Fig. 4. New outflow tract myocardium is derived from splanchnic mesoderm caudal and adjacent to the outflow. (A) Diagram of a stage 14 chick embryo showing the location of the secondary cardiac outflow field (yellow asterisk), which is located at this stage adjacent to the second arch (green). (B) Confocal image of a stage 22 chick embryo injected into the secondary heart field just caudal to the outflow tract with mitotracker at stage 14. Two new segments, a proximal fluorescent segment (asterisk) and a more distal non-fluorescent segment (star), were added to the primary tube between the time of injection at stage 14 and acquisition of this image at stage 22. The fluorescent spot (white arrow) cranial to the outflow tract shows the needle entry site for the mitotracker injection, which was originally posterior to the outflow tract. (C) The secondary heart forming region was removed from a stage 14 quail embryo and placed in the comparable site of a chick embryo at a similar stage. A transverse section at the level of the distal outflow myocardium shows two small vesicles with quail cells (QCPN antibody, brown) adjacent to the outflow tract (arrow). (D) Higher magnification of the vesicles reveals an inner lumen lined with quail cell epithelium, surrounded by MF20-positive (purple) myocardial cells. Scale bars: 100 µm.