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Neurite Outgrowth on
a
Step Gradient of Chondroitin
Sulfate Proteoglycan (CS-PG)
Diane
M.
Snow
*
and
Paul
C.
Letourneau
Department
of
Cell Biology and Neuroanatomy, University
of
Minnesota, Minneapolis, Minnesota
55455
SUMMARY
Sulfated proteoglycans (PGs) may play
a
significant role
in the regulation of neurite outgrowth. They are present
in axon-free regions of the developing nervous system
and repel elongating neurites in
a
concentration-depen-
dent manner
in
vitro.
The addition of growth-promoting
molecules, such
as
laminin, can modify the inhibitory
effect of PGs
on
neurite outgrowth (Snow, Steindler, and
Silver, 1990b). Substrata containing
a
high-PG/low-la-
minin ratio completely inhibit neurite outgrowth, while
normal, unimpeded outgrowth is observed
on
low-PG
/
high-laminin substrata. Therefore, different patterns of
neurite outgrowth may result from regulation of the ratio
of growth-promoting molecules to growth-inhibiting mol-
ecules. Using video microscopy, embryonic chicken dor-
sal root ganglia neurons (DRG), chicken retinal ganglia
neurons (RGC), and rat forebrain neurons
(FB)
were
analyzed
as
they extended processes from
a
substratum
consisting of laminin alone onto
a
step gradient of in-
creasing concentrations of chondroitin sulfate proteogly-
can (CS-PG) bound to laminin.
In
contrast to neurite
outgrowth inhibition that occurs
at
the border of a single
stripe of high concentration of CS-PG (Snow et al.,
1990b and this study), growth cones grew onto and up
CS-PG presented in
a
step-wise graded distribution.
Al-
though the behavior of the different cell types was
unique, a common behavior of each cell type was a de-
crease in the rate of neurite outgrowth with increasing
CS-PG concentration. These data suggest that appro-
priate concentrations of growth-promoting molecules
combined with growth-inhibiting molecules may regulate
the direction and possibly the timing of neurite out-
growth
in
vivo.
The different responses of different neuro-
nal types suggest that the presence of sulfated PG may
have
varying
effects
on
different aspects of neuronal de-
velopment.
Key
words:
chondroitin sulfate proteoglycan, neurite out-
growth, step gradient, growth cone, laminin.
INTRODUCTION
Studies of sulfated proteoglycans have shown a sig-
nificant role for these molecules in influencing the
outgrowth and/ or directional choices of elongat-
ing neurites (Brittis, Canning, and Silver
(
1992);
Carbonetto, Gruver, and Turner, 1983; Cole and
McCabe, 199
1
;
Fichard, Verna, Olivares, and
Saxod, 199
1;
Oohira, Matsui, and Katoh-Semba,
199
1;
Sheppard, Hamilton, and Pearlman, 199
1;
Snow, Steindler, and Silver, 1990a; Snow et al.,
Received January 31, 1991; accepted February 11, 1992.
Journal
of
Neurobiology,
Vol.
23,
No.
3, pp. 322-336
(1992)
0
1992 John Wiley
&
Sons,
Inc.
CCC
0022-3034/92/030322-
15$04.00
*
To
whom correspondence should be addressed.
322
1990b; Snow, Watanabe, Letourneau, and Silver,
1991; Verna, Fichard, and Saxod, 1989). Once
thought of mainly as extracellular matrix mole-
cules of cartilage and connective tissues, sulfated
proteoglycans are now known
to
be present in the
central nervous system (CNS) and peripheral ner-
vous system
(PNS)
in association with a variety of
cell types and may have numerous roles in the de-
veloping and the adult animal.
Certain sulfated proteoglycans (PG) are ex-
pressed in regions where neurons do not grow
in
vivo
and are inhibitory to neurite outgrowth
in vi-
tro
(Carbonetto et al., 1983; Cole and McCabe,
1991; Fichard et al., 1991; Oohira et al., 1991;
Snow et al., 1990a,b, 1991; Tosney and Land-
messer, 1985; Tosney and Oakley, 1990; Verna et
al., 1989). However, some studies show that cer-
tain PGs are expressed in rat (Sheppard et al.,
I99
1
:
Morigiwa and Silver, unpublished data) and
in chicken (Palmert, Kujawa, Caplan, and Silver,
1986) where neurons elongate
in vuo.
