 7 embryos. (D) Fluorescence recovery after photobleaching (FRAP) analysis of WT- MYL9-mRuby in erbb2:GFP;myl7:WT-MYL9-mRuby hearts. Right: quantifications of WT-MYL9-mRuby mobile fraction. n = 7 cardiomyocytes for each group. (E) F-actin staining in myl7:memGFP hearts treated with DMSO, PD168393, and AG1478. Arrowheads, the apical enrichment of F-actin. (F) Schematic of a dominant-negative human ERBB2 (DN-ERBB2). (G) myl7:DN-ERBB2-P2A-mCherry clones were confined to the compact layer at 96 hpf. (H) Quantifications of the spatial distributions of DN-ERBB2 clones at 72 and 96 hpf. CL CMs, compact layer cardiomyocytes; TL CMs, trabecular layer cardiomyocytes. [(E), (G), and (H)] n = 8 embryos for each stage. (I) Apical view and schematics of F-actin in WT and myl7:DN-ERBB2-P2A-GFP hearts. (J) Confocal image and statistics of trabeculae in myl7:WT-MYL9-mRuby (left), myl7:CA-MYL9-mRuby treated with DMSO (middle), and PD168393 (right) at 72 hpf. Arrowheads, trabeculae. (K) Confocal image showing DN-ERBB2-P2A-GFP+ clones in myl7:WT-MYL9-mRuby and myl7:CA-MYL9-mRuby hearts. (L) Quantifications of DN-ERBB2 clone distributions in (K). [(I) to (L)] n = 7 to 10 embryos for each group. Data are presented as mean ± SEM. Unpaired two-tailed Student’s t tests [(A) and (D)], one-way ANOVA (J), and two-tailed Mann-Whitney U tests [(H) and (L)] were applied to assess statistical significance. Scale bars, 20 μm.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Staining, Fluorescence, Dominant Negative Mutation, Clone Assay, Two Tailed Test, MANN-WHITNEY

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 4. erbb2 promotes trabeculation by triggering PI3K-mediated remodeling of the actomyosin network. (A) Confocal imaging of myl7:PH-AKT-GFP;erbb2:RFP hearts at 60 hpf. PH-AKT-GFP was enriched on the plasma membrane of erbb2:RFP+ cardiomyocytes, while its cytosolic distribution was observed in erbb2:RFP− cells. Insets are magnifications of boxed areas, and the dashed arrows indicate the position subjected to fluorescence intensity analysis. Right: fluorescence intensity profiles of PH- AKT-GFP. n = 10 embryos. (B) PD168393-treated myl7:PH-AKT-GFP;erbb2:RFP heart at 60 hpf. (C) Confocal image of myl7:PH-AKT-GFP;myl7:DN-ERBB2-P2A-mCherry heart at 60 hpf. (D) myl7:PH-AKT-GFP;erbb2:RFP hearts treated with LY294002 to inhibit PI3K signaling. [(B) to (D)] n = 8 embryos. (E) myl7:memGFP embryos, carrying myl7:actr2b- mRuby clones, were treated with DMSO (left) and PD168393 (right). Arrowheads denote the membrane localization of the actr2b-mRuby in delaminating cardiomyocytes. (F) Apical views of maximum intensity projections of F-actin in wild-type (WT) and myl7:arpin-mRuby hearts. F-actin is restricted to the lateral sides of cardiomyocytes in myl7:arpin-mRuby hearts. (G) Confocal images of myl7:memGFP (left) and myl7:arpin-mRuby;myl7:memGFP (right) hearts at 84 hpf. Arrowheads, trabeculae. (H) Apical views of F-actin network in DMSO and JLY-treated hearts. JLY decouples PI3K signaling from its ability to regulate actin dynamics. JLY, a combination of jasplakinolide (0.8 μM), latrunculin A (0.125 μM), and Y27632 (1 μM). [(E) to (H)] n = 6 to 10 embryos per group. Insets are enlarged views of boxed areas. [(F) and (H)] Dashed arrows indicate the intensity profile of F-actin. (I) Confocal images of 72 hpf myl7:WT-MYL9-mRuby;myl7:memGFP (left) and myl7:CA-MYL9-mRuby;myl7:memGFP (right) embryos treated with DMSO, LY294002, and CK666. Arrowheads indicate trabeculae. Quantifications of trabeculae are displayed on the right. n = 7 embryos per group. One-way ANOVA, followed by Tukey’s multiple comparisons test was used. Data are presented as mean ± SEM. Scale bars, 20 μm. (J) Schematic diagram showing erbb2 promotes actomyo- sin network remodeling through the PI3K-Arp2/3 axis.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Imaging, Clinical Proteomics, Membrane, Fluorescence, Clone Assay

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 5. Notch-mediated lateral inhibition suppresses trabeculation by reducing erbb2 expression. myl7:mCherry embryos were treated with 100 μM DAPT and DMSO from 84 to 96 hpf. Three independent replicates were performed, with each replicate containing approximately 1000 embryonic hearts. (A) RNA sequencing analysis re- vealed that erbb2 expression was significantly up-regulated following DAPT-treatment. (B) Gene set enrichment analysis (GSEA) revealed an increased expression level of key genes in the PI3K-AKT signaling pathway in Notch-inhibited hearts. Magenta, high expression; blue, low expression. (C) Confocal section (top) and maximum projec- tions (bottom) of Tp1:d2GFP;erbb2:RFP heart at 96 hpf. Note the mutually exclusive distributions of Tp1:d2GFP+ and erbb2:RFP+ cells. White arrowheads, Tp1:d2GFP+ cells; blue arrowheads, erbb2:RFP+ cells. (D) Maximum projections of Tp1:d2GFP;erbb2:RFP embryos treated with DMSO and DAPT. (E) erbb2:RFP embryos were injected with the myl7:NICD-P2A-GFP plasmid at the one-cell stage and imaged at 72 and 84 hpf. White arrowheads, NICD-P2A-GFP+ cells; blue arrowheads, erbb2:GFP+ cells. (F) Confocal images of myl7:PH-AKT-GFP hearts exposed to DMSO and DAPT. Insets are magnifications of boxed areas. (C to F) n = 10 embryos per group. (G) F-actin staining of erbb2:GFP hearts after treatment with either DMSO or DAPT. (H) Confocal images of myl7:WT-MYL9-mRuby (left) and myl7:DN-MYL9-mRuby (right) embryos treated with DMSO and DAPT. Notably, inhibiting Notch activity was unable to rescue the trabeculation defects in myl7:DN-MYL9-mRuby hearts. (I) Confocal images of myl7:WT-MYL9- mRuby (left), myl7:WT-MYL9-mRuby;myl7:NICD-P2A-GFP (middle), and myl7:CA-MYL9-mRuby;myl7:NICD-P2A-GFP (right) hearts. [(G) to (I)] n = 8 embryos per group. Scale bar, 20 μm. (J) Schematic illustration of the trabeculae formation paradigm: Initially, erbb2 is differentially expressed in the single-layer myocardium (left). Subsequently, erbb2 activates the PI3K-Arp2/3 axis, inducing cell tension heterogeneity and cardiomyocyte sprouting (middle). Last, the nascent trabeculae trigger Notch activity in adjacent cardiomyocytes, which then abolishes their erbb2 expression to confine these cardiomyocytes in the compact layer.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Inhibition, Expressing, RNA Sequencing, Injection, Plasmid Preparation, Staining, Activity Assay