neurons contain AChR-aggregating molecules antigenically related to agrin, and they are present at the time neuromuscular synapses are beginning to form. Discussion We find that the cell bodies of motor neurons stain with anti-agrin mAbs as does the synaptic basal lamina at neuromuscular junctions, and that the staining is concentrated in the Golgi apparatus, which processes proteins for secretion. We also provide evidence that motor neurons contain agrin-like AChR/AChE-aggregating molecules. Taken together, these findings support our hypothesis, as proposed by Nitkin et al. (1987), that motor neurons synthesize agrin or agrin-like AChR/AChE-aggregating molecules and release them at their axon terminals to become incorporated into the basal lamina of the synaptic cleft, and that these molecules account for the synaptic basal lamina's ability to induce and maintain AChR aggregates on muscle fibers. Our evidence that such molecules are present in the cell bodies of motor neurons in embryos and normal adults suggests further that they also account for the motor neuron's ability to cause the formation of postsynaptic specializations on mature muscle fibers. Our hypothesis does not rule out a role for other neuron-derived factors in the formation of the postsynaptic apparatus, such as regulating the levels of AChRs. In this regard, electromechanical activity, ARIA, and CGRP are discussed by Nitkin et al. (1987). An alternative interpretation of our observation that the cell bodies of motor neurons contain agrin-like molecules is that such molecules are produced and secreted by muscle fibers and/or Schwann cells and are taken up by motor neurons. However, there is no evidence that proteins secreted by Schwann cells, muscle fibers, or other target cells and internalized by Figure 5 (see facing page) Extracts of motor neuron-containing regions of the CNS of Torpedo, frog, and chick contain AChR-aggregating activity, which is immunoprecipitated by anti-agrin mAbs. Each of several different mAbs immunoprecipitated AChR-aggregating activity from the Torpedo electric lobe. mAb 6D4, which stains. neuromuscular junctions and motor neurons in Torpedo but not in frog and chick, immunoprecipitates AChR-aggregating activity from the electric lobe and spinal cord of Torpedo, but not from the spinal cords of the other two species. On the other hand, mAbs 5B1 and 3B5, which do stain neuromuscular junctions in frog and chick, also immunoprecipitated the activity in the extracts from these two species. Such findings would be expected if the active molecules were identical or closely related to the molecules that stain in motor neurons and at the neuromuscular junction. Data expressed as mean ± S.E.M.; the number of observations is given in parentheses above the bars. Our methods of measuring activity and of immunoprecipitation are given in Godfrey et al. 1984 and Reist et al. 1987. neurons appear in the neuron's Golgi apparatus, although certain plant lectins that bind tightly to membrane glycoproteins have been shown to appear in the Golgi apparatus of neurons after internalization (Gonatas et al. 1975). The hypothesis that agrin, an extracellular matrix molecule, mediates the nerve-induced formation and maintenance of AChR and AChE aggregates on muscle fibers raises many questions. For example, does agrin trigger the formation of the entire postsynaptic apparatus? The postsynaptic apparatus consists of several components in addition to the high concentration of AChRs and AChE. They include high concentrations of heparan sulfate proteoglycan and butyrylcholinesterase on the cell surface, of a 43 kD protein associated with the cytoplasmic domain of the AChR, and of cytoskeletal elements and infoldings of the plasma membrane (junctional folds). Studies are underway to determine whether agrin-induced patches of AChR and AChE have each of these components. Already, the patches have been found to contain a high concentration of heparan sulfate proteoglycan, butyrylcholinesterase, and the 43 kD protein (Smith et al. 1987; Wallace 1987). Are there agrin or agrin-like molecules at neuron-to-neuron synapses? Indeed, in unpublished experiments we have detected AChR-aggregating activity in extracts from regions of the Torpedo and frog brain that contain few motor neurons. The specific activity of such molecules from these regions of Torpedo brain was slightly above background, but the activity from frog brain was nearly as great as from the spinal cord, apparently too great to be accounted for by the presence of the relatively few motor neurons. The active molecules in the frog brain extracts were immunoprecipitated with anti-agrin mAbs, indicating that they are antigenically similar to agrin. We