neurite extension across regions of low cell-substratum...

DEVELOPMENTAL BIOLOGY 117.655-662 (1986)

Neurite Extension across Regions of Low Cell-Substratum Adhesivity: Implications for the Guidepost Hypothesis of Axonal Pathfinding

JAMES A. HAMMARBACK’ AND PAUL C. LETOURNEAU

According to the adhesive “guidepost” hypothesis, pioneer axons follow pathways marked by specific nonadjacent cells (guidepost cells). The hypothesis implies that high adhesivity between extending axons and guidepost cells facilitates axon extension across low-adhesivity tissues or spaces between guidepost cells. This study investigates the ability of a high-adhesivity substratum to promote axonal extension across a low-adhesivity substratum in vitro. Dissociated chick embryo dorsal root ganglion neurons are cultured on a substratum consisting of areas of high-adhesivity substratum- bound laminin (i.e., model adhesive guideposts) separated by a low-adhesivity agarose substratum. Increasing the cell- substratum adhesivity of these guideposts results in an increase in the percentage of neurites spanning a given width of the low-adhesivity substratum. Filopodial processes at the tips of neurites can extend over the low-adhesivity substratum. Apparently, filopodial contact with high-adhesivity guideposts enables neurites to extend across intervening low-adhesivity substrata. The maximum width of low-adhesivity substratum discontinuities spanned by some neurites in vitro is comparable to the distance between some putative guidepost cells in insects. Consistent with the adhesive guidepost hypothesis, these findings demonstrate neurite extension on a substratum of discontinuous cell-substratum adhesivity. (cl 1986 Academic Press, Inc.

INTRODUCTION

During development of invertebrate and vertebrate nervous systems, extending axons make stereotyped di- rectional changes (Keshishian and Bentley, 1983a,b; Lance-Jones and Landmesser, 1981a,b). The first axons taking these distinct paths have been called “pioneers” (Harrison, 1910; Bate, 1976). In some cases, pioneer axons in grasshoppers appear to extend along pathways that include specific non-adjacent cells called “guidepost cells” or “landmark cells” (Bate, 1976, 1978; Goodman cf uZ., 1982; Ho and Goodman, 1982; Keshishian and Bentley, 1983a). Elimination of a guidepost cell can block full development of an axonal pathway in the grasshopper (Bentley and Caudy, 1983). This suggests that the positioning of guidepost cells may be an important en- vironmental fact.or in growth cone navigation, though a significant portion of some insect axonal pathways is formed in the absence of putative guidepost cells (Berlot and Goodman, 1984).

The pioneer axons’ affinity for guidepost cells may be explained by increased adhesivity of the growth cone (i.e., the tips of extending neurites) to guidepost cells relative to neighboring tissue. Growth cones tend to form extensive lamellae when cultured on highly adhesive substrata but not on substrata of low adhesivity (Le-

’ To whom correspondence should be addressed: Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Mass. 01545.

tourneau, 1975). If this correlation holds for insect axons in v)ivo, the morphology of some pioneer growth cones indicates that guidepost cells are of high adhesivity and that lower adhesivity tissue exists between guidepost cells (Caudy and Bentley, 1986). These discontinuities in adhesivity would require pioneer growth cones to extend through low-adhesivity tissues in order to extend between high-adhesivity guidepost cells along their pathway; this is not consistent with the tendency for neurites to remain on the most adhesive substratum available (Letourneau, 19’77; Hammarback et uh, 1985). If the high adhesivity of the next guidepost cell could be detected by growth cones prior to axon extension over tissue of low adhesivity, growth cones may, in effect, remain in continuous contact with a substratum of high-adhesivity during axon extension.

Fine cell processes (filopodia) extending from the growth cone can extend across low-adhesivity substrata to contact distant high-adhesivity substrata. Filopodia can extend directly into liquid culture medium (Nakai and Kawasaki, 1959) whereas axon extension requires a solid substratum (Harrison, 1910). Filopodia can be more than 50 pm long; long enough to bridge the gap between some guidepost cells in viva (Taghert et ah, 1982).

If one assumes guidepost cells are of high adhesivity, filopodia may mediate axonal guidance on discontinuous pathways of guidepost cells by extending randomly over low-adhesivity substrata until a guidepost cell is en- countered.

0012-1606/86 $3.00 Copyright B 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

656 DEVELOPMENTAL BIOLOGY VOLUME 117,1986

Extending axon-like processes (neurites) can follow continuous pathways of artificial substrata that have greater adhesivity than the surrounding substratum (Letourneau, 1977). The basement membrane molecule laminin was shown to guide neurite outgrowth in a manner consistent with guidance by increased adhesivity (Hammarback et al., 1985). However, despite the popu- larity of the adhesive guidepost hypothesis, filopodial contact with distant adhesive guideposts has not been shown to enable neurites to extend across low-adhesivity substrata that otherwise would not be crossed.

