- Email: [email protected]
Modulators of neuronal migration and neurite growth
Modulators of neuronal migration and neurite growth Josef P. Kapfhammer University
of Zurich,
and Martin Zurich,
E. Schwab
Switzerland
A multitude of molecules have been identified over the past few years that promote neurite outgrowth in vitro. The concept that these molecules work mainly by providing an adhesive surface for neuronal growth cones has been challenged by evidence from recent experiments. Some of the substrate molecules have diverse actions on cell migration and neurite growth. In addition, there is now evidence that there are molecules that specifically inhibit growth cone locomotion. This has given rise to the hypothesis that growth cones integrate a variety of growth-promoting and inhibitory signals and translate them into directed locomotion.
Current
Opinion
in Cell Biology
1992, 4:863-868
Introduction
Adhesion
and neurite
Neuronal migration and neurite extension are basic mechanisms in development and regeneration of the nervous system. They depend on the availability of adequate substrates, typically provided by cellular surfaces or secreted molecules that constitute the extracellular matrix (ECM). In the last decade a variety of ECM and cell surface molecules have been identified that support and promote neuronal migration and neurite extension. In the nervous system most of these molecules are expressed in a spatially and temporally regulated manner, to form a complex pattern of widely overlapping expression domains, Many of these molecules support cell attachment as well as neurite outgrowth in zritro. The hypothesis that the pattern of neurite outgrowth is mainly detemlined by differential adhesion was originally widely accepted. Growth cones would always extend on the more adhesive substrate. Recent evidence, however, suggests that the mechanisms governing neuronal migration and neurite extension are much more complex. In particular, three new Iindings should be considered. First, the strength of adhesion between neuronal growth cones and their substrates shows little relation to neurite outgrowth and even neurite fasciculation. Second, some of the identified substrate molecules are multifunctional, i.e. they can promote or inhibit neurite outgrowth, depending on conditions. Lastly, molecules are now being characterized that specifically inhibit neurite growth.
In a classic tissue culture experiment, Letoumeau [l] demonstrated that outgrowing neurites choose between different substrates, preferring the more adhesive ones [ 21, Based on this and similar experiments, the concept of differential adhesion [3] became dominant in explaining pathfinding choices of neuronal growth cones and migrating neurons. This view has been challenged by recent experiments that used naturally occurring ECM molecules and cell adhesion molecules (CAMS) as substrates, instead of the artilicial substrates used in earlier experiments. For dorsal root ganglion growth cones it was shown that although the ECM molecule laminin is more potent than collagen for stimulating neurite outgrowth, it provides a weaker adhesive force [4], showing that the two activities are not directly related. Further evidence has recently been obtained from a study of neuronal migration on laminin and other ECM molecules [5-l. When a choice was provided, laminin was again preferred over other substrates by the migrating neurons and was the most effective substrate for neuronal migration. However, when assayed for adhesive forces it allowed only weak adhesion of neuronal cells. In fact, even when laminin was mixed with fibronectin at a tenth of the concentration, it actually decreased the adhesion of neurons to the substrate. Similar results were obtained for tenascin, which is a bad substrate for neuronal adhesion and spreading but supports neurite outgrowth [6,i’]. The opposite effect has been reported for a laminin isoform, s-laminin, and synthetic peptides containing an s-laminin-derived IRE (LeuArg-Glu) sequence [8*]. These peptides selectively me-
In this review we will briefly discuss these findings and propose a model of neuronal pathfinding that takes the combinatorial effects of growth promoting and inhibitory signals on neuronal growth cones into account.
Abbreviations adhesion molecule; CN%central nervous system; matrix; N-cadherin-neural cadherin; N-C-neural
CAM-cell ECM-extracellular @
Current
Biology
growth
Ltd
ISSN
0955-0674
CAM 863
864
Cell-to-cell
adhesion
and extracellular
matrix
diate attachment of NSC-34 cells (a cell !ine that has many properties of motoneurons), but inhibit neurite outgrowth from the same cells even in mixtures with ECM larninin. Lemmon et al. [9-l carried out a systematic study on the relation between adhesion of the growth cone, degree of neurite fasciculation and the rate of neurite outgrowth on defmed substrates. For the CAMS Ll and neural cadherin (N-cadherin), ECM laminin and the artificial substrate poly-lysine, they found that none of the three parameters were correlated, i.e. adhesion did not correlate with either growth rate or fasciculation and growth rate did not correlate with fasciculation. This indicates that adhesiveness, growth rate and fasciculation are probably independent and might involve different signalling and recognition mechanisms. It also challenges the widely held view [ 10,111 that the degree of neurite fasciculation is the result of an equilibrium between adhesive forces acting between growth cones and neurites on the one hand and growth cones, neurites and the substrate on the other. The different properties and effects of single substrate molecules described above could be due to different functional domains, neuronal receptors or response mechanisms. Indeed, ECM molecules and CAMS are large molecules, often with a defined domain structure that mediates various functional properties [12]. For the ECM molecule, tenascin, the ability for cell binding, neuronal migration and neurite outgrowth could be mapped to different