Endothelial differentiation gene (Edg) proteins are G-protein-coupled receptors activated by lysophospholipid mediators: sphingosine-1-phosphate (S1P) or lysophosphatidic acid. We show that in the CNS, expression of Edg8/S1P5, a high-affinity S1P receptor, is restricted to oligodendrocytes and expressed throughout development from the immature stages to the mature myelin-forming cell. S1P activation of Edg8/S1P5 on O4-positive pre-oligodendrocytes induced process retraction via a Rho kinase/collapsin response-mediated protein signaling pathway, whereas no retraction was elicited by S1P on these cells derived from Edg8/S1P5-deficient mice. Edg8/S1P5-mediated process retraction was restricted to immature cells and was no longer observed at later developmental stages. In contrast, S1P activation promoted the survival of mature oligodendrocytes but not of pre-oligodendrocytes. The S1P-induced survival of mature oligodendrocytes was mediated through a pertussis toxin-sensitive, Akt-dependent pathway. Our data demonstrate that Edg8/S1P5 activation on oligodendroglial cells modulates two distinct functional pathways mediating either process retraction or cell survival and that these effects depend on the developmental stage of the cell.

Interactions between recognition molecules on the surface of neuronal growth cones and guidance cues present in the local cellular environment are thought to account for the growth of neurites in the highly stereospecific manner that contributes to correct target cell innervation. In vitro assays have been used to identify candidate molecular components of this system, either directly by demonstrating their ability to promote neurite outgrowth, or indirectly by the ability of specific antibodies to inhibit neurite outgrowth. The role of the neural cell adhesion molecule (NCAM) in pathway finding is not fully understood. Some immunological studies support a positive role; others do not, and it has been reported that purified NCAM does not support neurite outgrowth. We have previously shown that an arbitrary biochemical index of neurite outgrowth, the relative level of immunoreactive neurofilament protein, is increased when human and rat dorsal root ganglion neurons are cultured on monolayers of cells expressing transfected human NCAM. But, the complexity of growth precluded a simple morphological analysis and we did not determine the 'dose-response' relationship between NCAM expression and neuronal response. Here, we report on the morphology of rat cerebellar neurons cultured on monolayers of 3T3 cells transfected with complementary DNAs encoding all of the main NCAM isoforms found in cells such as astrocytes, Schwann cells and skeletal muscle. The data indicate that both transmembrane and glycosyl-phosphatidylinositol linked NCAM isoforms are potent substrates for neurite extension. A critical threshold value of NCAM expression is required for increased neurite outgrowth. Above this threshold, small increases in NCAM induce substantial increases in neurite outgrowth.

Letter to the Editorwith phylogenetic tree analyses, allow at present for the Unified Nomenclature for the designation of eight subclasses into which all known (CEL)Sema-1a for C. elegans, etc.). Given the difficulty * Howard Hughes Medical Institute, Department of Molecular and in firmly establishing ortholog relationships among in-

The fusion of mononucleate precursor myoblasts to form the multinucleated skeletal muscle fibre is proceeded by a series of complex cell-cell interactions but the cell-surface molecules involved in these events have not been characterized. During myogenesis in vivo and in vitro, expression of the neural cell adhesion molecule (NCAM) undergoes an isoform transition that precisely correlates with terminal myoblast differentiation and myotube formation. Altered processing of RNA results in the replacement of the transmembrane NCAM (relative molecular mass, 145,000 (145K) in proliferating myoblasts by a predominant 125K NCAM form linked to glycosyl phosphatidylinositol in myotubes. We now report that mouse myoblasts transfected to constitutively express the human muscle-specific 125K glycosylphosphatidylinositol-linked NCAM isoform more readily fuse to form myotubes. This suggests that NCAM plays a part in myoblast fusion and that the isoform switch may promote this function.

Abstract. We have used monolayers of parental 3T3 cells and 3T3 cells expressing one of three transfected cell adhesion molecules (CAMs) (NCAM, N-cadherin, and L1) as a culture substrate for rat cerebellar neurons. A number of tyrosine kinase inhibitors have been tested for their ability to inhibit neurite outgrowth over parental 3T3 monolayers which we show to be partly dependent on neuronal integrin receptor function, as compared with neurite outgrowth stimuluted by the above three CAMs. Whereas genistein (100 gM), lavendustin A (20 gM), and tyrphostins 34 and 47 (both at 150 gM) had no effect on integrin dependent or CAM stimulated neurite outgrowth, the erbstatin analogue (10-15 #g/ml) and tyrphostins 23 and 25 (both at 150 gM) specifically inhibited the response stimulated by all three CAMs. CAM stimulated neurite outgrowth can be accounted for by a G-proteindependent activation of neuronal calcium channels; experiments with agents that directly activate this pathway localized the erbstatin analogue site of action upstream of the G-protein and calcium channels, whereas tyrphostins have sites of action downstream from calcium channel activation. These data suggest that activation of an erbstatin sensitive tyrosine kinase is an important step upstream of calcium channel activation in the second messenger pathway underlying the neurite outgrowth response stimulated by a variety of CAMs, and that this kinase is not required for integrindependent neurite outgrowth.