Growth Cone Guidance and Neuron Morphology on Micropatterned Laminin Surfaces

Home , Growth cone, Guidepost cells, Neurite, TECTA, Vitronectin

Journal of Cell Science 105, 203-212 (1993) 203 Printed in Great Britain © The Company of Biologists Limited 1993

Growth cone guidance and neuron morphology on micropatterned laminin surfaces

Peter Clark1,*, Stephen Britland2 and Patricia Connolly3,† 1Department of Anatomy and Cell Biology, St. Mary’s Hospital Medical School, Imperial College of Science, Technology and Medicine, Norfolk Place, London W2 1PG, UK 2Department of Neuropathology, Institute of Neurology, The National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, UK 3Department of Electronics and Electrical Engineering, University of Glasgow, Glasgow G12 8QQ, UK *Author for correspondence †Present address: Biosensors Group, BYK Gulden Italia S.p.A., Via Giotto 1, Cormano 1-20032, Milan, Italy

SUMMARY

Neurite growth cones detect and respond to guidance structures, but could be guided by isolated adhesive cues in their local environment that determine stereo- tracks. Growth cone morphology became progressively typed pathways during development and regeneration. simpler on progressively narrower single tracks. On Micropatterns of laminin (which was found to adsorb narrow period multiple parallel tracks (which did not preferentially to photolithographically defined hydro- guide neurite extension) growth cones spanned a phobic areas of micropatterns) were here used to model number of adhesive/non-adhesive tracks, and their mor- adhesive pathways that might influence neurite exten- phology suggests that lamellipodial advance may be sion. The responses of growth cones were determined independent of the substratum by using filopodia as a by the degree of guidance of neurite extension and also scaffold. In addition to acting as guidance cues, laminin by examining growth cone morphology. These parame- micropatterns also appeared to influence the production ters were found to be strongly dependent on the geom- of primary neurites and their subsequent branching. On etry of the patterned laminin, and on neuron type. planar substrata, dorsal root ganglion neurons were Decreasing the spacing of multiple parallel tracks of multipolar, with highly branched neurite outgrowth laminin alternating with non-adhesive tracks, resulted whereas, on 25 m tracks, neurite branching was in decreased guidance of chick embryo brain neurons. reduced or absent, and neuron morphology was typi- Single isolated 2 m tracks strongly guided neurite cally bipolar. These observations indicate the precision extension whereas 2 m tracks forming a 4 m period with which growth cone advance may be controlled by multiple parallel pattern did not. Growth cones appear substrata and suggest a role for patterned adhesiveness to be capable of bridging the narrow non-adhesive in neuronal morphological differentiation, but also high- tracks, rendering them insensitive to the smaller period light some of the limitations of growth cone sensitivity multiple parallel adhesive patterns. These observations to substratum cues. suggest that growth cones would be unresponsive to the multiple adhesive cues such as would be presented by Key words: axon, growth cone, neuron, guidance, laminin, oriented extracellular matrix or certain axon fascicle adhesion, motility, photolithography

INTRODUCTION siveness are crucial in guiding neuronal growth cones to distant targets in vivo. In frog spinal cord, the dorsal column Growth cones of extending axons and dendrites explore provides a track that specifically guides dorsal root gan- their local environment and are capable of detecting and glion (DRG) axons either rostrally or caudally after their responding to extrinsic cues that guide the direction of their entry into the spinal cord from the dorsal horn (Holder et extension. Cues involved in this guidance of neurite out- al., 1987). Motoneuron axon extension is believed to be growth include chemotropic gradients and specific substra- guided by the tissues that the axon encounters as it extends tum pathways (Dodd and Jessel, 1988; Hynes and Lander, peripherally (Lance-Jones and Dias, 1991; Tosney, 1991; 1992). Tang et al., 1992). Projection from the retina to the optic Local environmental cues influencing cell-substratum tectum appears to be via a preformed pathway, since sur- interactions are believed to be important in controlling gical rotation of the pathway region in embryos resulted in growth cone guidance. Recently, a