Fig. 3. At stage 16 HNK-1-positive cells express a myocardial phenotype. Double labeling by immunohistochemistry to show the relationship of HNK-1-positive cells (green) to myocardial cells (red) in the cardiac outflow and inflow tracts. The poles of the heart exhibit a gradient of HNK-1 and myosin protein expression during looping, as seen in transverse sections of the outflow and inflow poles of a stage 16 chick embryo double labeled for myosin. (A) The distal outflow tract shows HNK-1-positive/MF20-negative cells in the splanchnic mesoderm and HNK-1-negative/MF20-positive cells in the myocardium, separated by myocardium that is positive for both HNK-1 and MF20 (yellow). (B) The splanchnic mesoderm caudal to the attachment of the outflow tract with the pharynx (arrow) is HNK-1 positive/MF20 negative. (C) The atrium displays a gradient of overlapping protein expression, similar to that seen in the outflow myocardium.