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Fig. 6 Muscle pioneer cells are a subset of adaxial cells. (A,B) Transverse sections of ~22 h embryos in each of which an adaxial cell had been injected as in Fig. 2. Sections were immunostained with the 4D9 Engrailed antibody, which labels the muscle pioneers (green). One out of 7 injected adaxial cells became Engrailed-labeled muscle pioneers. (A) An adaxial cell (red) that became a superficial muscle cell that is not a muscle pioneer (green, arrowhead). (B) An adaxial cell (arrowhead, yellow due to double labeling in green and red fluorescence) that differentiated into an Engrailed-expressing muscle pioneer. (C) Transverse section through a 24 h embryo immunolabeled with the S58 myosin antibody (green) to mark the superficial muscle cells derived from adaxial cells and the 4D9 engrailed antibody (red) to label the muscle pioneers. The Engrailed-labeled muscle pioneers (arrowheads, bright red nuclei) are a subset of the superficial muscle cells. Other somite cells near the muscle pioneers also weakly express Engrailed (arrow, Hatta et al., 1991a), these cells are unlabeled by the S58 antibody and are not part of the superficial muscle cell population derived from adaxial cells. (D) Higher magnification view of the region of the myotome containing muscle pioneers. The two cells that express Engrailed at high levels (arrowheads, bright red nuclei), are also S58 positive (green). Another somite cell that weakly expresses Engrailed, not a muscle pioneer, does not express S58 and is not part of the adaxialderived superficial muscle cells (arrow, nucleus light red). In these transverse sections, dorsal is up. Scale bars: A-C, 50 μm, D, 10 μm.