a Ultrastructural analysis of the HO (insets, left: whole HO, right: a section stained with toluidine blue). Transmission electron microscopy of CM-like ultrastructures containing myofibrils with Z-bands (Z), mitochondria (M), glycogen granules (Gly) in myofibrils, lipid droplet (LD) and desmosomes (D). Scale bar: 400 nm. N nucleus, SM smooth muscle. Over n = 5, over three experiments. b Transmission electron microscopy of honeycomb sarcoplasmic reticulum (SR) structure (left) and wavy intercalated disc (ID; right) in ultrathin sections of CMs in the HOs. RER ribosomal endoplasmic reticulum, G Golgi complex. Scale bar: 500 nm; over n = 4, over three experiments. c Immunostaining to detect Tbx5 (green) and MHC (pink) (over n = 7, two experiments). The HO at day 11 and at day 15 (left panel, bright field, scale bar: 100 μm). Inset indicates hematoxylin and eosin-staining. Tbx5 expression (green) was positive in the left atrium (LA)-, right atrium (RA)- and left ventricle (LV)-like parts, MHC expression (pink) was strongly expressed in both LV- and RV-like parts, and a Tbx5-positive atria/ventricle (A/V) cushion-like structure was also detected (arrow). DAPI (blue) for nuclear staining.