An impor-
tant question, then, is how can sulfated PGs be in-
hibitory under some circumstances and seemingly
permissive for growth under others?
A
number of sulfated proteoglycans have been
shown to be involved in either the support of neu-
rite outgrowth or bind growth-promoting mole-
cules. For example, a complex of heparan sulfate
proteoglycan (HS-PG) and laminin promotes neu-
rite outgrowth as well as regeneration of neurites
(Chiu, Matthew, and Patterson, 1986; Hantaz-
Ambroise, Vigny, and Koenig, I987
)
.
Further evi-
dence that suggests a role for the HS-PG/laminin
complex in neurite outgrowth include the findings
that immunoreactivity for laminin and HS-PG are
co-localized on rat cerebral cortex astrocytes (Ard
and Bunge,
1988),
a rat Schwannoma neurite-pro-
moting factor is thought to be a complex of la-
minin and HS-PG (Davis, Manthorpe, Engvall,
and Varon, 1985) and identification of
a
growth-
promoting factor from corneal endothelial cell
conditioned media, in which the growth-promot-
ing activity was dependent upon complexing with
heparan sulfate proteoglycan (Lander, Fujii, and
Reichardt, 1985
).
Further, chondroitin sulfate pro-
teoglycan (CS-PG
)
binds to basic fibroblast growth
factor (FGF) (Walicke, 1988), cytotactin (Hoff-
man and Edelman, 1987). and laminin (Snow et
al., 1990b). Thus, a variety of cellular interactions
exist involving proteoglycans, complicating analy-
sis of the function of these molecules.
In
contrast to a possible growth-promoting role
for some sulfated PGs, a larger number of sulfated
PGs inhibit cell adhesion, cause growth cone col-
lapse
in vitro,
and/or inhibit or repel elongating
neurites (Carbonetto et al.,
1983;
Cole and
McCabe, 199
1
:
Davies, Cook, Stern, and Keynes,
1990: Fichard et al., 1991; Knox and Wells, 1979;
Oohira et al., 1991; Rich, Pearlstein, Weissmann,
and Hoffstein, 1981; Snow et al., 1990a,b, 1991;
Tosney and Oakley, 1990; Verna, 1985
).
Impor-
tantly, there
is
a correlation between the presence
of sulfated PGs
in vivo
and the absence of cell adhe-
sion or neurite outgrowth, i.e., in the roof plate of
the spinal cord (Snow et al., 1990a,b), the pelvic
girdle, and perinotochordal mesenchyme (Pett-
way, Guillory, and Bronner-Fraser, 1990; Tosney
and Oakley, 1990), the posterior somite (Stern and
Keynes, 1987; Tosney and Landmesser,
1985;
Tos-
ney and Oakley, 1990), epidermis (Fichard et al.,
199
1; Funderburgh, Caterson, and Conrad, 1987:
Verna, 1985; Vernaet al.,
1989),
and the roofplate
of
the hamster tectum (Snow
et
al., 1990a;
WU,
Silver, Schneider, and Jhaveri, I99
1
).
CS-PG, although present in regions of the brain
where neurites do not grow, is expressed in some
regions
of
the brain where neurons
do
grow.
CS-
PG is expressed in the subplate of the rodent fore-
brain (Palmert et al., 1986; Sheppard and Pearl-
man, 1990; Sheppard et al., 1991) a region in
which neurons actively extend axons (Shoukimas
and Hinds, 1978). Retinal ganglion cells (RGCs)
also extend processes in regions expressing CS-PG,
although immunostaining suggests that much less
CS-PG is present in the areas containing RGCs
than in areas that lie peripheral to the RGCs (Snow
et al., 199
1
).
In
vitro
data further suggests that inhi-
bition of RGC axon outgrowth by CS-PG is con-
centration dependent (Snow et al., 199
1
).