number of studies have the deflection of projection consistent with the direction of suggested that pre-formed pathways of differential adhe- rotation (Harris, 1989; Taylor, 1990). The nature of this 204 P. Clark, S. Britland and P. Connolly pathway is uncertain, though both NCAM (Silver and terning adhesivness for neurons (Kleinfeld et al., 1988). Rutishauser, 1984) and laminin (Cohen et al., 1987) have Recently, we have modified this technique by simplifying been implicated. NCAM pathways are also likely to be the fabrication procedure, thus allowing the manufacture of involved in guiding axons during chick limb innervation micropatterns of adhesion on fused quartz and standard (Tang et al., 1992) and cochlear development (Whitlon and glass. These patterns were used by us to examine the behav- Rutishauser, 1990). The presence of putative pathways of iour of non-neuronal cells (Britland et al., 1992; Clark et laminin appears to guide axonal outgrowth in a number of al., 1992). Here, we have examined the responses of the developing central and peripheral neural tissues (Rogers et growth cones of dissociated neurons in culture to patterns al., 1986; Riggott and Moody, 1987; Letourneau et al., of laminin of various geometries formed by its preferential 1988; Liesi, 1990). Three main mechanisms of axon guid- adsorbtion to micropatterns of hydrophobicity. These sub- ance by substratum adhesiveness operate: substratum pref- strata provide a convenient model system that has allowed erence, inhibition or repulsion of growth cones, and guide- the examination of the sensitivity of growth cones in detect- posting. In substratum preference, growth cones are faced ing and reacting to differentially adhesive cues. with a choice of adhesive (neuritogenic) or non-adhesive (non-neuritogenic but otherwise inert) substrata. Such adhesive substrata may be cell surface adhesion molecules MATERIALS AND METHODS (CAM), which may be highly specific, or patterned extracellular matrix material (ECM). Tracks of NCAM or Neuronal culture laminin might guide by substratum preference. The term Cerebral hemispheres of 7-day chick embryos were removed and ‘adhesiveness’ may not be entirely appropriate in some cleaned of meningeal membranes. The hemispheres were then instances. Substratum adhesiveness does not correlate with minced, incubated in 0.05% trypsin/0.2% EDTA in PBS for 15 neurite growth rate (Lemmon et al., 1992), and it has been min at 37°C, stopped with soybean trypsin inhibitor (Sigma, UK), suggested that laminin may act as an ‘anti-adhesive’ agent, triturated in serum-free medium (Gibco High Protein Hybridoma presumably promoting axon extension by increasing growth medium; Gibco, UK) supplemented with glutamine and antibiotics cone motility rather than growth cone-substratum adhesion as described previously (Clark et al., 1987) with a fire-polished Pasteur pipette, washed by centrifugation, and resuspended in (Gundersen, 1988; Calof and Lander, 1991), though a recent medium. Neurons were plated onto patterns in 6 cm Petri dishes study suggests that growth cone protrusions are stablised (106 per dish) and cultured in the serum-free Gibco Hybridoma by laminin substrata (Rivas et al., 1992). It is now clear medium at 37°C in a humidified atmosphere of 5% CO2. Some that the ‘adhesiveness’ of substrata may also be modulated neurons were cultured in Dulbecco’s MEM with 10% foetal calf by substances that actively inhibit or repulse growth cones. serum. The segmented distribution (to the posterior half of sclero- Dorsal root ganglion neurons were obtained from P1 neonatal tomes) of a repulsive agent is involved in the segmentation Balb/c mice. The mice were anaesthetised by cooling and killed of the developing peripheral nervous system (Keynes and by decapitation. Following sterilisation with ethanol, the skin was Cook, 1992). The presence of gradients of such repulsive opened to expose the vertebral column. The spinal ganglia were molecules is believed to be involved in the control of gan- exposed after laminectomy. Approximately 50 DRG were col- lected and then finely minced in divalent-free Hank’s balanced glion cell axon extension to the optic fissure in developing salt solution (HBS). Spinal cord cells were obtained from three retina (Snow et al., 1991) and in the formation of the appro- E13 foetal Balb/c mice. After removal from the chorionic sac, the priate pattern of connections of retinal ganglion cell axons foetuses were killed by decapitation and the spinal cord was