[Cholinesterase in the development of the chick limb : II. Enzyme activity and locomotory behavior of the presumptive cartilage cellsin vitro].

From our previous work we have put forward the hypothesis that cholinesterase activity in embryonic cells is related to morphogenetic movements. Therefore, the locomotory behavior of mesenchymal cells differentiating into cartilage by passing through a phase of Cholinesterase activity was analysedin vitro.Mesenchymal cores of chick limb buds stage 23/24 were partially disaggregated and cultured in plastic tissue culture dishes (Fig. 1). Within 31/2 to 5 days aggregates of mesenchymal cells differentiated into cartilage nodules surrounded by myoblasts (Figs. 2, 3 and 5). The cartilaginous nature of the nodules was confirmed by electron microscopy (Figs. 6 and 7). During the culture period serial photographs (24×24 mm) were taken (Tables 1-3). After formalin fixation the histochemical Cholinesterase reaction was carried out inside the culture dishes. Positive and negative cells were identified in the live serial photographs and their locomotory behavior was analysed.Initially the cells behaved like fibroblasts. Movements were regulated by contact inhibition, resulting in radial outward migration within the mesenchymal aggregates. In this first phase of development there was no cholinesterase activity. After 12 to 48 hours in culture however ChE-positive cells could be detected. Positive cells, appearing within a monolayer, detached from the bottom of the culture dish and crawled onto neighboring cells (Figs. 8a and b). In the periphery of the aggregates radial outward migration slowed down considerably. In the center short non-directional movements of positive cells could be observed, frequently leading to overlayering of cell bodies.In the third stage of development the ChE-positive cells stopped moving and transformed into cartilage cells (Fig. 9a and b). Finally, ChE-activity disappeared from the differentiated cartilage cells.From the difference in locomotory behaviour of negative and positive cells it is concluded that the appearance of Cholinesterase is accompanied by a change in the adhesive properties of the cells. An increase in cell adhesiveness enables the ChE-positive cells to detach from the bottom of the culture dish and to establish a new equilibrium of contact inhibition inside the cellular aggregates. This seems to be a prerequisite for the secretion of extracellular matrix and development of firm cell contacts. In vivo cartilage differentiation presumably also starts with an increase in cell adhesiveness in the presumptive cartilage cells. This provokes pseudopodial rearrangements leading to the condensation and demarkation of the cartilage anlagen. The change in adhesiveness is accompanied by Cholinesterase activity.