Neurite outgrowth
in
vitro
on different combina-
tions of either CS-PG, keratan sulfate proteoglycan
(KS-PG), or dermatan sulfate proteoglycan (DS-
PG) with laminin varies from maximal outgrowth
to complete inhibition, depending upon the ratio
of the sulfated proteoglycans to laminin in the mix-
tures (Snow et al., 1990b). When the concentra-
tion of laminin in a mixture with PG is low, the PG
acts as a molecular barrier to neurite outgrowth,
causing growth cones to stop and/or turn. Alterna-
tively, when a high concentration of laminin is
mixed with the PG, neurites grow across a PG/la-
minin substratum.
In the present study, we have developed a tech-
nique to produce a step gradient of CS-PG, bound
to a laminin substratum,
in
Llitro.
Using this para-
digm, different combinations of a growth-promot-
ing (laminin) and a growth-inhibiting molecule
(CS-PG) could be tested that include ratios inter-
mediate to the extremes of high PG/low laminin
and high laminin/low PG. Using video micros-
copy, the rate of outgrowth and outgrowth patterns
of chick dorsal root ganglia (DRG), chick retinal
ganglia (RGC), and rat forebrain (FB) neurites
were analyzed as they traveled from a substratum
consisting of only laminin onto the CS-PG step
gradient in the direction of increasing concentra-
tions of CS-PG bound to laminin.
Three conclusions can be drawn from these stud-
ies. First, growth cones that were unable to grow
onto CS-PG when confronted by a large step in
concentration of CS-PG bound to laminin (single
stripe assay) were able to elongate
on
the same con-
centration of CS-PG when it was presented as part
of a gradual step gradient. Second, elongation rates
decreased as growth cones grew up a concentration
step gradient of CS-PG. Third, each cell type stud-
324
Snow and Letourneau
ied displayed certain growth patterns unique to
that cell type.
Taken together, the above results indicate that
CS-PG, and possibly similar molecules, may be
present yet allow neurite outgrowth under some
conditions and be growth-inhibiting under others.
Further, responses to CS-PG may differ depending
upon the cell type. Interaction of growth cones
with a specific ratio
of
laminin, or other growth-
promoting/adhesive molecules (e.g., NCAM,
L1,
fibronectin) to CS-PG
(or
perhaps other proteogly-
cans or growth-inhibiting molecules) may provide
a mechanism for the regulation of the timing as
well as direction of neurite outgrowth in a given
region of the developing brain.
MATERIALS AND METHODS
Preparation of Tissue Culture Plates
Glass coverslips (24
X
30
mm) were washed with
20%
sulfuric acid, rinsed in distilled water, treated with
0.
I
A4
sodium hydroxide, rinsed again with distilled water and
0.1
M
phosphate-buffered saline (PBS), then air dried.
The coverslips were mounted with a mixture of warmed
paraffin, petroleum jelly, and bee’s wax
(
I
:
1
:
I
)
over
20-
mm holes drilled in the center of
60-
X
10-mm tissue
culture dishes (Falcon Labware, Oxnard, CA) and
stored until needed.
Preparation of Chondroitin Sulfate
Proteoglycan (CS-PG) Step Gradient
The preparation of the CS-PG step gradient is schema-
tized
in
Figure
1.
Laminin
(25-100
pg/ml) was applied
over an area of the glass coverslips
equal
to 3 14 mm2 at
4°C for
4
h
or
overnight. The coverslips were then
washed with Voller’s carbonate buffer (pH
9.6)
and al-
lowed to air dry. Strips
of
cellulose paper (Whatman
filter paper cut to
2
cm
X
400 pm) were soaked in
5
pl
of
I
.O
mg/ml chondroitin sulfate proteoglycan (from
bo-
vine nasal cartilage monomer; kindly provided by
L.
Culp, Case Western Reserve University) mixed with
rhodamine isothiocyanate (RITC,
5%),
then transferred
to the laminin-coated coverslip. Three parallel strips
were applied per coverslip and allowed to air dry. Con-
trol
plates received no further treatment.
(Note:
To
pro-
vide parallel experiments, it is
to
these
control plates that
neurite outgrowth on the
CS-PG
step
gradient is com-
pared since the experiments in Snow et al., 1990b used
CS-PG
stripes coated with laminin and not vice versa.
Importantly, either order
of
addition
of
CS-PG
and
la-
minin in the single stripe assay mulied in complete
inhi-
bition
of
neurite outgrowth.)