in the optic tectum (Baier and Bonhoeffer, 1992). Myelin- removed. The cords were washed in divalent-free HBS and finely associated inhibitory molecules repress regeneration of minced. The remainder of the procedure was common to both adult central neurons, but may be involved in development tissues. Minced tissue was transferred to a solution of 0.05% (Schwab and Schnell, 1991). The third mechanism, guide- trypsin in 0.2 mg ml- 1 EDTA (Flow Labs, UK) at 37°C for 30 posting (Palka et al., 1992), which has been described minutes. Trypsinisation was stopped by the addition of trypsin - 1 mainly in insects, could be considered a particular case of inhibitor (1 mg ml ) in medium as above. The cells were cen- substratum preference. In this case, the adhesive substra- trifuged and resuspended, by trituration, in the serum-free medium. For DRG neuron culture, the medium was supplimented tum is discontinuous, consisting of specific guidepost cells with 100 ng ml- 1 nerve growth factor (Sigma, UK). DRG cells along the pathway of axonal extension. The axon extends were plated at a density of approx. 5´ 103 cells cm- 2, and spinal after a guidepost cell is contacted by the filopodia of an cord cells at 1´ 104 cells cm- 2. Cells were incubated as above for exploring growth cone and cell-growth cone association is 24 (DRG cells) or 48 (spinal cord cells) hours. established. Axon guidance is the result of progressive extension from guidepost to guidepost. Laminin patterning A number of in vitro studies have examined the ability Patterns of hydrophobicity were made as described previously of patterns of adhesiveness to guide the outgrowth of neu- (Britland et al., 1992; Clark et al., 1992). Briefly, sulphuric rites. Laminin, the large ECM protein known to be capable acid/hydrogen peroxide-cleaned fused quartz or standard micro- of initiating and sustaining neurite extension (Lander et al., scope slide glass was spin-coated with photoresist, exposed to UV 1985), has been patterned using techniques of limited res- light through a chrome mask of the desired pattern, and devel- olution (electron microscope grids being the templates for oped to leave a pattern of photoresist and exposed glass. These patterns were immersed in 2% (v/v) dimethyldichlorosilane in the patterns; Hammarback et al., 1985, 1988; Gundersen, chlorobenzene, rinsed twice in chlorobenzene, and blown dry. The 1987), as has patterned nerve growth factor (Gundersen, resist pattern was then removed by rinsing in acetone and then 1985). A microfabrication technique has been used to water, resulting in a final pattern of methyl groups (the hydropho- micropattern amino groups to silica surfaces, thereby pat- bic surface) covalently coupled to the quartz surface. Repeat pat- Growth cone guidance by substratum adhesion 205 terns of equal sized hydrophobic lines and untreated quartz spaces though neurite outgrowth, when present, appeared less were made using masks of repeat spacings 4, 6, 12, 24 and 50 extensive in serum-containing cultures (not shown). No dif- m (i.e. feature sizes of half these values). Patterns were placed ference in patterning adhesiveness was found between in 6 cm Petri dishes, covered with 5 g ml- 1 laminin in PBS, and quartz and standard glass (not shown). Immunofluorescence incubated at 37°C for 2-3 hours, after which time excess laminin localisation of laminin revealed a confinement of staining solution was removed and cells added. Neurons were also cul- to the previously hydrophobic regions of the patterns (Fig. tured on laminin patterns made up of single 2 m lines, separated by 50 m and interupted with 50 m tracks every 500 m, 1C,D). In Fig. 1C it can be seen that the intensity of fluo- and on patterns that consisted of adjacent large hydrophobic and rescence is greater at the boundary with the non-silanated hydrophilic regions so that comparisons could be made at a bound- region. Control patterns (no laminin incubation) showed no ary. Cells were also seeded onto patterns of hydrophobicity that staining. had not been treated with laminin. Guidance of neurite extension Immunofluorescence localisation of laminin When neurons were cultured on repeating patterns of alter- After incubation in laminin solution as above, some patterns were nating lines of treated and untreated glass or quartz that had rinsed twice in 0.1% BSA in PBS, then either drained and pri- been exposed to laminin, the degree of alignment of neu- mary antibody added, or rinsed in PBS, fixed briefly in 4% rite outgrowth was dependent