Journal: Development (Cambridge, England)
Article Title: TBX5 is required for embryonic cardiac cell cycle progression
Figure Lengend Snippet: 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.
Article Snippet: Cryostat sections (14 μm) were rinsed with wash buffer (PBS with 1% Triton and 1% heat inactivated calf serum), and incubated at 4°C overnight, as indicated, with mouse anti-tropomyosin 1:50, mouse anti-troponin 1:20, mouse anti-fibrillin 1:50 (all from Developmental Studies Hybridoma Bank), mouse anti-MHC (Abcam) rabbit anti-fibronectin 1:50 (Sigma), rabbit anti- Beta-catenin all at 1:1000 (Sigma), rabbit anti-phosphohistone H3 1:50 (Upstate) and rabbit anti-cleaved caspase 3 1:50 (Cell Signaling).
Techniques: Immunostaining, Staining, Expressing, Transmission Assay, Derivative Assay, Transmission Electron Microscopy, Imaging