parts of the molecule using a panel of monoclonal antibodies [ 13.1. However, laminin activities for cell attachment and neurite outgrowth have been mapped to individual proteolytic fragments [I41 and can both be evoked by small synthetic peptides [ 151. Fibronectin cell-binding and neuronal-migration domains have also been mapped to the same site by monoclonal antibodies [16]. In these cases different activities could still be mediated by different signal transduction mechanisms on the cell surface. The ongoing molecular dissection of the active sites on CAMS and ECM molecules, together with the study of cellular receptors and signal transduction mechanisms in neurons and growth cones, will eventually clarify the interaction between the processes described above. The idea that adhesion is the single most important molecular property mediating neurite outgrowth and neurite fasciculation is oversimplified and can no longer be upheld. Cell adhesion and extracellular molecules can be multifunctional
matrix
If different sites exist on one molecule for adhesion, cell spreading, neuronal migration and neurite outgrowth, such a molecule can be expected to have diverse actions, depending on the micro-environment and the type of receptors expressed by the neuron. One example of such a multifunctional molecule is tenascin [ 13*,17]. Purified tenascin inhibits neuronal attachment in z&-o, although once the neurons are attached there is good neurite outgrowth [ 18,191. When a choice of substrate is
available, however, neurites prefer to extend on laminin and fibronectin instead of tenascin. In a recent paper it was shown that while tenascin in substrate-bound form supported neurite outgrowth, in soluble form it reduced outgrowth on several substrates, including laminin and tenascin itself [20-l. The temporal and spatial expression pattern of tenascin both during development and regeneration of peripheral nerves and migration of neural crest cells suggests a supportive role of tenascin in neurite outgrowth and neuronal migration [ 18,211. In the cerebellum, antibodies to tenascin interfere with granule cell migration 1221, although tenascin, according to an electron microscope study, is not present at the site of interaction between granule cells and the Bergmann glia [ 231. In rodent barrel field cortex, tenascin is transiently expressed in the forming barrel walls after arrival of the thalamic fibers [ 241. These descriptive and mostly correlative data will have to be complemented with experiments using delined site-specific antibodies in order to understand the complex action of this molecule. It seems likely that other ECM molecules that also have a typical domain structure, such as laminin, thrombospondin and fibronectin, can also be multifunctional. CAMS have complex functional interactions with each other as well. Ll, for example, has previously been shown to promote neurite outgrowth via homophilic interactions [ 251. However, anti-L1 antibodies completely block neurite outgrowth on another CAM, a,,onin-I, suggesting that Ll also acts as a neuronal receptor for axonin1 [26*]. The neural CAM (N-CAM), when present in the same membrane, can enhance homophilic binding of Ll [27]. Ll function thus appears to depend on the combination of other related molecules in its immediate environment, suggesting that it is also a multifunctional molecule. The mode of interactions between CAMS has recently been studied by Kintner [28-l. Expression of a truncated form of N-cadherin lacking the extracellular domain in Xenopus embryos drastically reduced cell adhesion mediated by several different cadherins (maternal cadherin, epithelial cadherin and N-cadherin). This is most probably caused by the competition of different kinds of cadherins for the same intracellular binding proteins, the catenins. The overexpressed cytoplasmic domain probably inhibits the function of the intact cadherins by binding the available catenins. These exper. iments also highlight the important fact that extracellular binding per se (mediated by the intact extracellular domains of the normally expressed cadherins) is not suf. ficient to generate cell adhesion, but that intracellular molecular interactions are also required. Inhibitors
of neurite
growth
Whereas most of the molecules discussed so far can promote neurite outgrowth, molecules that have an inhibitory action also exist, and they are able to arrest growth cone locomotion under otherwise favorable conditions. Inhibition in these cases takes place after firm focal adhesions have already formed between growth cone
Modulators
of neuronal
and inhibitory cell surface [29,30] and is very different from exposure to a non-adhesive surface. Inhibitors of neurite growth have been found and are now being purified from axonal surfaces and brain membranes [31] , posterior somites [32], differentiated oligodendrocytes and central nervous system (CNS) myelin [33] and membranes from the posterior optic tectum [341. In addition, inhibitory activities have also been found in several other systems (reviewed in [35]). Neurite growth inhibitors from oligodendrocytes were originally identified as part of the machinery involved in the failure of regeneration of nerve fibers in the adult CNS [361. The physiological role of these inhibitory molecules, however, is still unclear. Three recent studies (RJ Colello et nl., abstract, Society Neuroscience, 1991, 17:211; [37, 38.1) have now begun to tackle this problem. Schwab and Schnell [37] have looked at the development of the corticospinal tract in rats where the inhibitory molecules of myelin were neutralized in viz’0 by antibodies or where oligodendrocytes were deleted by neonatal X-ray treatment. In these animals, the area covered by the corticospinal tract was greatly enlarged, with corticospinal fibers running aberrantly in adjacent fiber tracts. These findings suggest that the expression of neurite growth inhibitors in areas that myelinate earlier than others can have a guidance role for late