on pattern spacing. Neurite formaldehyde in PBS, rinsed in PBS and primary antibody added. outgrowth on 4 and 6 m period patterns appears not to be After incubation in the primary antibody (1:40 affinity-purified affected, whereas that on patterns of larger period is guided, rabbit anti-laminin polyclonal antibody (Sigma, UK)) for 45 minutes, patterns were washed twice in 0.1% BSA in PBS and incu- the neurite elongation being oriented along the tracks of bated for 20 minutes in rhodamine-labelled goat anti-rabbit IgG adhesiveness for large distances (Fig. 2). Changes in direc- antibody. Patterns were washed with PBS, mounted in Vecta- tion of guided neurites were never observed, even on the mount (Vector Labs, UK), and examined and photographed using widest (25 m) tracks (Fig. 2F). These observations were a Zeiss Axioskop fluorescence microscope. borne out by those from the measurement of alignment of neurite outgrowth. Neurite outgrowth on 4 and 6 m period Determination of neurite alignment patterns is not significantly aligned, though that on patterns The degree of alignment of neurite outgrowth was determined as of greater period is, reaching complete alignment by 24 m previously (Clark et al., 1991). Chick embryo cerebral neurons period patterns (Fig. 3). were photographed under phase optics after 24 hours in culture. Though neurite outgrowth was not aligned by 4 m To determine whether or not neurite outgrowth was aligned on period patterns (2 m adhesive tracks separated by 2 m patterns, individual neurites (between 50 and 80 per sample) were non-adhesive tracks), single 2 m lines of adhesiveness scored as to the angle at which they intersected with an arc whose origin was the cell body and whose radius was 30 m. The pro- supported the adhesion and guided the neurite outgrowth of portion of neurites whose intersections fall within 45° of the chick embryo neurons (Fig. 4). These lines were 500 m alignment of the pattern was taken to be the measure of align- in length, separated by 50 m and they perpendicularly join ment. A population of neurites outgrowing randomly will be 50 m wide tracks. Neurites were seen to extend from these expected to have an alignment value of 0.5. Neurite guidance in wider zones to the 2 m lines, and vice versa (Fig. 4B). the direction of the pattern would give an alignment value sig- The neurite outgrowths of chick embryo brain neurons nificantly greater than 0.5, and guidance perpendicular to pattern (Fig. 5) and mouse embryo spinal chord neurons (not direction a value significantly less than 0.5. The significance shown) are highly aligned by 24 m period patterns. Dorsal (P<0.05) of measured differences in the degree of alignment was 2 root ganglion neuron neurite outgrowth is generally not determined by calculating chi from 2´ 2 contingency tables. aligned by 24 m patterns (though some individual neu- Some samples of cultured chick cerebral neurons were fixed in 4% formaldehyde in PBS at room temperature for 1 hour, washed rites do appear to be guided) (Fig. 5). in PBS, mounted under thin coverslips with aqueous mountant, and examined with differential interference contrast (DIC) optics. Laminin pattern geometry and DRG neurite Growth cones on various patterns were photographed using an oil branching immersion ´ 100 objective. On unpatterned laminin and on 24 m period laminin patterns, mouse DRG neurons typically produce an outgrowth of multipolar morphology, the individual neurites often RESULTS branching to form a dense arbor (Fig. 5A,B). In contrast, DRG neurite outgrowth is aligned by patterns of 50 m Laminin adsorption to micropatterned substrata period. The morphology of this outgrowth is typically bipo- The micrographs in Fig. 1 show the differences in adhe- lar, single neurites extending for large distances in oppo- siveness for chick embryo neurons between adjacent site directions along the tracks of laminin, without branch- hydrophobic and untreated areas of quartz glass surfaces. ing (Fig. 5E,F). It can be seen that, in the absence of laminin, neurons are more adherent to the untreated (hydrophilic) surface than Laminin patterns and growth cone morphology the adjacent treated (hydrophobic) surface, there being little On unpatterned laminin surfaces growth cones were typi- neurite outgrowth on either surface (Fig. 1A). After laminin cally wide (5-8 m) lamellar structures bearing a number treatment, adhesion is greater on the treated surface where of short (rarely longer than 6 m) filopodia (Fig. 6A). neurite outgrowth is also evident, whereas the untreated sur- Growth cones unguided by 4 m period laminin patterns face is less adhesive with little or no neurite outgrowth (Fig. (2 m lines and spaces) (in Fig. 6B neurite extension is 1B). This difference was found with and without serum, perpendicular to the pattern), maintained their lamellar 206 P. Clark, S. Britland and P. Connolly

