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Fig. 4. The timing of the cardiac differentiation program is altered in TBX5-depleted embryos. (A) RT-PCR analysis of the expression of heart-specific isoforms of MHC, troponin and tropomyosin; and skeletal muscle-specific genes MyoD and muscle actin, throughout early and mid-tadpole stages of development in CMO (`C') and T5MO (`T') stage-matched embryos. All samples are derived from a single batch of eggs, and identical results were achieved in at least two independent sets of experiments for each marker. EF1-Alpha was used as a loading control for all RT-PCR reactions. (B-I) Images depicting embryos injected with (B-E) CMO, or (F-I) T5MO and immunostained for Tmy, showing delayed expression of Tmy in the hearts of T5MO embryos. Shown are representative sibling embryos imaged at the indicated stages. White arrows denote expression of Tmy within the heart. (J-Q) Images of living cardiac actin:GFP transgenic embryos, showing a delay in the onset of cardiac actin expression in the heart. Representative sibling embryos obtained from a single batch of embryos were injected with (J-M) CMO or (N-Q) T5MO and imaged at the indicated stages. Shown is a representative pair of embryos, while identical results were observed in over 50 embryos. White arrows denote expression of GFP within the heart field. |
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Fig. 1. TBX5 is required for cardiac proliferation. Whole-mount antibody staining with tropomyosin (Tmy) of stage 37 (A) control morpholino (CMO) or (B) TBX5 morpholino (T5MO) embryos. Transverse heart sections through (C,E) stage 33 and (D,F) stage 37 embryos stained with Tmy, to mark cardiac tissue, and DAPI, to mark cell nuclei; (C,D) CMO-derived tissue; (E,F) T5MO-derived tissue. (G-L) Examples of proliferating cardiac cells in transverse heart sections from (G-I) CMO and (J-L) T5MO embryos sectioned through the cardiac region at stages 29, 33 and 37, as indicated. Proliferating cardiomyocytes are identified as those positive for Tmy (myofibrilis shown as green) and anti-phosphohistone H3 (pH3; localized to the nucleus and shown in red). (M-R) Examples of cardiac cells undergoing apoptosis in transverse heart sections from (M-O) CMO and (P-R) T5MO embryos at stages 29, 33 and 37, as indicated. Apoptotic cardiomyocytes are identified as those positive for Tmy (myofibrilis shown as green) and anti-cleaved caspase 3 (CC3; localized to the nucleus and shown in red). Quantification of results from (S) total cardiomyocyte cell numbers, (T) mitotic index, and (U) programmed cell death. In all cases, bars represent the average of at least three embryos: CMO, red bar and T5MO, blue bar. Error bars denote the standard deviation, and * denotes a statistically significant difference (at P<0.05) between CMO and T5MO embryos at a given stage. Results are derived from a single set of experiments, all experiments being repeated at least once with an independent batch of embryos. Scale bars: 50 μm. a, atrium; i, inflow tract; o, outflow tract; v, ventricle. |
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Fig. 5. TBX5 depletion leads to a disruption in cardiac myofibril structure. Cardiomyocyte structure in transverse sections through the hearts of (A,C,E,G) CMO or (B,D,F,H) T5MO stage 37 embryos, as detected by immunostaining for (A,B) cardiac troponin T (cTNT), (C,D) MHC, (E,F) actin or (G,H) Tmy. (I,K,M) Stage 37 CMO or (J,L,N) T5MO embryos double-immunostained for tropomyosin (green) and (I,J) fibronectin, (K,L) fibrillin or (M,N) β-catenin, all shown in red. Note increase in fibrillin staining on the walls of the chamber of T5MO hearts relative to CMO (compare panel K to L, white arrows) and ectoptic expression of fibronectin, shown by white arrow, in the dorsal portion of the heart in panel J relative to panel I. (O,P) High magnification confocal images of hearts from (O) CMO or (P) T5MO stage 37 embryos. Note that formation of organized cardiac muscle bundles in T5MO hearts is limited to a single cluster adjacent to the cardiac lumen. (Q-S) Representative transmission electron micrographs of transverse images of stage 37 embryos taken from (Q) CMO cardiac tissue or (R) T5MO cardiac tissue adjacent to the pericardial cavity and (S) T5MO cardiac tissue adjacent to the cardiac lumen. Cardiac muscle fibrils are shown pseudo-colored in yellow. Note that sarcomeres in T5MO hearts can only be identified adjacent to the cardiac lumen (compare R with S) and only found in concentric arrays. By contrast, CMO-derived hearts show both longitudinal and concentric arrays (compare Q with S). High-magnification TEM images reveal the ultrastructures of (T) CMO and (U) T5MO cardiac sarcomeres. Arrows denote A-bands. Note the smaller, non-continuous A-bands in the T5MO-derived sarcomeres (U). (V) Traces of the heart sections from CMO and T5MO embryos imaged by TEM are depicted schematically. Yellow circles represent the location of TEM imaging. Scale bars: 50 μm in A-N; 2 μm in Q-S; 0.2 μm in T,U. |
