Sequential Expression and Differential Function of Multiple Adhesion Molecules during the Formation of CerebeUar Cortical Layers

We have correlated the times of appearance of the neural cell adhesion molecule (N-CAM), the neuron-glia cell adhesion molecule (Ng-CAM), and the extracellular matrix protein, cytotactin, during the development of the chicken cerebellar cortex, and have shown that these molecules make different functional contributions to granule cell migration. Immunofluorescent staining showed distinct spatiotemporal expression sequences for each adhesion molecule. N-CAM was present at all times in all layers. However, the large cytoplasmic domain polypeptide of N-CAM was always absent from the external granular layer and was enriched in the molecular layer as development proceeded. Ng-CAM began to be expressed in the premigratory granule cells just before migration and later disappeared from cell bodies but remained on parallel fibers. Cytotactin, which is synthesized by glia and not by neurons, appeared first in a speckled pattern within the external granular layer and later appeared in a continuous pattern along the Bergmann glia; it was also enriched in the molecular layer. After we established their order of appearance, we tested the separate functions of these adhesion molecules in granule cell migration by adding specific antibodies against each molecule to cerebellar explant cultures that had been labeled with tritiated thymidine and then measuring the differential distribution of labeled cells in the forming layers. Anti-N-CAM showed marginal effects. In contrast, anti-Ng-CAM arrested most cells in the external granular layer, while anti-cytotactin arrested most cells in the molecular layer. Time course analyses combined with sequential addition of different antibodies in different orders showed that anti-NgCAM had a major effect in the early period (first 36 h in culture) and a lesser effect in the second part of the culture period, while anti-cytotactin had essentially no effect at the earlier time but had major effects at a later period (18-72 h in culture). The two major stages of cerebellar granule cell migration thus appear to be differentially affected by distinct adhesion molecules of different cellular origins, binding mechanisms, and overall distributions. The results indicated that local cell surface modulation of adhesion molecules of different specificities at defined stages and sites is essential to the formation of cerebellar cortical layers. Prominent feature during the development of the nervous system is the orderly formation of horizontal cellular and fibrous layers such as those found in the cerebeUar and the cerebral cortexes (Cowan, 1982). This morphogenetic process depends heavily on ordered cell migration in a radial direction, which depends in turn upon complex interactions among neurons and glia (Rakic, 1971). It has recently been suggested that the spatiotemporal regulation of adhesion among cells and substrates mediated by various adhesion molecules plays a regulatory role in determining the borders of histological layers in both neural and nonneural tissues, particularly by affecting cell movement (Edelman, 1985). To test this hypothesis in the central nervous system, it is useful to compare the functions of a set of adhesion molecules of different specificities, binding mechanisms, and cellular origins. By application of in vitro binding assays, two neuronal cell adhesion molecules, the neural cell adhesion molecule (N-CAM) ~ and the neuron-glia cell adhesion molecule (Ng-CAM), have been identified, isolated, and structurally analyzed (for review see Edelman, 1986). Each of these structurally and functionally different neuronal membrane glycoproteins has a distinct binding specificity and binding mechanism. Both can mediate neuron-neuron and neuron-glia interactions, but each contributes differentially to different functions in various regions of the nervous system, depending upon dynamic modulation of its local expression (Hoffman et al., 1986). A substrate adhesion molecule, cytotactin, that is synthesized by glia but not by neurons 1. Abbreviations used in this paper: EGL, external granular layer; IGL, internal granular layer; ld, large cytoplasmic domain of N-CAM; ML, molecular layer; N-CAM, neural cell adhesion molecule; Ng-CAM, neuron-glia adhesion molecule; PLZ, proliferative zone; PMZ, premigratory zone. © The Rockefeller University Press, 0021-9525/87/02/331/12 $1.00 The Journal of Cell Biology, Volume 104, February 1987 331-342 331 on M ay 2, 2017 D ow nladed fom Published February 1, 1987