Extracellular stiffness regulates cell fate determination and drives the emergence of evolutionary novelty in the teleost heart

Figures

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  Fig. 1.. Heart morphology, cellular composition, and extracellular stiffness in adult control, elnb knockdown, and elnb knockout zebrafish hearts. (A–F) Brightfield images of heart morphology and fluorescence images of immunostained sections of control (A and B), elnb knockdown (KD; C and D), and elnb knockout (KO; E and F) zebrafish. Green, cardiomyocytes; Magenta, smooth muscle. Scale bar, 200 µm. Arrowheads indicate ectopic cardiomyocytes formed in the BA. (G) Schematic representation of AFM-based extracellular stiffness measurement, guided by immunostaining of adjacent sections. (H) Quantification of extracellular stiffness in the BA and ventricle (V) of control hearts (BA: n = 31, N = 3; V: n = 33, N = 3), and in elnb KD and KO hearts. In elnb KD hearts, measurements were performed in smooth muscle-forming regions (BA-SM; n = 34, N = 3) and ectopic cardiomyocyte-forming regions (BA-Ectopic CM; n = 34, N = 3) in the BA, and the V (n = 39, N = 3). In elnb KO hearts, measurements were performed in BA-SM (n = 12, N = 3) and BA-Ectopic CM (n = 19, N = 3), and the V (n = 28, N = 3). Statistical significances were assessed using the Mann–Whitney test for control, and the Kruskal–Wallis test…

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  Fig. 2.. Heart morphology and cellular composition in adult Polypterus, and extracellular stiffness in adult Polypterus and developing zebrafish hearts. (A and B) Brightfield image of heart morphology (A) and fluorescence image of an immunostained section (B) of adult Polypterus heart. (Scale bar, 600 µm.) (C) Quantification of extracellular stiffness in the CA and V of adult Polypterus (CA: n = 35, N = 3; V: n = 37, N = 3). Statistical significance was assessed using Mann–Whitney test. N.S., P > 0.05. (D) Quantification of extracellular stiffness in the OFT, BA, and V in 2 dpf (OFT: n = 30, N = 3; V: n = 10, N = 3) and 4 dpf zebrafish control embryos (BA: n = 54, N = 3; V: n = 14, N = 3), and these in 2 dpf (OFT: n = 25, N = 3; V: n = 20, N = 3) and 4 dpf zebrafish elnb KO embryos (BA: n = 20, N = 3; V: n = 21, N = 3). Statistical significances were assessed using t test for 2 dpf control embryos and 4 dpf elnb KO embryos, and Mann–Whitney test for 4 dpf control embryos and 2 dpf elnb KO embryos. N.S., P > 0.05; ****P < 0.0001. (E and F) Immunostained section of 4 dpf zebrafish ltbp3 KD (E) and yap KD (F) hearts. (Scale bar, 50 µm.) (G) Quantification of extracellular stiffness in the BA…

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  Fig. 3.. Artificial stiffening of the extracellular environment induces ectopic cardiomyocyte formation in the BA of developing zebrafish embryos. (A) Schematic diagram of the indentation experiment. Local indentation was performed using a glass needle targeting the developing OFT in 60 hpf zebrafish embryos. (B and C) Fluorescence image of immunostained sections (B) and expression pattern of elnb (C) of control (mounting without indentation; B, N = 0/8) 96 hpf zebrafish embryos. (D and E) Fluorescence image of immunostained sections (D) and expression pattern of elnb (E) of indented (D, N = 5/7) 96 hpf zebrafish embryos. (F) Quantification of extracellular stiffness in the BA in control and indented 96 hpf zebrafish embryos. (B and D) Green, cardiomyocytes. Scale bar, 50 µm. Arrowheads indicate ectopic cardiomyocytes formed in the BA. (C and E) Scale bar, 200 µm. Arrowheads indicate elnb expression in the BA. A, atrium; BA, bulbus arteriosus; CM, cardiomyocytes; V, ventricle.