(A) Frontal view of an Nkx2.5cre/+; Rosa26Rtdtomato embryo at EHF stage. (A’) 3D reconstruction of the tdtomato signal in the cardiogenic area. Signal from tdtomato+ endothelial cells identified by shape was manually masked. See also Video 1. (B–G) Immunostaining for cTnnT (red) and Dapi (blue) showing six consecutive stages during cardiac differentiation (B–D) and HT morphogenesis (E–G). (B) At EHF cTnnt is initially not detectable. (C–D) During early somitogenesis, cTnnT signal becomes detectable in the cc. Insets in (B–D): magnification of single optical sections showing cTnnT localization and cell shape. (C’–G’ and E’’–G’’) Corresponding 3D renderings from cTnnT signal reconstruction. Red arrows in (E’–G’) highlight the dorsal closure of the HT. Yellow arrow in G’’ highlights the arterial pole (prospective RV). See also Video 2. (H) Quantification of the arterial pole/RV length in the open HT (41.4 ± 14.0 μm, n = 5) and after dorsal closure (109 ± 43.44 μm, n = 7), mean ±SD, p=0.0025. (I) Quantification of the cardiac volume at the different stages of HT development. (Initial cc: 1.63.106 μm3 ± 0.13, n = 4, cc: 2.89.106 ± 0.37 μm3, n = 3, transversal HT: 3.367. 106 μm3 ± 0.95, n = 5, open HT: 4.29.106 μm3 ± 1.08, n = 6, linear HT: 6.37. 106 μm3 ± 1.01, n = 5, mean ±SD). p-Values are indicated on the graph. (J) Immunostaining of an Nkx2.5eGFP embryo for PH3 (red) and Dapi (blue) at HT stage, showing proliferative cells in the ventricle. Scale bars: 100 μm.