Fig. 1. (A) At the border between a hydrophobic (upper area) and hydrophilic (lower area), without laminin treatment, there is greater chick embryo neuron attachment, but little neurite outgrowth on the lower hyrophilic region. Arrows indicate approximate border. (B) As with (A) but cells were seeded after laminin treatment. Cells show greater attachment and extend neurites on upper hydrophobic region. (C) and (D) Indirect immunofluorescence localisation of laminin on hydrophobic patterns. (C) Border between upper hydrophobic region, which stains positvely for laminin, and lower unsilanated hydrophilic region, which lacks stain. (D) A pattern of 8 m wide hydrophobic (silanated) lines, separated by 30 m unsilanated regions. Laminin staining is confined to hydrophobic lines. Bar in (A) (for A and B), 300 m. Bar in (C) (for C and D), 50 m. morphology though filopodia originating from the growth indicates that growth cone structures rarely cross to the non- cones, and lateral spines of the neurite, display a patterned adhesive surface. In fact, lamellar regions were never seen distribution corresponding to the laminin pattern (Fig. 6B). to be in contact with a boundary. Similarly, the neurites Filopodia are present at the most distal edge of the growth themselves are never seen at the boundaries. When neurite cone, their length indicating that they are bridging at least extension has occurred parallel and close to a boundary, the one non-adhesive strip, as are lamellar regions. distance between the neurite and the boundary remains rel- Patterns of 12 m period guide growth cones (see above) atively constant (approximately 5-7 m) (Fig. 6D,G,H). and, as seen in Fig. 6C, their morphology is altered. The There was no indication of a preference of growth cones growth cones, which are in contact with both boundaries for the areas adjacent to boundaries, since in a number of with adjacent non-adhesive strips, appear narrower and cases neurites have been seen to have turned at a bound- have fewer filopodia, which are confined to the distal end ary (not shown). (Fig. 6C). In contrast, growth cones of neurons cultured on 50 m repeat patterns (25 m lines and spaces) do not span the width of the adhesive strip, being in contact with DISCUSSION only one boundary with adjacent non-adhesive regions (Fig. 6D). Their morphology differs little from those on unpat- The effectiveness of patterned adhesiveness as a potential terned substrata. The growth cones of neurites extending in vivo axonal guidance cue has been established by in vitro along single 2 m lines have become extremely simplified, experiments. These studies have mainly used electron having become single filopodia (Fig. 6E) or narrow lamel- microscope grids as their pattern templates, and therefore lipodial structures tapering to a filopodium (Fig. 6F). were limited in resolution of feature size. Despite this lim- The examination of growth cones at boundaries between itation, it has been shown that adhesiveness per se adhesive and non-adhesive regions, such as on 50 m (Letourneau, 1975), and both patterned laminin (Hammar- period patterns (Fig. 6D) or on larger patterns (Fig. 6G,H), back et al., 1985, 1988; Gundersen, 1987) and nerve growth Growth cone guidance by substratum adhesion 207

Fig. 2. The dependence of orientation of chick embryo neurons neurite outgrowth on laminin pattern geometry. (A) Control, unpatterned laminin. (B) 4 m period pattern (2 m lines and spaces). (C) 6 m period pattern (3 m lines and spaces). (D) 12 m period pattern (6 m lines and spaces). (E) 24 m pattern (12 m lines and spaces). (F) 50 m period pattern (25 m lines and spaces). Bar, 200 m. factor (Gundersen, 1985), will guide neurite extension, and patterned adhesion may influence their ability to guide in testing the capability of chick autonomic ganglion neu- axonal growth efficiently. Surprisingly, laminin was found rons to pathfind by guidposting, Hammarback and to adsorb preferentially to the hydrophobic surface of the Letourneau (1986) have shown that neurites can bridge non- patterns, which consists of methyl groups covalently linked adhesive regions. via silane bonds