The use of ultraviolet-light to create microscopic protein pathways (Hammarback et al., 1985) is modified here to investigate the adhesive “guidepost” hypothesis. The ability of axons to extend on a discontinuous high-adhesivity substratum composed of the basement membrane protein laminin is measured. The affect of increasing the adhesivity of these artificial guideposts on the ability of axons to span low-adhesivity substratum is also examined.

MATERIALS AND METHODS

Cell culture. Chick dorsal root ganglia (DRG) from 8- to lo-day embryos were dissected and dissociated as described previously (Rogers et a,Z., 1983). Cells plated on patterned substrata were cultured in serum-free F-14 medium (Gibco) supplemented with 5 pg/ml insulin, 10 rig/ml B-nerve growth factor, 5 rig/ml sodium selenite, 100 pg/ml human transferrin (Sigma), 2 mMglutamine, and 1X antibiotic-antimycotic mixture (Gibco). Cultures were maintained in 5% CO8 and 95% room air in a humidified atmosphere. In some experiments, dissociated DRG or dissociated sympathetic chain ganglia were plated on palladium patterned agarose substrata (see below). Sympathetic ganglia were dissociated from 8- to lo-day embryos as described for DRG and plated in serum-free F-14 medium with the supplements listed above.

Substrata preparation. Discontinuous substrata were prepared by adsorption of laminin to discrete areas of a substratum composed of agarose and albumin. A mixture of 1.5% low gelling temperature agarose (Type VII, Sigma) and 1.0% w/v fatty-acid-free bovine serum albumin (Fraction V, Miles Scientific) is prepared by heating the agarose in dHz0 until melted, cooling the agarose to 43”C, and adding the bovine serum albumin (albumin). The melted agarose-albumin mixture is passed through a 0.45 pm filter (Millipore) and applied to acid-cleaned, dry-heat sterilized, glass coverslips resting in a loo-mm petri dish. Excess agarose-albumin is removed with suction at the edge of the petri dish. The agarose-albumin on the coverslips is gelled and dried overnight in a tissue culture hood. Electron microscope grids having a variety of grid-bar widths are

gently placed on the air-dried agarose-albumin-coated coverslips. These coverslips with grids are placed 40 cm from an ultraviolet light in a standard tissue culture hood and irradiated for 2 hr. The ultraviolet light is a Westinghouse Sterilamp 782L-30 which when new is reported by the manufacturer to emit 65 hw/cm’ at a distance of 1 m. The principal wavelength emitted is 2537 A. In uv-irradiated areas, the albumin is crosslinked and subsequently can adsorb laminin (see below). Laminin was applied to the uv-irradiated agarose-albumin coverslips in Voller’s carbonate buffer, pH 9.6. Laminin (generously provided by Dr. S. Palm and Dr. L. Furcht) was isolated from the Englebreth-Holm Swarm (EHS) tumor. The laminin isolation procedure is given in detail elsewhere (Palm and Furcht, 1983). The concentration of laminin applied to the uv-irradiated albumin de- pended on the particular experiment being performed. One hundred microliters of laminin was applied to square 18-mm coverslips for 1 hr at 37°C. Unbound laminin is aspirated from the coverslips and they are rinsed thoroughly with Ca2- Mg2-free phosphate-buffered saline.

In some experiments, agarose substrata patterned with palladium metal were used. Acid-cleaned coverslips were coated with 1.5% agarose. The agarose was gelled and dried onto the coverslips and electron microscope grids were placed on their surface. These coverslips with grids were placed in a vacuum-evaporator (Denton) and a 1.5-in. length of 0.008 in. diameter palladium wire was evaporated 5 in. from the coverslip surface.

Adhesion assays. Adhesion assays were performed on square 22-mm agarose-albumin-coated coverslips that were uniformly uv-irradiated for 2 hr by the sterilamp at a distance of 40 cm. A razor blade was used to cut a groove in the uv-irradiated agarose-albumin coating. The groove ran diagonally across the coverslips. On half of the coverslip 75 ~1 of 100 pg/ml laminin was applied, the diagonal groove kept the laminin from spilling over to the opposite half of the coverslip. After 1 hr at 37°C the coated half of the coverslip was rinsed with Voller’s carbonate buffer and the opposite half of the coverslip was coated with 40, 50, or 100 pg/ml laminin. The coverslips were incubated again for 1 hr, rinsed with Voller’s buffer, dH20, and then placed in 35 mm petri dishes. Dissociated dorsal root ganglia cells were plated on these coverslips (approximately 125,000 cells/coverslip) in 1.5 ml of Ham’s F-12 containing 10% fetal calf serum and 10 rig/ml B-nerve growth factor. The cells were cultured in 5% CO, at 37°C.