developing tEKt.5 In this study it was also observed that the number of branches entering gray matter areas was increased. Myelin and neurite growth inhibitors are predominantly present in white matter, but to a lesser degree also expressed in gray matter. The finding of an enhanced branching of fibers in gray matter areas under conditions where neurite growth inhibitors are absent or neutralized would be compatible with a role of neurite growth inhibitors in restricting branching and sprouting of fibers in spinal cord gray matter. A similar restriction has also been shown in the brain, After early postnatal lesions in hamsters, retinal fibers cross the midline of the optic tectum and secondarily innervate the other side of the superior colliculus [ 391. Such recrossing fibers, however, are mostly restricted to the most superficial layer of the tectum and only sparsely enter the deeper stratum opticum. This correlates with the presence of differentiated oligodendrocytes in the deeper, but not the superficial, layer at this time [40]. In the presence of the neutraizing antibody against neurite growth inhibitors from oligodendrocytes, however, retinal fibers course and terminate in the deeper stratum opticum as well [38.1. This is an example of neurite growth inhibitors affecting the sprouting and termination pattern of fibers in gray matter, and it raises the possibility that the inhibitors could also be involved in the regulation of sprouting and rearrangement of terminals in the adult CNS. A similar Function of neurite growth inhibitors from oligodendrocytes has been suggested by a recent study of axon numbers in nomlal and myelin free optic nerves (RJ Colello et nl., abstract, Society Neuroscience, 1991, 17:211>. After stimulation of the retinal ganglion cells with basis tibroblast growth factor (bFGF) in 10 day old rats, axon numbers increased and showed considerable fluctuation along the length of optic nerves where myelination was prevented by neonatal X-ray treatment. This finding again implies
migration
and neurite
Kapfhammer and Schwab
growth
that myelination and the presence of neurite growth inhibitors could prevent sprouting of nerve fibers in vivo.
RS2 Coordinated cytoskeletal reaction Growth cone
cone
and motility
Fig. 1. Schematic drawing showing some of the types of receptors that affect growth cone extension. RI1 and RI2 are receptors for inhibitors of neurite growth, not yet characterized on the molecular level. RSI and RS2 are receptors for substrate molecules of the extracellular matrix, e.g. integrins. RCI and RC2 are receptors for cell adhesion molecules (CAMS) e.g. immunoglobulin domain CAMS. RF1 and RF2 are receptors for soluble factors, e.g. the trk family of receptor tyrosine kinases. IPI and IP2 are the intrinsic properties of the neuron and growth cone, e.g. modification by connections with local interneurons versus influences from projection neurons, The different receptors influence a limited number of second messenger pathways that eventually affect the motile state of the cytoskeleton (straight arrows). In addition, activation of one type of receptor might influence the properties of other receptors (curved arrows).
Neurite growth inhibitors from oligodendrocytes are an example of a late onset inhibitory molecule. Other inhibitors or repulsors of neurite growth, however, are present early in development and probably involved in axonal pdthhnding and target specificity. In the developing chick optic tectum, an inhibitory activity has been identified that specifically affects temporal but not nasal retinal fibers [ 341, The most attractive concept for such a molecule would be its presence as a gradient in the optic tectum where it could direct retinal fibers to their appropriate target cells [ 411. Baier and Bonhoeffer [42**] tested the activity of gradients of the repulsive factor in an in rftt-o model, where inhibitory posterior tectal membranes were added to non-inhibitory anterior tectal membranes to produce shallow spatial gradients of inhibitory activity. Temporal retinal fibers were inhibited by gradients of as little as 1% per 25 urn (which approximately corresponds to the distance covered by a single growth cone). A distribution of the inhibitory activity from O-100 % would cover a distance of 2.5 mm, roughly the size of the optic tectum at the appropriate developmental stage. This result is most remarkable particularly
865
866
Cell-to-cell
adhesion
and extracellular
matrix
with respect to the finding that laminin, deposited as a shallow gradient, has no effect on growth cone guidance
tein can be expressed on one branch of an axon that has already completed growth, but still be absent on an extending branch of the same neuron. However, irf the complex irz sia situation it is still very unclear what the potential inhibitor of neurite growth recognized by Thy-l might be.
[43,441. So far little is known about receptors for inhibitors of neurite growth. The first example for a molecule that is required on the neuronal side for a specific inhibition of neurite outgrowth is the immunoglobulin superfamily member, Thy-l. Tiveron et al. [45**] transfected Thy-l into NGl15-401L cells, a neuronal cell line that does not normally express this protein. Outgrowth of the transfected Thy- 1-1 neurons was inhibited on monolayers of astrocytes that had been cultured for 2 months or longer, whereas the Thy-l - cells grew out well on this substrate. Neurite outgrowth on other cellular substrates (e.g. fibroblasts, Schwann cells) was similar for both cell lines. The inhibition is most probably mediated by the binding of Thy-l to a heterotypic ligand, because some of the tested cellular substrates do express Thy1 on their surface, but do not inhibit outgrowth of the Thy- 1 + neurons. Addition of soluble Thy-l to block the l&and on the astrocyte surface or addition of anti-Thy1 antibodies blocking the neuronal Thy-l reverses the inhibition and allows neurite outgrowth of the Thy-l + neurons on the cultured astrocytes.
