JCS03252 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Detection of heart and skeletal muscle specific antigens by different antibodies. Cryosections derived from skeletal and heart muscle were reacted with antibodies against desmin (A, B), cTnT (C, D), skTnI (E, F), fast MyHC (clone My32) (G, H), and pan-MyHC (clone MF20) (I, J) to demonstrate the specificity of different antibodies. The desmin and the pan-MyHC antibody (MF20 clone) react both with skeletal and cardiac muscle. The antibody against cTnT detects the cardiac TnT isoform whereas the antibody binds to the skeletal isoform of TnI. Fast MyHC is only found in skeletal but not in heart muscle (My32 clone).

• Supplemental Figure 2 -

  Fig. S2. Triple labeling of transplanted newt cardiomyocytes. Cells were either labeled with DiI (A), Hoechst (C) or infected with an Adenovirus expressing GFP (B) before transplantation. Some experiments shown in this study were performed using triple-labeled cells. The DiI labeling appeared to be superior compared to other labeling techniques because it yielded signals, which were stronger and more stable during the course of the experiments than GFP and Hoechst-derived signals. Bars, 50 μm.

• Supplemental Figure 3 -

  Fig. S3. Rapid down-regulation of heart muscle-specific genes in newt cardiomyocytes transplanted into regenerating limbs. DiI labeled cardiomyocytes (red fluorescence) were transplanted into a regenerating limb 5 days after amputation and subjected to a double staining for desmin (green fluorescence) and MyHC (blue fluorescence) after 1 (A-D) and 15 days (E-H). Newt cardiomyocytes maintained the expression of the intermediary filament protein desmin in the majority of transplanted cells (A, B and inserts within) but rapidly lost the expression of MyHC (MF20 staining). 1 day after transplantation no MyHC staining was detectable in cardiomyoblasts transplanted into the limb blastema. At the bottom of the images (C, D and inserts within) a MyHC-positive myotube is visible, which served as an endogenous control for MyHC staining. After 15 days transplanted cells started to re-express MyHC (dark blue fluorescence in (G) and light blue staining in (H). Note that MF20 detects both skeletal and cardiac MyHC isoforms). Hoechst-counterstain was used to label all nuclei on the sections. The nuclei are shown in yellow after a false color transformation of the blue Hoechst fluorescence. Bars, 50 μm.

• Supplemental Figure 4 -

  Fig. S4. Rapid down-regulation of heart muscle-specific genes in newt cardiomyocytes transplanted into regenerating hearts. DiI labeled cardiomyocytes (red fluorescence) were transplanted into a regenerating heart and subjected to staining for cTnT (green fluorescence) after 1 day. Transplanted newt cardiomyocytes located within the regenerating heart tissue rapidly lost expression of cTnT. Note that the expression of cTnTn is also down-regulated within the damaged heart region (indicated by a dotted yellow line). Hoechst-counterstain was used to label all nuclei on the sections. The position of the cluster of DiI-labeled cardiomyocytes is indicated by a dotted white line. Bars, 50 μm.

• Supplemental Figure 5 -

  Fig. S5. Newt cardiomyocytes transdifferentiate into cartilage cells in regenerating newt limbs. Immunofluorescent detection of DiI labeled cardiomyocytes (red fluorescence) with the cartilage-specific LE-Lectin (green fluorescence). Hoechst counterstain (blue fluorescence) was used to label all nuclei on the sections. Sections were taken from the regenerating limb 35 days after amputation at different planes. The section shown in (A, C, E, G) was derived from a proximal position while the section displayed in (B, D, F, H) was taken from the distal part of the regenerating limb representing the developing toe buds. Bars, 50 μm.