After 3 hr of culture, 1 ml of medium (37°C) was added to each dish and they were rotated on a rotatory shaker for 1 min at 100 rpm. The coverslips were rinsed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 20 min at room temper-

HAMMARBACK AND LETOURNEAU Neurites Extend m Guideposts 657

ature, rinsed with dHa0, and mounted in Elvanol. The cells in 10 consecutive 16X objective fields parallel to and on each side of the groove were counted. The fields counted were two 16X objective fields distant from the diagonal groove.

Immunochemistry. Immunochemistry was performed on laminin patterned coverslips. Coverslips with cells were fixed with 4% paraformaldehyde for 20 min, rinsed, and incubated with 1 mg/ml albumin in PBS for 15 min at room temperature. The preparation and character- ization of affinity-purified rabbit antibodies against laminin has been described previously (Rogers et al., 1983). Anti-laminin (ODz8,, 0.336) was diluted l/100 in PBS containing 1 mg/ml albumin and 80 ~1 was applied to each coverslip for 1 hr at 37°C in a humidified cham- ber. Controls were incubated with rhodamine-conjugated rabbit anti-mouse IgG fraction (4 mg/ml antibody, Cappel) at a l/1000 dilution. The coverslips were rinsed by immersion in large volumes of PBS followed by re- moval of excess PBS by touching the coverslip edges to a paper towel; this procedure was repeated three times. A further rinse of each coverslip in three consecutive 5- ml PBS rinses lasting 15 min each with periodic agitation was performed. The secondary antibody was a peroxi- dase-conjugated goat anti-rabbit IgG antibody (Cappel) diluted l/250. The secondary was applied and unbound antibody rinsed off exactly as for the primary antibody. Finally, the coverslips were rinsed in 50 mMTris buffer, pH 7.6. Four milliliters of 0.5 mg/ml diaminobenzidene (DAB) in Tris buffer containing 0.3 pi/ml of 30% HzOe was added to each 35-mm dish containing a coverslip. Experimentals and controls were incubated for approximately 5 min at room temperature. The reaction was stopped by dilution with dHBO and the coverslips mounted in Elvanol.

Measurement of neurite extension across low-adhesivity substratum. The ability of growth cones to extend across low-adhesivity substratum separating spots of adsorbed laminin was measured by culturing dissociated DRG for 2 days on uv-patterned laminin-exposed substrata (see above). Immunochemistry was performed on these cultures to localize adsorbed laminin. The number of axons crossing a given width of low-adhesivity substratum was recorded as was the number of growth cones that had extended to the edge of the high-adhesivity laminin-coated agarose-albumin squares corresponding to the squares of electron microscope grids. At sites where immunochemistry indicated contaminating laminin was present on the unirradiated agarose-albumin or a non-nerve cell had spanned this low-adhesivity substratum, axons and growth cones were not counted.

Measurement of jilopodia length. The length of filopodia extending on uv-irradiated laminin-coated agarose-albumin was measured using an Optomax System

IV automated image analyzer (Optomax, Inc., Hollis, N.H.). The neurons had been cultured for 2 days and fixed with 2% glutaraldehyde in PBS for 1 hr. The number of filopodia per growth cone was also recorded.

Time-lapse video microscopy. Crossing of low-adhesivity substratum by living cells was observed by culturing dissociated DRG cells on coverslips mounted over holes drilled in 50-mm petri dishes. A mixture of bees wax, white petrolatum, and paraffin (1:l:l) was melted and used to seal the coverslips into petri dishes. The melted agarose-albumin mixture was applied to the mounted coverslips and they were patterned using ultraviolet light and treated with 100 pg/ml laminin as described above. The cells were cultured overnight in F- 12 buffered with Hepes and supplemented as for the F- 14 medium described above. The cultures were then placed on a Zeiss IM microscope stage heated to 37°C with an air-stream incubator (Nicholson Precision In- struments). A SIT camera (Model 65; Dage-MTI, Inc.) and a time-lapse video recorder (Panasonic NV-8030) were used to record the motility of the extending neurites. Pictures were taken through the microscope during the recording using Pan-X film and a SLR camera.