Conclusion There is now a substantial list of molecules that affect neurite growth, including the substrate molecules discussed above and soluble neurotrophic and chemotropic factors. It has become clear that these molecules do not necessarily promote neurite growth in a simple fashion; some affect it in more diverse ways, and some even have an inhibitory action. For ECM molecules and CAMS, adhesion, neurite growth and fasciculation can be affected independently by different parts of one molecule and/or mediated by activation of different second messenger pathways on the receiving side (470,481. Activation of second messenger systems and actions on cytoskeletal components have been poorly studied, and deeper insights are urgently needed. Integrins, for example, which function as receptors for several ECM molecules, display considerable molecular diversity and can interact with various second messenger systems (reviewed in [49]). ECM molecules, CAMS and neurite growth inhibitors are present in ztljo in widely overlapping patterns. Thus, the environment of the growth cone can be viewed as a composition of different domains that express multiple molecules. Any single ECM molecule,
A possible function of Thy-l as a receptor-like molecule for an inhibitory signal would be consistent with the developmental expression pattern of Thy-l in the CNS. For several fiber systems it was shown that Thy-l protein is expressed on axons only after growing fibers have reached their targets [ 46.1. Remarkably, Thy- 1 pro-
Domain
Fig. 2. Three growth cones making extracellular matrix (ECM) substrate ECM molecule 52 and the second the upper domain, because it has has the receptor for the inhibitor present in the upper domain. The upper domain. fc) The growth cone override the two signals from the
II,
Sl,
52,
Cl,
c2
different choices at a domain boundary. The growth cones are extending on a domain expressing the molecule Sl and the cell adhesion molecule (CAM) C2 and approach a domain where the second CAM Cl plus the neurite growth inhibitor II are also expressed. (a) The growth cone will cross into no receptor for the inhibitor but a receptor for the additional CAM Cl (RCl). fb) The growth cone (Rll), but also has receptors for both the CAMS (RCl and RC2) and ECM molecules (RSI and RS2) positive signals will therefore override the inhibitory signal and the growth cone will extend on the will not extend on the upper domain because the inhibitory signal for which it has the receptor will ECM molecule and CAM.
Modulators
of neuronal
CAM or inhibitory molecule may be present in several domains, the difference between domains being in the particular combination of molecules present. Different types of growth cones express specific sets of receptors for various molecules; the most important functional classes of receptors are indicated in Fig. 1. Combinatorial actions, with amplifying or counteracting intracellular effects are likely to be relevant, as the different receptors influence a limited number of second messenger pathways. This eventually results in the integration of the complex environmental signals to regulate the motility and direction of advance of the growth cone. Hypothetical examples of growth cones that make pathway choices by integrating environmental signals are shown in Fig. 2. A l&-o studies have proven to be extremely useful in identifying particular molecules, characterizing their main actions and investigating their interactions with related molecules and receptors. In order to understand neuronal pathfinding in situ, they will have to be complemented by in zv’rlostudies using defined antibodies or receptor blocking agents, gene ablations and transgenic animals. Such studies will increase our understanding of the behavior of growth cones in their natural environment.
Acknowledgements We thank Dr Philippe Douville for critical reading of the manuscript and Dr Brigitte Ktihricht for help with the drawings.
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XI’I: GP. KI\‘~RO BP. MC)RRI~~ RJ: The Surface Glycoprotcin Thy-l Is Excluded from Growing Axons during Development: a Study of the Expression of Thy-l during rluogenesis in Hippocampus and Hindbrain. De~,e/o~,,lr,ll 1199I, 112:161~176. Shows that. for two CNS projrction systems, axon:kt Thy- I protein is expressed only after tihers have reached their targets. Results are corn patible with ;I possible role of Thy I ;LS a receptor for inhibitor acti\iv. ;ls yet undefined -17. .
IXIHER17’ I’, ASHTON SV, IMCXXX SE, WALSH FS: Morphoreguhtory Activities of N-CAM and N-Cadherin Can be Accounted for by G Protein-dependent Activation of L-type and N-type Neuronal Caz+ Channels. Cell 1991, 67:21-33. Demonstration in PC12 cells that the morphotogicat changes towards the neuronal phcnqx induced by N-CAM and N-Cadhcrin involve trJnsmenlhrane signalting \ia Ca’+ channels and G proteins. This 4Tect of the two CAMS is thus due to the acthation of second messenger bystems. Remarkably, similar changes induced by NGF require gene transcription.
of
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SCHI:CH I’. LOHSE MJ, ScttACtINI:R M: Neural CeU Molecules Influence Second Messenger Systems. 19x9, 3:1%20.
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ME: AxonaI Regeneration in the Rat by an Antibody Against Myetin-associInhibitors. Naftrre 1990, 343:26+272.
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HYNES RO: Integrins: Versatility. in CeU Adhesion. Cell 1997. 69:l
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JP, BANDTUJW CE: Inhibitors RetI Neurmci 1993. 116:in press.
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SCHNELL L, SCHWAB Spinal Cord Produced ated Neurite Growth
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Modulation, l-25.
