a Immunostaining for Mlc2v (red) and Mlc2a (green) in the HOs (n = 30, over three independent experiments). Left: a whole HO. Middle: high-resolution images of the atrium- and ventricle-like regions corresponding to the insets in left panel. Right: embryonic hearts from E10.5 and E12.5. Scale bar: 50 μm. See also Supplementary Fig. 6a. b Whole-mount immunostaining of Mlc2v (red) and Mlc2a (green) in the HOs. Distinct Mlc2a-positive atrial cells were detected in the HO (over n = 6, over three independent experiments). Scale bar: 50 μm. c The HOs (over n = 3, over two experiments) possessed mainly two parts (top panel; a section stained with toluidine blue) with cells exhibiting features of the atrial myocytes (high density of mitochondria dispersed in cells and secretory vesicles, left) and features of the ventricular myocytes (well-structured sarcomeres with Z band and myofibrils containing glycogens, right). These morphological features are similar to those of the in vivo embryonic hearts. M mitochondria, SV secretory vesicles, G Golgi complex, N nucleus, RER ribosomal endoplasmic reticulum. A (yellow), atrium; V (red), ventricle in the insets. Scale bar: 400 nm. d Gene expression pattern of atrium- and ventricle-like structures in HOs. The HOs cultured for 13 days (n = 3) were surgically dissected to each atrium- and ventricle-like parts for RNA-seq analysis. Specific genes showing the similar chamber expression patterns in HOs and E11.5H. See also Supplementary Fig. 6b. Source data are provided as a source data file. e Ventricular- and atrial-specific gene expression profiles in dissected HOs and E11.5H based on the 951 differentially expressed genes (−1 < log2-fold change > 1, adjusted P value < 0.05) between atria and ventricles of E11.5H.