RESULTS

Neurites can extend across narrow gaps of agarose- albumin substratum, of low cell-substratum adhesivity, separating laminin substrata of higher adhesivity (Fig. 1). These patterned substrata are created by uv irradiation of agarose-albumin-coated glass partially masked by electron microscope grids. The selective adsorbtion of laminin onto uv-irradiated agarose-albumin creates square areas of laminin-coated substratum corresponding to openings in the electron microscope grid that al- lowed ultraviolet light to hit the agarose-albumin. The narrow lanes of agarose-albumin substratum, protected from uv irradiation by the electron microscope grid-bars, adsorb less laminin, as indicated by immunochemistry (not shown). These unirradiated lanes retain the low- adhesivity of agarose-albumin that has not been exposed to laminin. The spatial relationships of cells to agarose- albumin and uv-irradiated agarose-albumin can be observed directly because uv-irradiated agarose-albumin is darker than unirradiated agarose-albumin (Fig. 1). Immunochemistry resulted in a subtle change in color of the uv-irradiated areas exposed to laminin and al- lowed precipitates of laminin that occasionally contam- inated the unirradiated agarose to be identified (not shown).

The low cell-substratum adhesivity of agarose-albumin is evident where this substratum is contiguous with uv-irradiated agarose-albumin that has adsorbed laminin. Adherent neurons are only found on the laminin

658 DEVELOPMENTAL BIOLOGY VOLUME 117, 1986

FIG. 1. Dissociated DRG cells cultured on areas of adhesive laminin substrata separated by nonadhesive agarose substrata. An agarose- albumin mixture was dried onto glass coverslips that were then masked with electron microscope grids and uv irradiated. After uv irradiation, grids are removed and laminin is applied to the substrata. The rela- tionship of cells to the uv-irradiated areas can be seen because these areas are phase-dark relative to the unirradiated areas. If the unirradiated gaps between uv-irradiated substrata are not too wide (top photo; IO-Frn gaps), neurites can extend across them. If these gaps are wider, neurites do not extend across them (bottom photo; 40-pm gaps). Cells do not attach or extend neurites on unirradiated agarose-albumin alone.

treated uv-irradiated substratum and not on the agarose-albumin substratum (Fig. 1). The absence of neurons and neurites on agarose-albumin does not appear to be caused by toxicity because neurons are found at the border of agarose-albumin substrata and neurites remain extended across narrow expanses of agarose- albumin (Fig. 1).

Time-lapse video microscopy indicates several distinct steps in neurite extension across low-adhesivity substrata (Fig. 2). First, many filopodia are extended and retracted over the agarose-albumin substratum from growth cones on laminin coated areas. The filopodia observed extending over the agarose-albumin substratum are in constant motion. Eventually, some filopodia ex-

FIG. 2. Photographs of a living growth cone extending across low- adhesivity agarose-albumin from laminin-coated uv-irradiated agarose-albumin. In the top photograph, growth cones “A” and “IS” extend filopodia and lamellae over the unirradiated agarose-albumin. Growth cone B has retracted in the middle photograph while growth cone A has extended a filopodium (F) across the lo-pm gap of unirradiated agarose-albumin to contact the distant phase-dark area of laminin-coated uv-irradiated agarose-albumin. The neurite then ex- tends across the gap with a rounded-up growth cone that spreads out after contacting the distant high-adhesivity substratum. This neurite is then used by other growth cones to cross the unirradiated agarose- albumin (C).

HAMMARBACK AND LETO~JRNEAU New&es Extend on Guideposts 659

TABLE 1 NEURITE CROSSINGS OF LOW-ADHESIVITY AGAROSE SUBSTRATA SEPARATING HIGH ADHESIVITY LAMININ SUBSTRATA

Neurites crossed/non-crossed (percentage crossed)*

Laminin applied to uv-agarose-albumin substratum” (fig/ml) 10-15

Width of nonadhesive substratum (bm)

17-22 27-32 34-39 44-49

40 21139 (54) ND 5180 (6) O/48 (0) O/48 (0) 50 192/311(62) 23157 (40) 13/94 (14) 29/122(24) 41153 (3)

100 1871234 (80) 23154 (43) 84/l% (47) 1171249 (47) 38/202 (19)

’ One hundred microliters of laminin at the designated concentration was applied to 22 mm square coverslips bearing electron microscope grid patterns of uv-irradiated agarose-albumin. Three coverslips were prepared for each laminin concentration reported. Dissociated DRG cells were cultured on these substrata for 2 days in serum-free medium before counting the number of neurites crossing the low-adhesivity agarose-albumin substratum from high-adhesivity laminin-coated uv-irradiated agarose-albumin. See Materials and Methods for further details.