J Kapfhammer and M Schwab, Brain Research Institute, Zurich. August.Forel-Str. I. CH - 8029 Zurich, Switzerland.
and
Clniversity
of
of Zurich,
and Martin Zurich,
E. Schwab
Switzerland
A multitude of molecules have been identified over the past few years that promote neurite outgrowth in vitro. The concept that these molecules work mainly by providing an adhesive surface for neuronal growth cones has been challenged by evidence from recent experiments. Some of the substrate molecules have diverse actions on cell migration and neurite growth. In addition, there is now evidence that there are molecules that specifically inhibit growth cone locomotion. This has given rise to the hypothesis that growth cones integrate a variety of growth-promoting and inhibitory signals and translate them into directed locomotion.
Current
Opinion
in Cell Biology
1992, 4:863-868
Introduction
Adhesion
and neurite
Neuronal migration and neurite extension are basic mechanisms in development and regeneration of the nervous system. They depend on the availability of adequate substrates, typically provided by cellular surfaces or secreted molecules that constitute the extracellular matrix (ECM). In the last decade a variety of ECM and cell surface molecules have been identified that support and promote neuronal migration and neurite extension. In the nervous system most of these molecules are expressed in a spatially and temporally regulated manner, to form a complex pattern of widely overlapping expression domains, Many of these molecules support cell attachment as well as neurite outgrowth in zritro. The hypothesis that the pattern of neurite outgrowth is mainly detemlined by differential adhesion was originally widely accepted. Growth cones would always extend on the more adhesive substrate. Recent evidence, however, suggests that the mechanisms governing neuronal migration and neurite extension are much more complex. In particular, three new Iindings should be considered. First, the strength of adhesion between neuronal growth cones and their substrates shows little relation to neurite outgrowth and even neurite fasciculation. Second, some of the identified substrate molecules are multifunctional, i.e. they can promote or inhibit neurite outgrowth, depending on conditions. Lastly, molecules are now being characterized that specifically inhibit neurite growth.
In a classic tissue culture experiment, Letoumeau [l] demonstrated that outgrowing neurites choose between different substrates, preferring the more adhesive ones [ 21, Based on this and similar experiments, the concept of differential adhesion [3] became dominant in explaining pathfinding choices of neuronal growth cones and migrating neurons. This view has been challenged by recent experiments that used naturally occurring ECM molecules and cell adhesion molecules (CAMS) as substrates, instead of the artilicial substrates used in earlier experiments. For dorsal root ganglion growth cones it was shown that although the ECM molecule laminin is more potent than collagen for stimulating neurite outgrowth, it provides a weaker adhesive force [4], showing that the two activities are not directly related. Further evidence has recently been obtained from a study of neuronal migration on laminin and other ECM molecules [5-l. When a choice was provided, laminin was again preferred over other substrates by the migrating neurons and was the most effective substrate for neuronal migration. However, when assayed for adhesive forces it allowed only weak adhesion of neuronal cells. In fact, even when laminin was mixed with fibronectin at a tenth of the concentration, it actually decreased the adhesion of neurons to the substrate. Similar results were obtained for tenascin, which is a bad substrate for neuronal adhesion and spreading but supports neurite outgrowth [6,i’]. The opposite effect has been reported for a laminin isoform, s-laminin, and synthetic peptides containing an s-laminin-derived IRE (LeuArg-Glu) sequence [8*]. These peptides selectively me-
In this review we will briefly discuss these findings and propose a model of neuronal pathfinding that takes the combinatorial effects of growth promoting and inhibitory signals on neuronal growth cones into account.
Abbreviations adhesion molecule; CN%central nervous system; matrix; N-cadherin-neural cadherin; N-C-neural
CAM-cell ECM-extracellular @
Current
Biology
growth
Ltd
ISSN
0955-0674
CAM 863
864
Cell-to-cell
adhesion
and extracellular
matrix
diate attachment of NSC-34 cells (a cell !ine that has many properties of motoneurons), but inhibit neurite outgrowth from the same cells even in mixtures with ECM larninin. Lemmon et al. [9-l carried out a systematic study on the relation between adhesion of the growth cone, degree of neurite fasciculation and the rate of neurite outgrowth on defmed substrates. For the CAMS Ll and neural cadherin (N-cadherin), ECM laminin and the artificial substrate poly-lysine, they found that none of the three parameters were correlated, i.e. adhesion did not correlate with either growth rate or fasciculation and growth rate did not correlate with fasciculation. This indicates that adhesiveness, growth rate and fasciculation are probably independent and might involve different signalling and recognition mechanisms. It also challenges the widely held view [ 10,111 that the degree of neurite fasciculation is the result of an equilibrium between adhesive forces acting between growth cones and neurites on the one hand and growth cones, neurites and the substrate on the other. The different properties and effects of single substrate molecules described above could be due to different functional domains, neuronal receptors or response mechanisms. Indeed, ECM molecules and CAMS are large molecules, often with a defined domain structure that mediates various functional properties [12]. For the ECM molecule, tenascin, the ability for cell binding, neuronal migration and neurite outgrowth could be mapped to different parts of the