to standard glass or fused quartz. This pref- Here, we have developed a method for micropatterning erential adsorption was unexpected, since our earlier expe- laminin that provides experimental substrata that model rience with these patterns had suggested that cell attach- substratum preference. The limit of resolution of this ment factors in serum (presumably vitronectin and method will be the limit of resolution of standard pho- fibronectin) adsorb poorly to the hydrophobic surfaces, cell tolithographic techniques, i.e. feature size of 1-2 m. We adhesion being far greater on untreated glass (Britland et have used such patterns to examine how the geometry of al., 1992; Clark et al., 1992). However, this property of 208 P. Clark, S. Britland and P. Connolly

1.0 present study, we found that the growth cones of chick embryo brain and mouse embryo spinal cord neurons were 0.8 consistantly guided by 12 m tracks of laminin separated by 12 m non-adhesive tracks (24 m period), suggesting 0.6 that they are unable to bridge this distance. In contrast, we found that mouse neonatal dorsal root ganglion neuron 0.4 growth cones were often not guided by 24 m period pat- alignment 0.2 terns but were by 50 m patterns, i.e. they are able to bridge 12 m, but not 25 m, non-adhesive regions. Although 4 0.0 m period patterns (2 m tracks of laminin separated by control 4 6 12 24 50 2 m non-adhesive lines) were unable to guide the out- patternpattern period period (µm) ( m) growth of chick embryo brain neurons, single 2 m tracks Fig. 3. The measured degree of alignment of neurite outgrowth of (i.e. separated by 50 m) are able to sustain adhesion and chick embryo neurons as a function of pattern period. The promote bipolar outgrowth of neurites. Similarly, the guid- horizontal broken line indicates the 0.5 value of alignment ance of BHK and MDCK cells was markedly reduced on representing random outgrowth of neurites. 2 m period repeat patterns of adhesiveness (Clark et al., 1992), but the cells were aligned by single 2 m adhesive lines (unpublished results). Therefore, the proximity of laminin has provided precise micropatterns to which neu- adjacent adhesive areas reduces guidance by tracks. It rons selectively adhere and which promote neurite out- would seem that in order to guide neurite extension, a track growth. Immunofluorescence localisation of laminin must be separated from such adjacent regions by a distance suggests that there may be increased accumulation close to greater than the distance over which a growth cone can boundaries as a result of diffusion effects at these sites. Any extend exploring protrusions. increase in laminin concentration at these areas does not The present study has shown that tracks of adhesion as appear to lead to the preferential accumulation of growth narrow as 2 m and as wide as 25 m can precisely guide cones, since turning of neurites from boundaries was axonal extension over large distances. However, as dis- observed. This is consistent with the previously observed cussed above, a growth cone will only be constrained by a lack of haptotactic guidance of growth cones on gradients track of adhesiveness if the track is separated from any adja- of laminin (McKenna and Raper, 1988). cent regions of adhesiveness by greater than the bridging The ability of a pattern to guide neurite outgrowth was distance of the particular growth cone, i.e. the length of found to be strongly dependent on its geometry. Narrow (4 protrusion produced by the growth cone. These data have and 6 m period) patterns did not orient outgrowth, wider indicated some important limitations of growth cones period patterns did. This is likely to reflect the ability of response to local environmental influences. Hammarback growth cones to produce protrusions that are able to bridge and Letourneau (1986) showed that autonomic neuron non-adhesive regions. Growth cones of autonomic ganglion growth cones could extend across relatively large non-adhe- neurons have been shown to be capable of bridging non- sive regions and suggested that this ability is important in adhesive regions 30-40 m wide and occasionally 50 m the guidepost hypothesis of guidance of axonal outgrowth. wide (Hammarback and Letourneau, 1986). Similarly, a Guideposting in higher animals has not been observed strong dependence on pattern geometry was found in the (Palka et al., 1992). Our observations indicate that the dis- guidance of fibroblastic and epithelial cells, where bridging tances that avian and mammalian central neurons are able across non-adhesive tracks was seen to affect the ability of to bridge may be too small to allow guideposting to be an the substratum to align cells (Clark et al., 1992). In the effective mechanism of guidance, since these distances are