’ “Neurites crossed” are those crossing a given width of low-adhesivity substratum separating areas of high-adhesivity substratum. “Non- crossed” neurites are those with growth cones at the border between low-adhesivity substratum and high-adhesivity substrata. “Percentage crossed” equals lOO[Neurites Crossed/(Neurites Crossed + Non-crossedj].

tend completely across a given width of agarose-albumin substratum and become immobile in the plane of focus of the adhesive substratum. Then, a narrow rounded-up growth cone rapidly crosses the agarose-albumin. The filopodia that initially crossed the agarose-albumin are still intact. Immediately upon crossing the nonadhesive substratum, the growth cone flattens and spreads on the laminin-treated uv-agarose-albumin. The extent of growth cone spreading is indicative of cell-substratum adhesivity; the narrow rounded-up growth cone is typical of growth cones on low-adhesivity substrata while the broad flattened growth cone is typical of growth cones on high-adhesivity substrata (Letourneau, 1979). The neurite that now spans the agarose-albumin serves as a bridge for other neurites enabling them to cross rapidly (Fig. 2).

As the amount of laminin applied to electron microscope grid patterns of uv-irradiated agarose-albumin is increased, the percentage of neurites spanning a given width of agarose-albumin substratum is increased (Ta- ble 1). For a given amount of laminin applied to the

substratum, the percentage of neurites crossing the nonadhesive substratum decreases as the width of nonadhesive substratum is substantially increased. How- ever, the percentage of neurites crossed did not always decrease for small increases in the width of non-adhesive substratum (Table 1). It is possible that changes in the geometry of laminin guideposts that coincide with changes in the width of nonadhesive substratum may account for some of this variability. These coincident changes in shape of the laminin guideposts results from the fact that electron microscope grids having different grid bar widths tend to have open areas with slightly different geometries.

In addition to increasing the percentage of neurites crossing a given width of nonadhesive substratum, increasing the amount of laminin applied to uv-irradiated agarose-albumin increases the adhesivity of this substratum as detected by the resistance of DRG cells to detachment from the substratum by shearing forces (Table 2). The apparent importance of filopodia in neurite extension across the narrow gaps of low-adhesivity aga-

TABLE 2 LAMININ CONCENTRATION EFFECTS ON DRG CELL ADHESIVITY TO UPIRRADIATED AGAROSE-ALBUMIN

Laminin applied to left half/ laminin applied to right half

of agarose-albumin-coated coverslip (fig/ml)

I Cells bound to left

half of coverslip

II Cells bound to right

half of coverslip

III Column I/column II

(x I!I SE, N = 3)

30/100 132 2494 0.088 f 0.027 501100 639 2262 0.400 f 0.120

100/100 3742 3935 0.967 + 0.032

Note. Agarose-albumin-coated coverslips were uv irradiated for 2 hr at a distance of 40 cm from a bactericidal lamp. Each half of these coverslips was then exposed to 100 ~1 of the designated concentration of laminin for 1 hr. DRG cells were plated on these coverslips for 3 hr and then placed on a rotatory shaker for 30 set at 100 rpm. The number of cells remaining bound to each half of the coverslips is reported in Column I and Column II (see Materials and Methods for sampling method). Column III is the ratio of cells in Column I to cells in Column II.


TABLE 3 FILOPODIAL LENGTH AND NUMBER PER GROWTH CONE ON LAMININ TREATED UV-IRRADIATED AGAROSE-ALBUMIN SUBSTRATA

Laminin applied to Average f SE

uv-agarose- albumin Filopodial length Filopodia/growth Filopodia Growth cones

substratum (rg/ml) (A cone measured counted

40 12.2 f 1.1 4.620.9 116 25

50 12.7 f 1.1 5.6 + 1.1 141 25

100 11.7 * 1.0 5.0 f 1.0 124 25

Note. One hundred microliters of the designated concentration of laminin was applied to uv-irradiated agarose-albumin on 22-mm square coverslips for 1 hr. Dissociated DRG cells were cultured on these substrata (triplicate coverslips for each concentration of laminin) for 2 days and then fixed with glutaraldehyde. Filopodia were measured using an automated image analyzer. See Materials and Methods for further details.

rose-albumin suggested that the correlation between adhesivity and ability to cross agarose-albumin may be related to the ability to form filopodia as substrate adhesivity is increased. Filopodial length and the number of filopodia per growth cone were measured on 40, 50, and 100 pg/ml laminin-exposed uv-irradiated agarose- albumin (Table 3). No significant difference in length or number of filopodia was found. However, we did not measure the turnover rates of filopodia on these substrata.

