DISTRIBUTION OF NESTIN IN THE DEVELOPING MOUSE LIMB BUD IN-VIVO AND IN MICRO-MASS CULTURES OF CELLS ISOLATED FROM LIMB BUDS

Early skeletal muscle development is accompanied by changes in the com position of the cytoskeleton. In this report we analyze the distributi on of the intermediate filament nestin in the developing mouse limb bu ds in vivo and in mesenchymal cells isolated from limb buds in vitro. The subcellular distribution of nestin mRNA and protein in muscle cell s was also analyzed. We find a shift in nestin expression during early limb bud development. At embryonic day 11 (E11), low levels of nestin (protein) were expressed in the mesenchymal cells of the developing l imb bud. Later, nestin mRNA and protein were down-regulated in the mes enchymal condensations undergoing chondrogenesis (E12 and E13), but re mained expressed predominantly in the ectodermal cells and in the diff erentiating myoblasts. At E18, only muscle fibres, endothelial cells a nd nerves were nestin positive. This shift in expression was reproduce d in vitro, in micro-mass cultures of mesenchymal cells. In E11 cultur es, nestin protein was initially expressed in all cells. Upon formatio n of cartilage foci (after 2-3 days in culture), nestin immunoreactivi ty was not observed in cartilage, and low levels were detected in the cells located between the foci. A subpopulation of mono- and multinucl eated cells, peripheral to the cartilage nodules, expressed the muscle -specific intermediate filament desmin protein together with high leve ls of nestin protein. The proportion of nestin-expressing cells could be changed by addition of specific signalling molecules. Insulin-like growth factors I and II (IGF I and II) increased the number of nestin- positive cells, while basic fibroblast growth factor (FGF) reduced the number of nestin-expressing cells. Finally, we present evidence for a different subcellular localization of nestin protein and mRNA: the mR NA is predominantly located in the ends of the muscle cell, whereas th e protein is found in the central region. Intracellular localization o f nestin mRNA may constitute an additional level of regulation of the cytoskeleton during muscle development.