Figure 1. Loss of snai1b leads to cardiomyocyte (CM) extrusion, disrupting myocardial wall integrity.

(A–B”) Single-plane images of Tg(myl7:BFP-CAAX) snai1b^+/+ (A) and snai1b^-/- (B) hearts at 50 hpf. Close-up of boxed areas (A’, B’) and schematic (A”, B”). (C–D’) 3D surface rendering of the myocardium of Tg(myl7:BFP-CAAX) snai1b^+/+ (C, C’) and snai1b^-/- (D, D’) embryos at 50 hpf. CM extrusions are clearly observed in snai1b^-/- embryos (magenta arrowheads in B, B’, B”, D, D’). (E) More CMs are extruding in Tg(myl7:BFP-CAAX) snai1b^-/- embryos compared with snai1b^+/+ and snai1b^+/- siblings at 50 hpf (snai1b^+/+, n = 20; snai1b^+/-, n = 23; snai1b^-/-, n = 24). (F–H) Blocking cardiac contractions with tnnt2a MO leads to a reduced number of extruding CMs in snai1b^-/- embryos, comparable with uninjected snai1b^+/+ embryos. (F–G’) 3D surface rendering of the myocardium of snai1b^+/+ (F) and snai1b^-/- (G) uninjected embryos and snai1b^-/- embryos injected with tnnt2a MO (G’). (H) Fewer CMs are extruding (magenta arrowheads in G) in snai1b^-/- embryos injected with tnnt2a MO (n = 14) compared with uninjected snai1b^-/- (n = 6) and snai1b^+/+ (n = 9) embryos at 50 hpf. (I–L) 3D surface rendering of the myocardium showing snai1b^+/+ donor-derived CMs in a snai1b^+/+ (I) or snai1b^-/- (J) heart, and snai1b^-/- donor-derived CMs in a snai1b^+/+ heart (K). (L) The percentage of donor-derived CMs that extrude is higher when snai1b^-/- donor-derived CMs are in snai1b^+/+ hearts (n = 8) than when snai1b^+/+ donor-derived CMs are in snai1b^+/+ (n = 5) or snai1b^-/- (n = 14) hearts. (M–P) Overexpression of snai1b specifically in CMs partially rescues the CM extrusion phenotype in snai1b^-/- embryos. 3D surface rendering of the myocardium of a snai1b^-/- embryo (M), and snai1b^-/- embryo overexpressing snai1b under a myl7 (N) or a fli1a (O) promoter. (P) Fewer CMs are extruding (magenta arrowheads) in snai1b^-/- embryos (n = 19) overexpressing snai1b in CMs (N, P) compared with snai1b^-/- embryos (M, P, n = 38), and this number is comparable to that in snai1^+/+ embryos (n = 24). The number of extruding CMs does not change in snai1b^-/- embryos (n = 16) when snai1b is overexpressed in endothelial cells (fli1a) (O, P). Plot values represent means ± S.D.; p-values determined by one-way ANOVA followed by multiple comparisons with Dunn test (E, H, L, P). Scale bars: 20 µm. V: ventricle; A: atrium; ap: apical; ba: basal; n: number of embryos.

Figure 1—figure supplement 1. Generation of snai1b mutants.

(A) Schematic of the promoter-less snai1b allele. Two gRNAs (red lines) were used to generate a snai1b mutant allele lacking 1300 bp upstream of the start codon. (B) Relative mRNA levels of snai1b are significantly reduced in snai1b^-/- hearts at 48 hpf, whereas snai1a expression levels appear unchanged, indicating lack of transcriptional adaptation by the paralogue; n = 4 biological replicates, 30 embryos each. (C–D’) Single-plane images of 50 hpf snai1b^+/+ (C) and snai1b^-/- (D–D’) hearts, with 50% of snai1b^-/- hearts exhibiting cardiac looping defects (D’). (E) Unlooped and looped snai1b^-/- hearts display a similar number of extruding CMs (snai1b^+/+, n = 14; looped snai1b^-/-, n = 14; unlooped snai1b^-/-, n = 14). (F) A higher proportion of the extruding CMs in snai1b^-/- hearts are located in the atrioventricular canal compared with the ventricle and atrium (snai1b^+/+, n = 20; snai1b^-/-, n = 24). (G, H) Single–plane images of 50 hpf snai1b^-/- hearts overexpressing snai1b under a myl7 (G) or fli1a (H) promoter. (I, J) Frames from time-lapse imaging of the beating heart of a snai1b^-/- embryo to visualize extruding cardiomyocytes (CMs) at 52 (I), 58 (J), and 70 (K) hpf; extruded CMs are found in the pericardial cavity (arrowheads in J and K). Plot values represent means ± S.D.; p-values determined by Student’s t-test (B) or by one-way ANOVA followed by multiple comparisons with Dunn test (E, F). Scale bars: 20 µm (C–D’, G–K). BRE: transcription factor IIB response element; V: ventricle; A: atrium; n: number of embryos; FC: fold change. All Ct values are listed in Supplementary file 2.

Figure 1—figure supplement 2. Increased cardiomyocyte (CM) extrusion in snai1b^-/- larvae.