Laminin does not appear to increase the adhesivity of unirradiated agarose-albumin. Unirradiated agarose- albumin remains incapable of supporting cell attach- ment even after exposure to 100 pg/ml laminin, the highest concentration tested (Fig. 1). Also, neurite extension does not occur on unirradiated agarose-albumin exposed to 40, 50, or 100 pg/ml laminin. A neurite or neuron was occasionally found on unirradiated agarose- albumin. However, immunochemistry later showed streaks of contaminating laminin at these sites (not shown). Thus, the correlation between the amount of laminin applied to uv-patterned substrata and the ability

of neurites to cross agarose-albumin is not related to a measurable increase in adhesivity of the unirradiated agarose-albumin.

The ability of neurites to extend on discontinuous substrata prepared by an alternate method is similar to their ability to extend on laminin-patterned substrata. Discontinuous substrata were constructed of palladium evaporated onto dried agarose partially masked by electron microscope grids. The palladium substratum sup- ports neurite outgrowth without being coated with laminin. Thus, the low-adhesivity agarose substratum that separates palladium substrata is never exposed to laminin. Despite the absence of laminin, the ability of DRG cell neurites to cross from one palladium surface to an- other is comparable to that found in uv-patterned agarose-albumin substrata exposed to 100 pug/ml laminin. Sympathetic ganglion neurites extended as well as DRG neurites on discontinuous palladium substrata (Table 4).

DISCUSSION

Our results demonstrate neurite outgrowth on discontinuous pathways of substratum-bound molecules

TABLE 4 DRG AND SYMPATHETIC GANGLION NEURITE CROSSINGS OF LOW-ADHESIVITY ACAROSE SUBSTRATUM FROM PALLADIUM SUBSTRATUM’

Neurites crossed/Non-crossed (percentage crossed)*

Width of low-adhesivity substratum (rm)

Cell type O-10 10-20 20-30 30-40 40-50 50-60 60-70

Dorsal root ganglion Sympathetic ganglion

17714 (98) 605/25(96) 182/104 (64) 29156 (34) 21/258(g) N.D. O/70 (0) 77/9 (90) 71/41 (63) N.D. 77/135 (36) l/23 (4) o/35 (0) N.D.

“Substrata were patterned by evaporating palladium metal onto agarose-coated coverslips masked by electron microscope grids; three coverslips were made for each cell type. Neurons were obtained by dissociation of dorsal root ganglia (DRG) or sympathetic chain ganglia. Dissociated cells were cultured on these substrata for 2 days in serum-free medium before counting the number of neurites crossing the IOW- adhesivity agarose substratum from high-adhesivity palladium substratum. See Materials and Methods for further details.

* “Neurites crossed” are those crossing a given width of low-adhesivity agarose substratum separating areas of high-adhesivity palladium substratum. “Non-crossed” neurites are those with growth cones at the border between low-adhesivity agarose and high-adhesivity palladium substrata. “Percentage crossed” equals lOO[Neurites Crossed/(Neurites Crossed + Non-crossed)].

HAMMARBACK AND LETOURNEAU Neurites Extend on Guideposts 661

that enhance cell-substratum adhesivity. Thus, if the adhesivity of the alternative substratum is sufficiently low, appropriately spaced adhesive guideposts could guide neurite outgrowth. The 40-50 pm discontinuities of low-adhesivity agarose-albumin that are spanned by some DRG neurites are comparable in width to the apparent discontinuities of low adhesivity spanned by pioneer axons in the grasshopper (Caudy and Bentley, 1986).

The direct observations of neurites extending across low-adhesivity substrata show filopodia responding dif- ferently to contact with low-adhesivity versus high-adhesivity substrata. Growth cones extend over low-adhesivity agarose-albumin after their filopodia have spanned the low-adhesivity substratum to contact an adhesive area (Fig. 2). Time-lapse movies have previously shown that filopodia contact and bend other neurites suggesting that filopodia adhere sufficiently strongly to transfer force generated in the filopodia, growth cone, or neurite (Nakai, 1960; Wessells et al., 1980). Filopodial contact with adhesive substrata may lower the requirement for substratum adhesivity of the growth cone proper and may support the tension of the neurite while the growth cone advances.

Increasing the adhesivity of the discontinuous adhesive areas increases neurite extension on these pathways. This raises the possibility that filopodia may be capable of detecting relatively small changes in substratum adhesivity. The adhesivity of DRG cells to uv-irradiated agarose-albumin is increased by application of higher concentrations of laminin. However, the relevance of DRG cell body adhesivity to filopodial adhesion is not well established. Because of technical difficulties, we were not able to control for effects of increased adhesivity on the neurite, growth cone, or filopodia that occur prior to filopodial contact with distant adhesive guideposts. We do know that filopodia length and number per growth cone do not change significantly over the range of substratum-adhesivity tested. We can conclude that, within the range of substratum adhesivity tested, increasing adhesivity results in increased neurite extension on substrata of discontinuous adhesivity.