have not observed anti-agrin mAb staining in nonmotor neurons or at neuron-toneuron synapses. It may well be that agrin-like molecules are produced by many types of neurons to both induce and maintain the formation of postsynaptic specializations at their synapses but are too low in concentration to be detected by our staining techniques. Do agrin-like molecules play a role in the aggregation of cell surface proteins at sites other than synapses? We have previously reported that, in frog, anti-agrin mAbs stain the external surface of axonal membranes at nodes of Ranvier (Reist et al. 1987), sites where sodium channels are aggregated. We also have reported that the basal lamina between capillary endothelium and astrocyte endfoot processes in the CNS stains (Magill-Solc and McMahan, submitted); the astrocyte endfoot processes have a high concentration of potassium channels (Newman 1986). Clearly, the discovery of agrin may have several interesting consequences. Acknowledgments These studies were supported by National Institutes of Health grant NS14506, grants from the Wills Foundation, the Weingart Foundation, Mr. Keith Linden, and the Isabelle M. Niemela fund. C. Magill-Solc was funded by a National Science Foundation predoctoral fellowship and by NIH training grant NS07158. REFERENCES Anglister, L., and McMahan, U.J. Basal lamina directs acetylcholinesterase accumulation at synaptic sites in regenerating muscle. Journal of Cell Biology 101:735-743, 1985. Burden, S.J.; Sargent, P.B.; and McMahan, U.J. Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of nerve. Journal of Cell Biology 82:412-425, 1979. Dohrmann, U.; Edgar, D.; Sendtner, M.; and Thoenen, H. Muscle-derived factors that support survival and promote fiber outgrowth from embryonic chick spinal motor neurons in culture. Developmental Biology 118:209-221, 1986. Godfrey, E.W.; Nitkin, R.M.; Wallace, B.G.; Rubin, L.L.; and McMahan, U.J. Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells. Journal of Cell Biology 99:615-627, 1984. Gonatas, N.K.; Steiber, A.; Kim, S.U.; Graham, D.I.; and Avrameas, S. Internalization of neuronal plasma membrane ricin receptors into the Golgi apparatus. Experimental Cell Research 94:426-431, 1975. Landmesser, L., and Morris, D.G. The development of functional innervation in the hindlimb of the chick embryo. Journal of Physiology 249::301-326, 1975. Magill, C.; Wallace, B.G.; and McMahan, U.J. Molecules similar to agrin are concentrated in motor neurons. Soc. Neurosci. Abstr. 13:106.4, 1987. Magill-Solc, C., and McMahan, U.J. Motor neurons contain agrin-like molecules. Journal of Cell Biology, submitted. McMahan, U.J., and Slater, C.R. The influence of basal lamina on the accumulation of acetylcholine receptors at synaptic sites in regenerating muscle. Journal of Cell Biology 98:1453-1473, 1984. Newman, E.A. High potassium conductance in astrocyte endfeet. Science 233:453454, 1986. Nitkin, R.M.; Smith, M.A.; Magill, C.; Fallon, J.R.; Yao, Y.-M.M.; Wallace, B.G.; and McMahan, U.J. Identification of agrin, a synaptic organizing protein from Torpedo electric organ. Journal of Cell Biology 105:2471-2478, 1987. Reist, N.E.; Magill, C.; and McMahan, U.J. Agrin-like molecules at synaptic sites in normal, denervated and damaged skeletal muscles. Journal of Cell Biology 105: 2457-2469, 1987. Smith, M.A.; Wallace, B.G.; Yao, Y.-M.M.; Schilling, J.W.; Snow, P.; and McMahan, U.J. Purification and characterization of agrin. Soc. Neurosci. Abstr. 13:106.5, 1987. Smith, M.A.; Yao, Y.-M.M.; Reist, N.E.; Magill, C.; Wallace, B.G.; and McMahan, U.J. Identification of agrin in electric organ extracts and localization of agrin-like molecules in muscle and central nervous system. Journal of Experimental Biology 132:223-230, 1987. Wallace, B.G. Aggregating factor from Torpedo electric organ induces patches containing acetylcholine receptors, acetylcholinesterase, and butyrylcholinesterase on cultured myotubes. Journal of Cell Biology 102:783-794, 1987. Wallace, B.G.; Nitkin, R.M.; Reist, N.E.; Fallon, J.R.; Moayeri, N.N.; and McMahan, U.J. Aggregates of acetylcholinesterase induced by acetylcholine receptor-aggregating factor. Nature 315:574-577, 1985. Glycoproteins That Regulate Axon Growth L.F. Reichardt, J.L. Bixby, D.E. Hall, M.J. Ignatius, Department of Physiology/Howard Hughes Medical Institute The central question that motivates our research is how do neurons extend processes to their targets and establish appropriate synapses during development and regeneration? Neurons of one type develop reproducibly in different embryos, and molecules in their extracellular environment are clearly important for directing much of this development. In particular, molecules that regulate growth cone