(A–F) 3D surface rendering of the heart at 78 and 100 hpf. snai1b^-/- larvae (B, E) exhibit more extruding CMs (arrowheads) compared with snai1b^+/+ larvae (A, D); quantification (C, F) (C, snai1b^+/+, n = 6; snai1b^-/-, n = 10; F, snai1b^+/+, n = 12; snai1b^-/-, n = 10). Plot values represent means ± S.D.; p-values determined by Student’s t-test. Scale bars: 20 µm. V: ventricle; A: atrium; n: number of embryos.

Figure 1—figure supplement 3. Wild-type like cardiomyocyte (CM) numbers in snai1b mutants.

(A–A”’) TUNEL assay analysis indicates that extruding CMs in snai1b^-/- embryos are not apoptotic. Maximum intensity projection of Tg(myl7:BFP-CAAX) snai1b^+/+ (A) and snai1b^-/- (A’–A”) hearts at 50 hpf. Close-up of extruding CM (arrowhead) labelled with DAPI, but not with TUNEL (A”). (B, B’) Maximum intensity projections of Tg(myl7:H2B-GFP) snai1b^+/+ (B) and snai1b^-/- (B’) hearts at 52 hpf. (C) The total number of CMs does not change significantly in snai1b^-/- hearts (n = 12) compared with snai1b^+/+ hearts (n = 12). Plot values represent means ± S.D.; p-value determined by Student’s t-test. Scale bars: 20 µm (A–A’, B–B’); 10 µm (A’’). V: ventricle; A: atrium; n: number of embryos.

Figure 1—figure supplement 4. snai1b mutants exhibit reduced cardiac trabeculation.

(A–C’) Single-plane images and inner 3D surface rendering of trabecular cardiomyocytes (CMs) (asterisks in A) in 100 hpf snai1^-/- (C, C’) and snai1b^+/+ (A, A’) larvae. (B–D) Fewer delaminating (B) and trabecular (D) CMs are observed in snai1b^-/- larvae compared with snai1b^+/+ larvae at 78 (snai1b^+/+, n = 6; snai1b^-/-, n = 8) and 100 (snai1b^+/+, n = 6; snai1b^-/-, n = 9) hpf. Plot values represent means ± S.D.; p-values determined by Student’s t-test. Scale bars: 20 µm. V: ventricle; A: atrium; n: number of embryos.

Figure 1—figure supplement 5. Altered cardiomyocyte (CM) morphology and function in snai1b mutants.

(A, B, F, G) Maximum intensity projection of Tg(myl7:HRAS-EGFP) snai1b^+/+ (A, F) and snai1b^-/- (B, G) hearts at 52 (A, B) and 74 (F, G) hpf. At both timepoints, snai1b^-/- embryos exhibit reduced CM apical cell surface (C, H), increased CM aspect ratio (D, I), and reduced ventricular volume (E, J) compared with snai1b^+/+ embryos. (C, H) snai1b^+/+, N = 190 at 52 hpf, N = 173 at 74 hpf; snai1b^-/-, N = 195 at 52 hpf, N = 178 at 74 hpf. (D, I) snai1b^+/+, N = 183 at 52 hpf, N = 162 at 74 hpf; snai1b^-/-, N = 188, N = 170 at 74 hpf. (E, J) snai1b^+/+, n = 32 at 52 hpf, n = 28 at 74 hpf; snai1b^-/-, n = 27 at 52 hpf, n = 26 at 74 hpf. (K–P) snai1b mutants exhibit no differences in heart rate (K), ventricular ejection fraction (L), or ventricular fractional shortening (M) compared with snai1b^+/+ embryos at 52 hpf (K, snai1b^+/+, n = 28; snai1b^-/-, n = 29; L, snai1b^+/+, n = 33; snai1b^-/-, n = 35; M, snai1b^+/+, n = 28; snai1b^-/-, n = 35), but exhibit reduced heart rate (N), ventricular ejection fraction (P), and ventricular fractional shortening (O) compared with snai1b^+/+ larvae at 74 hpf (N, snai1b^+/+, n = 26; snai1b^-/-, n = 28; P, snai1b^+/+, n = 27; snai1b^-/-, n = 24; O, snai1b^+/+, n = 24; snai1b^-/-, n = 20). Plot values represent means ± S.D.; p-values determined by Mann–Whitney U test. Scale bars: 20 µm. V: ventricle; A: atrium; n: number of embryos; N: number of CMs.

Figure 1—video 1. Extruding cardiomyocytes (CMs) in snai1b^-/- hearts detach from the myocardium and are visible in the pericardial cavity.

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(A) Time-lapse imaging of a Tg(myl7:HRAS-EGFP) snai1b^+/+ heart from 52 to 70 hpf. No CMs were observed in the pericardial cavity (three embryos). (B) Time-lapse imaging of a Tg(myl7:HRAS-EGFP) snai1b^-/- heart from 52 to 70 hpf. Extruding CMs (arrowheads) detach from the myocardium and can be observed in the pericardial cavity for several hours (three embryos). Scale bars: 20 µm.