(A, B): Phenotypes of snai1b knockdown at 48hpf, lateral view. Black arrows: heart valve. (C–F): Changes in heart shape in myl7::GFP embryos injected with control (Ctl) and snai1b MO at 48hpf and 72hpf. A, atrium; V, ventricle. (G): Quantification of phenotypes produced by snai1b morphants at 48hpf, with or without snai1b mRNA at different concentrations. (H, I): Heart endocardium in flk::GFP embryos injected with Ctl and snai1b MO at 72hpf. White arrows: heart. Scale bars: 100µm.

(A–J): myl7 expression marking myocardial precursor cells in zebrafish embryos. (A, C, E, H): embryos injected with Ctl MO develop normally at 15, 18, 24, and 48hpf. (B, D, F, I): embryos injected with snai1b MO display cardiac fusion delays (B, D, F) and band-like heart shape at 48hpf (I). Myl7 expression (black arrows) is reduced at 15 and 18hpf (B, D). (G, J): embryos injected with both snai1b MO and mRNA display partial rescue of heart defects. (K–M): rescue of heart defects (arrowhead) in embryo injected with snai1b MO and mRNA at 48hpf.

Transverse sections of myl7::GFP (green) transgenic embryos immunostained for Fn (red). DAPI (blue) staining indicates nuclei. (A, B): Fn fibrils around myocardial precursors and across the midline in control embryos. (C, D): Fn levels decrease in snai1b MO injected embryos. (E, F): Fn levels are restored in embryos injected with α5β1 integrin protein. (B, D, and F): Magnified view of dashed rectangle of A, C, and E, respectively. Scale bars: 50µm.

In situ hybridization for indicated genes. (A–C): Expression pattern of α5 integrin in different embryos at 15hpf, dorsal view. (D): different types of phenotypes displayed in injected embryos at 20hpf. Graph shows phenotypic ratios of injected embryos at the same stage. Percentage of embryos of type 3 between embryos with snai1b MO and those with both snai1b MO and 25pg α5β1 integrin protein was statistically significant (P = 0.023, t-test). Ctl means Ctl MO, MO means snai1b MO.

Snai1b expression is related to myocardial precursors and Fn at 14–15 hpf. (A–C): In situ hybridization for indicated genes. (A): myl7 expression pattern. (B): snai1b expression pattern. (C): Fn expression pattern. (A–C) lateral view, (D): snai1b expression in cardiac precursors overlaps with myl7 (Brown). (E): itga5 was co-expressed with snai1b (Brown).

Efficiency of snai1b MO in injected embryos. (A–F): different concentrations of snai1b MO inhibited the expression of Snai1b-GFP fusion protein in zebrafish at 7 hpf. (A, C, E): Magnified view of rectangle in B, D, and F, respectively. (G): quantification of phenotypes produced by different concentrations of snai1b MO at 48 hpf.

Heart defects induced by snai1b knockdown are not due to apoptosis or endodermal defects. (A–C): TUNEL assay shows increased apoptosis in the snai1b MO injected embryos (B) compared to controls (A). Co-injection of p53 MO inhibits apoptosis (C) at 15 hpf. White arrows: cardiac precursor regions. (D–F): myl7 expression was decreased in snai1b MO injected embryos (E) compared to controls (D), and p53 MO did not rescue myl7 expression at 15 hpf. (G): q-PCR showed the myl7 expression do not change at 15 hpf (H, I): endoderm formation was not influenced, as judged by the expression of the endoderm marker foxa2 at 26 hpf.

Snai1b modulates Fn assembly, but does not affect the expression of fn. (A, B): fn expression was not changed at 16 hpf. (C): q-PCR showed the increased fn expression at 18 hpf. Table 1: phenotypes caused by exogenous Fn co-injected at 18 hpf.

Some integrins are not regulated by snai1b. (A-H): In situ hybridization for indicated genes. (A-D): β1 integrin expression was not changed by snai1b knockdown at 15 hpf. (A, C): dorsal view, (B, D): lateral view. (E-H): β3a and β3b integrin expression was not detected at 15 hpf (E, F), but at 72 hpf (G, H). (I): qRT-PCR showed that α8 and αV integrin transcription was not regulated by snai1b at 15 hpf. (J): qRT-PCR showed that α5 integrin transcription was up-regulated by snai1b overexpression, but not β1 integrin transcription at 15 hpf. The increased of α5 integrin transcription was statistically significant (P=0.007, t-test).

α5 integrin mRNA rescues cardiac defects induced by Snai1b knockdown. (A): different types of phenotypes displayed in injected embryos at 19 hpf. Graph shows phenotypic ratios of injected embryos at the same stage. Ctl means Ctl MO, MO means snai1b MO.

Loss of Snai1b resulted in abnormal Fibronectin matrix assembly. Lateral view, (A): Fn assembly was normal in the control embryo, (B): Fn assembly was disrupted in the morphant, especially in the posterior somites.