molecule using a panel of monoclonal antibodies [ 13.1. However, laminin activities for cell attachment and neurite outgrowth have been mapped to individual proteolytic fragments [I41 and can both be evoked by small synthetic peptides [ 151. Fibronectin cell-binding and neuronal-migration domains have also been mapped to the same site by monoclonal antibodies [16]. In these cases different activities could still be mediated by different signal transduction mechanisms on the cell surface. The ongoing molecular dissection of the active sites on CAMS and ECM molecules, together with the study of cellular receptors and signal transduction mechanisms in neurons and growth cones, will eventually clarify the interaction between the processes described above. The idea that adhesion is the single most important molecular property mediating neurite outgrowth and neurite fasciculation is oversimplified and can no longer be upheld. Cell adhesion and extracellular molecules can be multifunctional
matrix
If different sites exist on one molecule for adhesion, cell spreading, neuronal migration and neurite outgrowth, such a molecule can be expected to have diverse actions, depending on the micro-environment and the type of receptors expressed by the neuron. One example of such a multifunctional molecule is tenascin [ 13*,17]. Purified tenascin inhibits neuronal attachment in z&-o, although once the neurons are attached there is good neurite outgrowth [ 18,191. When a choice of substrate is
available, however, neurites prefer to extend on laminin and fibronectin instead of tenascin. In a recent paper it was shown that while tenascin in substrate-bound form supported neurite outgrowth, in soluble form it reduced outgrowth on several substrates, including laminin and tenascin itself [20-l. The temporal and spatial expression pattern of tenascin both during development and regeneration of peripheral nerves and migration of neural crest cells suggests a supportive role of tenascin in neurite outgrowth and neuronal migration [ 18,211. In the cerebellum, antibodies to tenascin interfere with granule cell migration 1221, although tenascin, according to an electron microscope study, is not present at the site of interaction between granule cells and the Bergmann glia [ 231. In rodent barrel field cortex, tenascin is transiently expressed in the forming barrel walls after arrival of the thalamic fibers [ 241. These descriptive and mostly correlative data will have to be complemented with experiments using delined site-specific antibodies in order to understand the complex action of this molecule. It seems likely that other ECM molecules that also have a typical domain structure, such as laminin, thrombospondin and fibronectin, can also be multifunctional. CAMS have complex functional interactions with each other as well. Ll, for example, has previously been shown to promote neurite outgrowth via homophilic interactions [ 251. However, anti-L1 antibodies completely block neurite outgrowth on another CAM, a,,onin-I, suggesting that Ll also acts as a neuronal receptor for axonin1 [26*]. The neural CAM (N-CAM), when present in the same membrane, can enhance homophilic binding of Ll [27]. Ll function thus appears to depend on the combination of other related molecules in its immediate environment, suggesting that it is also a multifunctional molecule. The mode of interactions between CAMS has recently been studied by Kintner [28-l. Expression of a truncated form of N-cadherin lacking the extracellular domain in Xenopus embryos drastically reduced cell adhesion mediated by several different cadherins (maternal cadherin, epithelial cadherin and N-cadherin). This is most probably caused by the competition of different kinds of cadherins for the same intracellular binding proteins, the catenins. The overexpressed cytoplasmic domain probably inhibits the function of the intact cadherins by binding the available catenins. These exper. iments also highlight the important fact that extracellular binding per se (mediated by the intact extracellular domains of the normally expressed cadherins) is not suf. ficient to generate cell adhesion, but that intracellular molecular interactions are also required. Inhibitors
of neurite
growth
Whereas most of the molecules discussed so far can promote neurite outgrowth, molecules that have an inhibitory action also exist, and they are able to arrest growth cone locomotion under otherwise favorable conditions. Inhibition in these cases takes place after firm focal adhesions have already formed between growth cone
Modulators
of neuronal
and inhibitory cell surface [29,30] and is very different from exposure to a non-adhesive surface. Inhibitors of neurite growth have been found and are now being purified from axonal surfaces and brain membranes [31] , posterior somites [32], differentiated oligodendrocytes and central nervous system (CNS) myelin [33] and membranes from the posterior optic tectum [341. In addition, inhibitory activities have also been found in several other systems (reviewed in [35]). Neurite growth inhibitors from oligodendrocytes were originally identified as part of the machinery involved in the failure of regeneration of nerve fibers in the adult CNS [361. The physiological role of these inhibitory molecules, however, is still unclear. Three recent studies (RJ Colello et nl., abstract, Society Neuroscience, 1991, 17:211; [37, 38.1) have now begun to tackle this problem. Schwab and Schnell [37] have looked at the development of the corticospinal tract in rats where the inhibitory molecules of myelin were neutralized in viz’0 by antibodies or where oligodendrocytes were deleted by neonatal X-ray treatment. In these animals, the area covered by the corticospinal tract was greatly enlarged, with corticospinal fibers running aberrantly in adjacent fiber tracts. These findings suggest that the expression of neurite growth inhibitors in areas that myelinate earlier than others can have a guidance role for late developing