A quantitative study of the segmental distribution of somitic cells in the developing chick limb bud using laser-scanning confocal microscopy.

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Our aim was to map the segmental distribution of somitic cells in the limb before and after the fusion of these cells into myotubes. Somitic cells of the brachial somites were labeled by injection of DiI and DiO into the somitoceles of embryonic Day 2 (E2) embryos. The quantitative distribution of dye-labeled cells from injected somites was examined in whole mounts of E4 wing buds and in sections of E5 wing buds using confocal microscopy. Dye derived from cells of anterior brachial somites 16 and 17 was highly concentrated in the anterior half of E4 wings, whereas dye from posterior brachial somites 20 and 21 was concentrated in the posterior half of the wing. Not more than 14% (on average) of the dye in the wing was found outside the preferred half. Dye was equally dispersed in the anterior and posterior halves of the wing when the middle two somites contributing to the wing musculature, numbers 18 and 19, were injected. The total amount of dye in the whole limb at E4 was not significantly different after injection of similar amounts of dye into either pair of brachial somites 16/17, 18/19, or 20/21. In E5 embryos the distribution of labeled cells in the newly formed muscle masses, identified with antibody 2H2 to myosin light chains, was examined in cryostat cross-sections of the wing. Dye was more widely distributed in the wing than at E4. As much as 32% (on average) of the dye was found in the muscle mass outside the preferred half after administration of dye into the anterior or posterior pair of somites. We conclude that the branchial somites contribute similar numbers of cells to the wing musculature and that the segmental origin of these cells is rigidly maintained during their lateral migration into the limb. Cells from adjacent segments then "mix" when they fuse to form myotubes at E5. In addition, we found a significantly greater amount of dye per unit of muscle mass in the proximal compared with distal parts of the limb. We argue that this finding is due to division of myogenic cells during the course of their migration in the wing.