The adhesivity of unirradiated agarose-albumin does not appear to be increased by exposure to increasing concentrations of laminin. It is likely that an increase in adhesivity of unirradiated agarose-albumin would facilitate neurite extension on discontinuous substrata resulting in the observed correlation between substratum adhesivity and neurite extension. However, extension of neurites on unirradiated agarose-albumin is very rare at all concentrations of laminin tested. An obvious increase in the amount of laminin bound to the 40 wg/ ml versus the 100 pug/ml laminin-exposed unirradiated agarose-albumin is not found by immunochemistry.

Even very high cell density was incapable of promoting neurite extension onto unirradiated agarose-albumin substrata during 2 days of cell culture. On discontinuous palladium substrata, low-adhesivity agarose was not exposed to laminin. On these substrata, neurite extension was as great as neurite extension on discontinuous laminin substrata. Therefore, neurites can extend across narrow lanes of agarose that have not been exposed to laminin. It should be noted, however, that the agarose of palladium substrata was not gelled with albumin and it was exposed to vacuum and heat during palladium evaporation.

It is interesting that the average filopodial length of DRG growth cones is much shorter than the width of low-adhesivity agarose-albumin gaps that can be crossed. Less than 1% of the filopodia are greater than 30 pm but more than 50 pm of low-adhesivity agarose- albumin substratum is occasionally spanned. As men- tioned above, the time-lapse studies indicate growth cones cross the low-adhesivity substratum after a filopodia spans the width of the low-adhesivity substratum (Fig. 2). A corollary is that only the growth cones having filopodia longer than the required distance may be able to cross low-adhesivity gaps. Growth cones with shorter filopodia may cross only by remaining in contact with other neurites that have previously crossed the low-adhesivity gaps. Thus, filopodial length could determine the discontinuous substratum pathways available to a growth cone in viva. The DRG and sympathetic ganglion cell types have approximately the same filopodia lengths and are approximately equal in their ability to extend on discontinuous substrata. In vivo, the adhesivity of tissue surrounding guidepost cells may be great enough to support a limited amount of neurite extension. Thus, growth cones may be expected to span greater widths of low-adhesivity substratum to reach adhesive substrata in vivo than we found in vitro.

The geometry of the laminin guideposts may be significantly different than in vivo guidepost geometry. The guidepost cell may present a smaller target for extending axons than the laminin guideposts do in our model. Other factors being equal, the efficiency of successful crossings of low-adhesivity terrain would then be greater in our model than in vivo. Even for narrow gaps of nonadhesive substratum, the efficiency of neurite crossings of nonadhesive substrata is not close to the 100% that may be required for successful development of axonal pathways in vivo. The low efficiency may be a result of the many continuous adhesive pathways that are available to an extending axon in our model besides the discontinuous adhesive pathway over the low-adhesivity agarose-albumin substratum. Heterogeneity in the DRG cell pop- ulation of neurites undoubtedly contributes to the lower efficiency as well. In any case, our model does not provide


direct evidence for the guidepost hypothesis operating in specific developmental systems. The model does verify the potential for axonal extension on pathways of discontinuous adhesivity that is implied by the adhesive “guidepost” hypothesis.

The methodology applied here may be applicable to the study of non-nerve cell motility. Harris has dem- onstrated that a variety of transformed cells are able to extend across discontinuous adhesive substrata (Harris, 1973). Filopodial or lamellipodial protuberances of invasive cells could contact distant basement membrane structures that contain laminin and other molecules promoting cell adhesion. These may be important steps in the invasion of basement membrane bounded tissues by metastatic cells. The cells could then move to environments supporting cell proliferation. The non- nerve cells in our cultures were qualitatively less able to span the low-adhesivity substratum than neurons; this may be related to the ability to extend filopodia or to the increased tensions that must be stabilized by adhesive contacts of non-nerve cells (Harris et al., 1981).

We are grateful for the expert technical assistance of Terri Shattuck and Janet Balson. This work was supported by National Institutes of Health Grants (HD 19950 and HD 17192), the Spinal Cord Society, and a National Science Foundation Grant (PCM 8203855) awarded to P.C.L.

REFERENCES

BATE, C. M. (1976). Pioneer neurons in an insect embryo. Nature (Lun- don) 260, 54-56.

BATE, C. M. (1978). Development of sensory systems in arthropods. In “Handbook of Sensory Physiology, Vol. IX” (M. Jacobson, ed.). Springer Verlag, New York.

