Studies on extracellular matrix components that promote neurite outgrowth.

Abstract

Studies on Extracellular Matrix Components That Promote Neurite Outgrowth A.D. L ANDER, K. TOMASELLI, A.L. CALOF, AND L.F. REICHARDT Departmel'lt of Physiology, Division of Neurobiology, University of California, San Francisco, San Francisco, California 94143 An important determinant in the development of mul- ticellular organisms is the extracellular matrix (ECM) upon which cells attach, migrate, and differentiate. It is likely that a class of substances that affect neuronal de- velopment will be found to be associated with the ECM . During early stages of neuronal development, strong spatial and temporal correlations are seen be- tween the appearance of fibronectin and migration of granule cells to form the external granule cell layer in the cerebellum (Hatten e t al. 1982) and migration of neural c rest cells to form the variety of tissues derived from the crest (Thiery et al. 1982). When cerebellar granule cells or neural c rest cells are cultured in vitro, their abilities to adhere to and migrate on fibroncc tin substrata correlate temporally with their migratory be- haviors in vivo (Hatten et al. 1982; Rovasio e t al. The ECM is also important in later stages of neuronal development. The interaction between a g rowth cone and its substratum can determine the rate at which it grows a nd the paths it follows in vitro (Letourneau 1975). In vivo, axons may also follow routes deter- mined by the substratum (Katz and Lasek 1979). In some cases , this reflects the association of axo ns with already oriented cells, e.g., radial glia in the cerebel- lum (Rakic 1974) and pioneer fibers in Daphnia (Lev- inthal et al. 1976). There are several facto rs that, when bound to culture substrata, stimulate outgrowth from particular classes of neurons. Of these, fibronectin and laminin (Akers et al. 1981 ; Baron-Van Evercooren et al. 1982) are known components of the ECM in vivo. One group of factors is derived from cultured cells (Collins 1978; Adler et al. 1981 ; Coughlin et al. 1981 ; Lander et al. 1982), and, when attac hed to a substratum, these fac- tors promote profuse and rapid neurite outgrowth . Whe n tested o n sympathetic neurons , neurite outgrowth is seen even in the absence of nerve growth factor (NGF). The properties of these factors are discussed in this paper. secreted by corneal endothelial cells in vitro (Fujii et al. 1982; Lander ct al. 1982) . PC12 cells and rat sympa- thetic neurons exte nd neurites on the ECM even in the absence of NGF, and process outgrowth is no t pre- vented by preincubation of ECM with antiserum to NGF or inclusion of NGF antiserum in the culture medium. The behavior of neurons o n ECM was unusual in other ways (Table l). Neurites appeared earlie r and grew more rapidly on ECM than o n polylysine-coated plastic. Within 6 hours after plating onto ECM , more than 80% of the neurons had neurites, many of them several cell diameters in length. In contrast, neurons plated o nto polylysine and cultured with NGF had very few processes by 6 hours or even 12 hours , although most cells extended neurites by 24 hours . Although neurites appeared early and grew rapidly o n ECM in the absence of NGF, their rate of growth slo wed dramatically after 24 hou rs and cell viability (estimated by morphological criteria) fell steadily there- after. By 72 hours, less than 20% of the neurons ap- peared alive; none survived over 5 da ys. Massive cell death could be avoided only if NGF was present in the culture medium. T hen, good viability was maintained for over a week, the longest time assayed. Thus, ECM can substitute for NGF in inducing short-term process outgrowth, but not in maintaining long-term neuronal viability . Table I. Comparison of the Response of Neurons to NGF and ECM Hours after plating Substratum Medium (a) Percent neurite outgrowth Poly lysine ECM +NGF -NGF (b) Percent neuronal survival Poly lysine +NGF -NGF ECM Rat sympathetic neurons cultured with NGF on polylysine-coated tissue culture plastic and without NGF on ECM were fixed at various times after plating. Neurite outgrowth was measured by counting ran- dom fields and detennining the percentage of presumptive neurons wi1h neurites. Survival was estimated crudely as the percentage of presumptive neurons lacking morphological signs of cell death or in· jury . including cell swelling, loss of adhesiveness. retraction of neurites. and accumulation of intracytoplasmic granules. Over 100 neurons were counted for each point shown above. RESULTS Extracellular Matrix Promotes Neurite Outgrowth Our attention was originally drawn to the ECM by observations that PC12 (pheochromocytoma) cells, rat sympathetic neurons, and other neuronal cell types ex- hibit profuse and rapid process outgrowth on the matrix