tEKt.5 In this study it was also observed that the number of branches entering gray matter areas was increased. Myelin and neurite growth inhibitors are predominantly present in white matter, but to a lesser degree also expressed in gray matter. The finding of an enhanced branching of fibers in gray matter areas under conditions where neurite growth inhibitors are absent or neutralized would be compatible with a role of neurite growth inhibitors in restricting branching and sprouting of fibers in spinal cord gray matter. A similar restriction has also been shown in the brain, After early postnatal lesions in hamsters, retinal fibers cross the midline of the optic tectum and secondarily innervate the other side of the superior colliculus [ 391. Such recrossing fibers, however, are mostly restricted to the most superficial layer of the tectum and only sparsely enter the deeper stratum opticum. This correlates with the presence of differentiated oligodendrocytes in the deeper, but not the superficial, layer at this time [40]. In the presence of the neutraizing antibody against neurite growth inhibitors from oligodendrocytes, however, retinal fibers course and terminate in the deeper stratum opticum as well [38.1. This is an example of neurite growth inhibitors affecting the sprouting and termination pattern of fibers in gray matter, and it raises the possibility that the inhibitors could also be involved in the regulation of sprouting and rearrangement of terminals in the adult CNS. A similar Function of neurite growth inhibitors from oligodendrocytes has been suggested by a recent study of axon numbers in nomlal and myelin free optic nerves (RJ Colello et nl., abstract, Society Neuroscience, 1991, 17:211>. After stimulation of the retinal ganglion cells with basis tibroblast growth factor (bFGF) in 10 day old rats, axon numbers increased and showed considerable fluctuation along the length of optic nerves where myelination was prevented by neonatal X-ray treatment. This finding again implies
migration
and neurite
Kapfhammer and Schwab
growth
that myelination and the presence of neurite growth inhibitors could prevent sprouting of nerve fibers in vivo.
RS2 Coordinated cytoskeletal reaction Growth cone
cone
and motility
Fig. 1. Schematic drawing showing some of the types of receptors that affect growth cone extension. RI1 and RI2 are receptors for inhibitors of neurite growth, not yet characterized on the molecular level. RSI and RS2 are receptors for substrate molecules of the extracellular matrix, e.g. integrins. RCI and RC2 are receptors for cell adhesion molecules (CAMS) e.g. immunoglobulin domain CAMS. RF1 and RF2 are receptors for soluble factors, e.g. the trk family of receptor tyrosine kinases. IPI and IP2 are the intrinsic properties of the neuron and growth cone, e.g. modification by connections with local interneurons versus influences from projection neurons, The different receptors influence a limited number of second messenger pathways that eventually affect the motile state of the cytoskeleton (straight arrows). In addition, activation of one type of receptor might influence the properties of other receptors (curved arrows).
Neurite growth inhibitors from oligodendrocytes are an example of a late onset inhibitory molecule. Other inhibitors or repulsors of neurite growth, however, are present early in development and probably involved in axonal pdthhnding and target specificity. In the developing chick optic tectum, an inhibitory activity has been identified that specifically affects temporal but not nasal retinal fibers [ 341, The most attractive concept for such a molecule would be its presence as a gradient in the optic tectum where it could direct retinal fibers to their appropriate target cells [ 411. Baier and Bonhoeffer [42**] tested the activity of gradients of the repulsive factor in an in rftt-o model, where inhibitory posterior tectal membranes were added to non-inhibitory anterior tectal membranes to produce shallow spatial gradients of inhibitory activity. Temporal retinal fibers were inhibited by gradients of as little as 1% per 25 urn (which approximately corresponds to the distance covered by a single growth cone). A distribution of the inhibitory activity from O-100 % would cover a distance of 2.5 mm, roughly the size of the optic tectum at the appropriate developmental stage. This result is most remarkable particularly
865
866
Cell-to-cell
adhesion
and extracellular
matrix
with respect to the finding that laminin, deposited as a shallow gradient, has no effect on growth cone guidance
tein can be expressed on one branch of an axon that has already completed growth, but still be absent on an extending branch of the same neuron. However, irf the complex irz sia situation it is still very unclear what the potential inhibitor of neurite growth recognized by Thy-l might be.
[43,441. So far little is known about receptors for inhibitors of neurite growth. The first example for a molecule that is required on the neuronal side for a specific inhibition of neurite outgrowth is the immunoglobulin superfamily member, Thy-l. Tiveron et al. [45**] transfected Thy-l into NGl15-401L cells, a neuronal cell line that does not normally express this protein. Outgrowth of the transfected Thy- 1-1 neurons was inhibited on monolayers of astrocytes that had been cultured for 2 months or longer, whereas the Thy-l - cells grew out well on this substrate. Neurite outgrowth on other cellular substrates (e.g. fibroblasts, Schwann cells) was similar for both cell lines. The inhibition is most probably mediated by the binding of Thy-l to a heterotypic ligand, because some of the tested cellular substrates do express Thy1 on their surface, but do not inhibit outgrowth of the Thy- 1 + neurons. Addition of soluble Thy-l to block the l&and on the astrocyte surface or addition of anti-Thy1 antibodies blocking the neuronal Thy-l reverses the inhibition and allows neurite outgrowth of the Thy-l + neurons on the cultured astrocytes.
