A scanning electron microscope study of the early limb-bud in normal andtalpid3mutant chick embryos

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Abstract
Scanning electron microscope studies, supported by transmission electron microscope and light microscope observations, have been made of the wing-bud apex in the region of the apical ectodermal ridge (AER) in both normal and talpid3 mutant embryos. In normal embryos: the ectoderm consists of two layers, the periderm and the basal layer, resting upon a basal lamina. The external surface of the periderm cells bears numerous villi, especially numerous at the cell boundaries. In the AER the cells of the basal layer are compacted into a fan-shaped form, remaining as a single layer, though displacement of their nuclei gives the appearance of a stratified epithelium; the periderm cells tend to round up and many are necrotic. Microfilaments and microtubules are present in greater numbers in the AER than in other ectoderm cells and are orientated along the long axis in the very narrow elongated cells which comprise the middle of the ridge. The mesenchymal cells of the mesoderm tend to be flattened, with extensive flattened surface areas separated from each other by long edges of lamellar cytoplasm, from which many long filopodial extensions arise. Often the cells are elongated, with the filopodia arising predominantly at the anterior and posterior ends. In talpid3 embryos: when these were compared with normals no differences were detected in the general ectoderm or in the AER, but in the mesoderm a statistical analysis revealed significant differences in the occurrence of sections through very fine cytoplasmic processes in transmission electron microscope micrographs. Taken in conjunction with visual examination of scanning electron microscope pictures the differences suggested that talpid3 cells have filopodia distributed more extensively around the cell, but that these extensions do not extend so far through the intercellular spaces or produce such fine terminal arborizations as in normal mesenchyme. At the ectodermal/mesodermal boundary there is a web of extracellular material under the basal lamina, separated from the underlying mesoderm by a very narrow gap in normal embryos. Fine cytoplasmic extensions from the mesenchyme cells extend across the gap towards the basal lamina and many are in contact with it. The gap is wider in talpid3 wing-buds and a statistical analysis confirmed that far fewer filopodia approach closely to the basal lamina or make contact with it. The significance of these findings in relation to problems of AER formation and activity and motility of mesenchymal cells in the wing-bud is discussed.