Fig. 4. Chick embryo neurons on patterns that include single 2 m lines of laminin. These lines guide neurite outgrowth from cell bodies on the lines themselves, and growth cones originating from cell bodies on the intervening 50 m adhesive track (arrow in B). Neurites are also seen to leave the 2 m tracks onto the large intervening track (small arrowhead in B). Bar, 200 m. Growth cone guidance by substratum adhesion 209

though we have previously shown that chick embryo cerebral neurons, unlike non-neuronal cells, were insensitive to ultrafine topography (Clark et al., 1991). Similarly, when a growth cone encounters a fasciculated bundle of neurites, it will be presented with multiple parallel adhesive cues on a scale that the present observations suggest would not guide neurite extenstion, i.e. the adhesive cues would not prevent extension across or around the fascicle (unless a single neurite or small bundle of neurites in the fascicle presented a separate, specific adhesive cue). We contend that topographic cues (Curtis and Clark, 1992) and persis- tence of neurite extension (Katz, 1985) play an important part in maintaining the linear order and preventing random tangling in axon fascicles. It must be noted, however, that the nature of their substratum can influence the morphology of growth cones, both in vitro and in vivo, by altering the number and length of protrusions (Letourneau, 1979; Wilson and Easter, 1991; Payne et al., 1992). This phe- nomenon will also be an important factor in determining the sensitivity with which growth cones respond to guidance cues in their environment. The observed morphology of growth cones on laminin (both patterned and unpatterned) correlates with their impaired ability to bridge distances greater than 6 m, since filopodia longer than 6 m were rarely seen. Similarly, the ability to bridge distances smaller than 6 m renders them insensitive to the cues provided by smaller period patterns. At single boundaries between non-adhesive and adhesive regions, the morphology of growth cones, where lamellar regions and neurites were never in contact with a boundary, suggests that growth cone advance (i.e. the advance of the lamellipodium) is inhibited in the direction of filopodia in contact with the boundary. Lamellipodial advance would appear to require established, stable filopodia, as has been suggested previously (Heidemann and Buxbaum, 1991; Rivas et al., 1992). This requirement may, in fact, be absolute, since laminin substratum is available to the edge of the pattern boundaries, but lamellopodia are not able to advance to the edge. This results in the neurite itself being formed at a distance from the boundary. On narrow multiple parallel tracks, lamellipodial regions of growth cones appear to be in contact with the non-adhesive regions, which suggests that lamellopodial advance is directed by filopodia and may be independent of cell-substratum adhesion (possibly in a similar manner to the spreading of fibroblasts on topographically discontinuous surfaces; Fig. 5. DRG neuron morphology. (A) and (B) Neonatal mouse Rovensky et al., 1991); i.e. the nature, or indeed presence, DRG cell on unpatterned laminin having multipolar morphology. of a substratum may be unimportant for lamellipodial exten- (C) and (D) DRGs on 24 m pattern (12 m lines and spaces) sion, so long as there is a scaffold of filopodia on which show multipolar morphology with little guidance. (E) and (F) advance can be supported. The progressive simplification DRGs on 50 m period pattern (25 m lines and spaces) have bipolar morphology, with neurites guided over long distances of growth cone morphology on increasingly narrower adhe- without branching. sive tracks has shown the ability of these motile structures to conform to the available preferred substratum. This adap- tation appears to be as a result of the inhibition of as small as, if not smaller than, many cells themselves. It filopodium formation except at the distal edges of the would also seem that many growth cones may be incapable growth cones (i.e. in the direction of the track). Time-lapse of responding to the multiple parallel adhesive cues pro- observation of growth cone advance on various patterns vided by aligned fibrillar extracellular matrix material (see may, in the future, reveal differences in the efficiency of Clark et al., 1992). The observed guidance of neurites by morphologically distinct growth cones in neurite extension, aligned collagen gels in vitro (Ebendal, 1976) is likely to which may have relevance in vivo. Micropatterns of laminin be the result of another anisotropic property of these gels, provide a mechanism for specifically altering growth cone 210 P. Clark, S. Britland and P. Connolly