BENTLEY, D., and CAUDY, M. (1983). Pioneer axons lose directed growth after selective killing of guidepost cells. Nature (London) 304, 62- 65.

BERLOT, J., and GOODMAN, C. (1984). Guidance of peripheral pioneer neurons in the grasshopper: Adhesive hierarchy of epithelial and neuronal surfaces. Science 223,493-496.

CAUDY, M., and BENTLEY, D. (1986). Pioneer growth cone morphologies reveal proximal increases in substrate affinity within leg segments of grasshopper embryos. J. Neurosci. 6,364-379.

GOODMAN, C. S., RAPER, J. A., Ho, R. K., and CHANG, S. (1982). Path- finding by neuronal growth cones during grasshopper embryogenesis. In “40th Symposium, Society for Developmental Biology” (S. Sub- telny and P. Green, eds.), pp. 275-316. Alan R. Liss, New York.

HAMMARBACK, J. A., PALM, S. L., FURCHT, L. T., and LETOURNEAU, P. C.

(1985). Guidance of neurite outgrowth by pathways of substratum- adsorbed laminin. J. Neurosci. Res. 13,213-220.

HARRIS, A. K. (1973). Behavior of cultured cells on substrata of variable adhesiveness. Exp. Cell Res. 77,285-297.

HARRIS, A. K., STOPAK, D., and WILD, P. (1981). Fibroblast traction as a mechanism for collagen morphogenesis. Nature (London) 290,469- 251.

HARRISON, R. G. (1910). The outgrowth of the nerve fiber as a mode of protoplasmic movement. J. Exp. 2001. 9,787-848.

Ho, R. K., and GOODMAN, C. S. (1982). Peripheral pathways are pi- oneered by an array of central and peripheral neurones in grasshopper embryos. Nature (Lmdm) 297,404-406.

KESHISHIAN, H., and BENTLEY, D. (1983a). Embryogenesis of peripheral nerve pathways in grasshopper legs. I. The initial nerve pathway to the CNS. Dew. Biol. 96, 89-102.

KESHISHIAN, H., and BENTLEY, D. (1983b). Embryogenesis of peripheral nerve pathways in grasshopper legs. II. The major nerve routes. Dev. Biol. 96,103-115.

LANCE-JONES, C., and LANDMESSER, L. (1981a). Pathway selection by chick lumbosacral motoneurons during normal development. Proc. R. Sot. London B. 214,1-18.

LANCE-JONES, C., and LANDMESSER, L. (1981b). Pathway selection by embryonic chick motoneurons in an experimentally altered envi- ronment. Proc. R. Sot. Lmdm B. 214.19-52.

LETOURNEAU, P. C. (1975). Possible roles for cell-to-substratum adhesion in neuronal morphogenesis. Dev. Biol. 44,77-91.

LETOURNEAU, P. C. (1977). Regulation of neuronal morphogenesis by cell-substratum adhesion. In “Society for Neuroscience Symposium” (W. M. Cowan and J. A. Ferrendelli, eds.). pp. 67-81. Society for Neuroscience, Bethesda.

LETOURNEAU, P. C. (1979). Cell-substratum adhesion of neurite growth cones and its role in neurite elongation. Exp. Cell Res. 124, 127-138.

NAKAI, J. (1960). Studies on the mechanism determining the course of nerve fibers in tissue culture. II. The mechanism of fasciculation. 2. Zellfbrsch. Mikrosk. Amt. 51, 427-449.

NAKAI, J., and KAWASAKI, Y. (1959). Studies on the mechanism determining the course of nerve fibers in tissue culture. I. The reaction of the growth cone to various obstructions. 2. Zellforsch Mikrosk. Amt. 51,108122.

PALM, S. L., and FURCHT, L. T. (1983). Production of laminin and fibronectin by Schwannoma cells: Cell-protein interactions in vitro and protein localization in peripheral nerve in viva .J. Cell Biol. 96, 1218-1226.

ROGERS, S. L., LETOURNEAU, P. C., PALM, S. L. MCCARTHY, J. B., and FURCHT, L. T. (1983). Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin. Dev. Biol. g&212-220.

TAGHERT, P., BASTIANI, M., Ho, R. K., and GOODMAN, C. S. (1982). Guid- ance of pioneer growth cones: Filopodial contacts and coupling re- vealed with an antibody to lucifer yellow. Dev. Biol. 94, 391-399.

WESSELLS, N. K., LETOURNEAU, P. C., NUTTALL, R. P., LUDUENA-AN- DERSON, M., and GEIDUSCHEK, J. M. (1980). Responses to cell contacts between growth cones, neurites and ganglionic non-neuronal cells. J. NeurocytoL 9,647-664.

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