Conclusion There is now a substantial list of molecules that affect neurite growth, including the substrate molecules discussed above and soluble neurotrophic and chemotropic factors. It has become clear that these molecules do not necessarily promote neurite growth in a simple fashion; some affect it in more diverse ways, and some even have an inhibitory action. For ECM molecules and CAMS, adhesion, neurite growth and fasciculation can be affected independently by different parts of one molecule and/or mediated by activation of different second messenger pathways on the receiving side (470,481. Activation of second messenger systems and actions on cytoskeletal components have been poorly studied, and deeper insights are urgently needed. Integrins, for example, which function as receptors for several ECM molecules, display considerable molecular diversity and can interact with various second messenger systems (reviewed in [49]). ECM molecules, CAMS and neurite growth inhibitors are present in ztljo in widely overlapping patterns. Thus, the environment of the growth cone can be viewed as a composition of different domains that express multiple molecules. Any single ECM molecule,
A possible function of Thy-l as a receptor-like molecule for an inhibitory signal would be consistent with the developmental expression pattern of Thy-l in the CNS. For several fiber systems it was shown that Thy-l protein is expressed on axons only after growing fibers have reached their targets [ 46.1. Remarkably, Thy- 1 pro-
Domain
Fig. 2. Three growth cones making extracellular matrix (ECM) substrate ECM molecule 52 and the second the upper domain, because it has has the receptor for the inhibitor present in the upper domain. The upper domain. fc) The growth cone override the two signals from the
II,
Sl,
52,
Cl,
c2
different choices at a domain boundary. The growth cones are extending on a domain expressing the molecule Sl and the cell adhesion molecule (CAM) C2 and approach a domain where the second CAM Cl plus the neurite growth inhibitor II are also expressed. (a) The growth cone will cross into no receptor for the inhibitor but a receptor for the additional CAM Cl (RCl). fb) The growth cone (Rll), but also has receptors for both the CAMS (RCl and RC2) and ECM molecules (RSI and RS2) positive signals will therefore override the inhibitory signal and the growth cone will extend on the will not extend on the upper domain because the inhibitory signal for which it has the receptor will ECM molecule and CAM.
Modulators
of neuronal
CAM or inhibitory molecule may be present in several domains, the difference between domains being in the particular combination of molecules present. Different types of growth cones express specific sets of receptors for various molecules; the most important functional classes of receptors are indicated in Fig. 1. Combinatorial actions, with amplifying or counteracting intracellular effects are likely to be relevant, as the different receptors influence a limited number of second messenger pathways. This eventually results in the integration of the complex environmental signals to regulate the motility and direction of advance of the growth cone. Hypothetical examples of growth cones that make pathway choices by integrating environmental signals are shown in Fig. 2. A l&-o studies have proven to be extremely useful in identifying particular molecules, characterizing their main actions and investigating their interactions with related molecules and receptors. In order to understand neuronal pathfinding in situ, they will have to be complemented by in zv’rlostudies using defined antibodies or receptor blocking agents, gene ablations and transgenic animals. Such studies will increase our understanding of the behavior of growth cones in their natural environment.
Acknowledgements We thank Dr Philippe Douville for critical reading of the manuscript and Dr Brigitte Ktihricht for help with the drawings.
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42. BAI~R II. B~NIIOI:I;FI~R F: Axon Guidance by Gradients of a .. Target Derived Component. Scietrir 1992. 255:t’2--17i. Sbon:s that an inhibitor? factor cleri\til from the optic tectum is :ic tive when present as a shalknv spatial gradient. Inhibitor moleculrs may act in \TF subtle nzys. e.g. directing axons hy rheir gradient-like dislribution. 43.
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TI~I;R~N M-C. BAKI~ONI E. R~‘EIIO F%P. CA>KXIII:I’ AM, SI%IE> JP. GHOS\ZII) F. MOKRI~ R: Selective Inhibition of Neurite Outgrowth on Mature Astrocytes by Thy-l Glycoprotein. Na/~tre 1992. 355:74i-7iX. Presents rhe tirst candidate for 3 ncuronal rcceplor molecule nith ;I specitic inhibitor? acti\iF. After trsnsfection of Thy- 1. a neuronal cell line sho\ved much reduced outgrowth from astroqes that bad been cultured for extended time periods. This inhibitory erect could he :tholisbed by addition of sotuhle Thy- I protein to the astnqTes or by antibodies blocking Thy-1 function. 16. .
of a Neuronal Embryonic Chick
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IXIHER17’ I’, ASHTON SV, IMCXXX SE, WALSH FS: Morphoreguhtory Activities of N-CAM and N-Cadherin Can be Accounted for by G Protein-dependent Activation of L-type and N-type Neuronal Caz+ Channels. Cell 1991, 67:21-33. Demonstration in PC12 cells that the morphotogicat changes towards the neuronal phcnqx induced by N-CAM and N-Cadhcrin involve trJnsmenlhrane signalting \ia Ca’+ channels and G proteins. This 4Tect of the two CAMS is thus due to the acthation of second messenger bystems. Remarkably, similar changes induced by NGF require gene transcription.
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SCHI:CH I’. LOHSE MJ, ScttACtINI:R M: Neural CeU Molecules Influence Second Messenger Systems. 19x9, 3:1%20.
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and
Clniversity
of