Fig. 6. DIC photomicrographs of chick embryo cerebral neuron growth cones on various laminin surfaces. (A) Control, unpatterned laminin. (B) 4 m period pattern. The strips marked L indicate the appoximate level of 2 m laminin lines in the region of the growth cone. (C) Growth cone being guided on a 12 m period pattern. Broken lines indicate the confines of the 6 m track guiding this growth cone. (D) 25 m laminin track (indicated by broken lines) guiding growth cone extension. (E) and (F) Simple growth cones on isolated 2 m tracks. (G) and (H) Growth cones of neurites extending parallel to the boundaries between adjacent large areas of adhesive (lower) and non-adhesive (upper) substratum. morphology without altering the composition of the sub- of adhesiveness perpendicularly, in the absence of other stratum. cues, the direction taken would be expected to be randomly It is clear that tracks of adhesiveness are capable of pro- right or left; for example, DRG axons encountering the viding a guidance cue that can steer axon extension for large dorsal column in the frog spinal cord (Holder et al., 1987). distances. This type of cue is, however, a bidirectional one. Other cues, such as chemotactic or haptotactic gradients, Turning or meandering of growth cones is possible, partic- will be required at such ‘T-junctions’ to provide specific ularly on wider tracks. Where a growth cone meets a track directionality. In order to provide this directionality in vivo, Growth cone guidance by substratum adhesion 211 it is probable that the cues need only be short and/or tran- by isolated narrow tracks of neuritogenic substratum. It sient, as persistance of locomotion will maintain direction seems likely that in vivo a growth cone will remain con- of extension (Katz, 1985). It must be noted, however, that strained to an isolated track of preferred substratum until McKenna and Raper (1988) were unable to demonstrate either its target is reached or another cue over-rides the sub- haptotactic guidance of neuronal growth cones on gradients stratum cue. Tracks of preferred substratum could guide of substratum-bound laminin, in vitro, though gradients of axon extension with directionality, in the absence of gradi- inhibitory molecules are believed to guide axons in vitro ents, where there is a clear spatial relationship between the and in vivo (Snow et al., 1991; Baier and Bonhoeffer, direction of the track and the point at which it can be 1992). encountered by a growth cone. Growing axons may be fun- We noted that mouse neonatal DRG cells cultured on nelled into a track such that, because of persistance of loco- unpatterned laminin (or on a 24 m period laminin pattern, motion, only one direction of extension is possible. Guid- which does not guide neurite outgrowth) had a multipolar ance by tracks of preferred substratum is only one of a neurite outgrowth, a dense arbor emanating from a single number of possible cues involved in neurite guidance in neuron as a result of branching of primary neurites. When vivo, but the present and previous in vitro studies clearly these same cells were cultured on 50 m period patterns, show that this may operate in diverse and widespread devel- which oriented their neurite outgrowth, the outgrowth was, opmental processes to provide a precise and effective mech- in many cases, bipolar. Single neurites extended from oppo- anism of controlling axon extension. site ends of the cell body, usually without forming any branches. Mature DRG cells in vivo have a pseudounipo- We thank Yash Bhasin and Barry Crook at St. Mary’s, Henry lar mophology, though a bipolar morphology is a normal Goulding and Stephen Durr at the Institute of Neurology, and Joan intermediate developmental stage (Pannese, 1974). Our Carson at Glasgow University for their excellent technical assis- observations could indicate that the extracellular environ- tance. ment may contain distributed information that influences the morphological differentiation of these neurons. 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