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Figure 1. Division of embryonic cardiomyocytes. (A) Stage-33/34 embryo, Tropomyosin (red) in the cardiac tube, phH3 (green), DNA in nuclei (Hoechst, blue). Arrowhead: dividing cardiomyocyte (phH3 and Tropomyosin positive). (B) Total number of Tropomyosin-positive cardiomyocytes at increasing embryonic-stages (n ≥ 3 embryos/stage). (C) Percentage of dividing cardiomyocytes (both phH3 and Tropomyosin positive) at increasing stages. |
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Figure 2. Cell division is not required for cardiomyocyte differentiation. Embryos stained for phH3 expression after incubation without (A) or with HUA (B) from stage-13. (C) Percentage of phH3-positive cells in the epidermis are decreased after HUA treatment vs. untreated control (matched areas, n ≥ 3 separate embryos ± SD; *P < 0.001). Transverse sections of stage-33/34 embryos after incubation of embryos in normal media (D and D′) or media containing HUA (E and E′), Tropomyosin expression (red), phH3 (green), and DNA in nuclei (Hoechst, blue); §Myotome is also positively labelled by tropomyosin. Note: Stage-29/30 embryos after control (F and H) or HUA (G and J) incubation showing TIc (F and G) or Nkx2.10 (H and J) expressions. (K) Heart size in control or HUA-incubated embryos as measured by TIc and Nkx2.10 expression area (see Figure 4A–H for examples). |
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Figure 4. Xic1 is required for heart formation. TIc expression in Xic1Mo injected embryos at stage-29/30 (A–D) and stage-33/34 (E–H), demonstrating the range of cardiac phenotypes from normal expression of TIc (D and H, score = 3) to no expression of TIc (A and E, score = 0). (J) Graph summarizing the percentage of stage-29/30 embryos with hearts of sizes 0 to 3, as assayed by TIc expression, following injection with 20 ng of CTRMo or Xic1Mo and rescued by co-injection with Xic1mRNA (30 pg). n = 58–130 embryos per injection. Transverse sections of stage-33/34 embryos, after injection with CTRMo (K and K′) or Xic1Mo (L and L′), Tropomyosin (red), phH3 (green), DNA in nuclei (Hoechst, blue). §Myotome is also positively labelled by tropomyosin. (M) Total number of cardiomyocytes (blue or red) and the proportion of phH3 cardiomyocytes (green) in five separate stage-33/34 embryos, injected with either CTRMo or Xic1Mo. (N) Average total number of cardiomyocytes (blue or red), and the number of dividing cardiomyocytes (green) from embryos shown in graph M (*P = 0.022, n = 5). |
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Supplementary Figure 4: (A) Representative pictures of transverse sections of stage 33/34 embryos showing different heart phenotypes, after injection with either CTRMo (A-C and enlarged box: A’-C’) or Xic1Mo (D-F and enlarged box: D’-F’), Tropomyosin (red), phH3 (green), DNA in nuclei (Hoechst, blue). |