motility are believed to play a central role in the establishment of specific axonal pathways. In many vertebrate and invertebrate species, growth cones are influenced by contacts with the surfaces and secreted products of other neuronal and nonneuronal cells (see Bentley and Caudy 1983; Raper et al. 1984; Kapfhammer and Raper 1987). A major challenge in developmental neurobiology is to identify these molecules and their neuronal receptors. Experiments in vitro have provided insights into the molecular mechanisms of neuronal process outgrowth. Three distinct classes of proteins that promote axonal outgrowth have been described: diffusible molecules, such as trophic factors and chemotropic agents (cf. Davies et al. 1987; Lumsden and Davies 1986), constituents of the extracellular matrix (ECM) (cf. Manthorpe et al. 1983), and cell adhesion molecules (CAMs) (Chang et al. 1987; Bixby et al. 1987, 1988; Lagenauer and Lemmon 1987; Neugebauer et al. 1988; Tomaselli et al. 1988a; Seilheimer and Schachner 1988). Several glycoprotein constituents of the ECM have been localized in embryos at positions appropriate for influencing neuronal behavior in vivo and have also been shown to interact with neurons in vitro. Several of these, notably fibronectin (FN), laminin (LN), and collagen types I and IV, promote neuronal adhesion and axon growth (reviewed in Lander 1987). The effects of FN and the two collagens on growth cone motility are modulated by appropriate trophic factors, such as nerve growth factor (NGF) (cf. Campenot 1977). At least one of these ECM glycoproteins, tenascin, has fascinating inhibitory effects on the adhesion and migratory behavior of many cells (Crossin et al. 1986; Tan et al. 1987; Chiquet-Ehrismann et al. 1988). Others, e.g., vitronectin (VN) and thrombospondin, are likely to be products of cells contacted by neurons, but have at present no well-defined functions in neuronal development (cf. O'Shea and Dixit 1987). Among these ECM constituents, the glycoprotein LN is particularly interesting because it has dramatic stimulatory effects on neuronal survival, process outgrowth, and expression of neurotransmitters (cf. Manthorpe et al. 1983; Lander et al. 1983; Edgar et al. 1984, 1988; Calof and Reichardt 1985; Acheson et al. 1986). Studies of LN expression suggest that it is likely to influence axon growth and other aspects of neuronal differentiation during development and regeneration in vivo. LN immunoreactivity has been detected where axonal pathways are established in several regions of both the central and peripheral nervous systems (Liesi 1985a, b; Rogers et al. 1986; Cohen et al. 1987; Letourneau et al. 1988; McLoon et al. 1988). During peripheral nerve regeneration, growth cones of neurons contact channels of basal lamina that contain LN (e.g., Schwab and Thoenen 1985). An antibody to a LN-proteoglycan complex partially inhibits neurite outgrowth on sections of sciatic nerve in vitro and appears to inhibit the regeneration of sympathetic fibers in vivo (Sandrock and Matthew 1987a, b). Neuronal responses to LN and other ECM constituents must depend on specific cell surface receptors. A superfamily of cell surface heterodimers, called integrins, has recently been identified on a variety of adherent cells, and these have been shown to function as receptors for several ECM constituents, including FN, VN, fibrinogen, von Willebrand factor, LN, several collagens, and at least one cell surface protein, I-CAM, an immunoglobulin homolog (cf. Staunton et al. 1988; reviewed in Hynes 1987; Ruoslahti and Pierschbacher 1987). Each of these receptors appears to be a dimeric complex in which one of several homologous a subunits associates noncovalently with a ẞ subunit to form a functional receptor. At present, three integrin a/ß heterodimer families, distinguished by distinct but homologous ẞ subunits (B1, B2, and ß3) have been identified (for nomenclature, see Hynes 1987). Members of the ẞ1 and ẞ3 families are distributed on a wide variety of cell types, while ẞ2 heterodimers appear to be restricted to cells in the hematopoietic lineage. The binding specificity of an integrin receptor depends on the particular combination of a and ẞ subunits. Some integrin a/ß heterodimers appear specific for one ECM protein (e.g., the FN receptor, aFN/ẞ1). Other heterodimers bind several ligands (e.g., the platelet ECM receptor, aь/ßз). The function of each heterodimeric receptor depends on the presence of divalent cations (cf. Cheresh et al. 1987; Marlin and Springer 1987), and those a subunits that have been sequenced (αFN, άVN, αIIb, αLFA-1, αMac-1) contain three to four putative divalent cation binding sites (compared in Pytela 1988). Divalent cation-dependent binding |