Other controls consisted of
laminin stripes superimposed on a laminin background
to rule out mechanical influences. For step gradient
plates,
a
second dose
(3-5
pl)
of CS-PG was applied
to
the cellulose strips after they had completely dried from
the first application. This resulted in some of the second
application of CS-PG “spilling” over the edge of the
border of the first stripe of CS-PG. The application pro-
cess was repeated three times per strip. Following the last
application
of
CS-PG, the cellulose strips were com-
pletely air dried and removed from the dish.
The result of the above procedure was a step gradient
of CS-PG bound to a laminin-containing substratum
where CS-PG increased in concentration in a step-wise
manner (Fig.
2).
The step gradient was detected using
epifluorescence microscopy, which showed that the
center of each stripe was brightest and fluorescence de-
creased with distance from the center toward each edge
of the CS-PG stripe. The width of the outer two tiers on
each side of the central strip was variable, but ranged
between
30
and
60
pm. Each CS-PG step gradient con-
sisted of a stripe of five tiers denoted throughout this
document as PG, (outer tiers
on
each side of the stripe),
PG, (between outer tier and center of stripe on each
side), and PG, (single center stripe approximately
400
pm wide).
Following preparation ofthe step gradient on the cov-
erslip, the tissue culture dishes were covered with media
and stored at
37°C
in a
5%
C02/95%
air incubator for
1-2
h
to preserve fluorescence while the retinal tissue
was dissected (see dissection protocol below).
Characterization of the CS-PG Step
Gradient
The concentration of CS-PG
in
each tier was determined
in
two ways. The first method estimated the amount
of
CS-PG bound to each tier of the step gradient by quanti-
tative fluorescence measurements and comparisons to
the binding of known concentrations of CS-PG bound to
the substratum by a single application. In this case, the
step gradient was made without the addition of RITC,
and the bound CS-PG was labeled with either
of
two
antibodies to chondroitin sulfate:
CS56
(
Avnur and
Geiger,
I985),
which recognizes undigested
CS
chains
or
3-B-3 (Couchman, Caterson, Christner, and Baker,
I984), which recognizes chondroitin-0-, 4-, and
6-sul-
fated “stubs” following digestion with chondroitinase
ABC. Dishes were incubated
in
PBS with
5%
normal
goat serum (NGS) for
30
min, followed by incubation
with primary antibody overnight at 4°C. The dishes were
washed four times with PBS/NGS, followed by incuba-
tion with biotinylated goat anti-mouse IgG
(
I:
100)
for 2
h
at 25”C, then four washes with PBS (no serum), and a
final incubation with streptavidin-Texas Red without
serum
(
1
:
100)
for
2
h
at 25°C. The dishes were washed
four times with PBS and coverslipped. Importantly, the
dimensions of the fluorescent pattern that resulted from
immunolabeling closely resembled the pattern that re-
sulted when RITC was mixed with the CS-PG before
making the gradient.
Fluorescence was quantitated by measuring the fluo-
Neurite Outgrowth on
a
CS-PG
Step
Gradient
325
LN
(50ug/rnl)
4
hrs;
4'C
air dry
CS-PG
RlTC
II
high mag
of
one stripe
(1
rng/rnl)
air
dry
I
CS-PG
+
RlTC
(5111,
3x)
add
E6
retina
ynts
J
incubate
24
hrs
immunolabel video tape
Schematic diagram of the protocol used to make the CS-PG/laminin step gradient;
Figure
1
see Materials and Methods for details.
rescence of each tier using a silicon-intensified target
(SIT)
camera
(65
MK
I1
MTI; Dage-MTI, Michigan
City,
IN;
kindly provided by M. Atkinson, University
of
Minnesota) with a constant gain and voltage setting,
connected to an inverted microscope (IM-35; Carl Zeiss,
Thornwood,
NY).
Images were recorded onto a Pana-
sonic TQ-2026F optical disc time-lapse video recorder
from a Quantex QX-7 Image Processor using the soft-
ware program
ICOS.
The fluorescence of processed
images
of
the three CS-PG tiers were compared to re-
corded images of the fluorescence emitted from known
concentrations of
CS-PG
bound with a single applica-
tion. Each measurement was an average of six readings
from several areas of the tiers.
Detection
of
laminin within the step gradient was de-
termined in a similar manner using rabbit anti-laminin
polyclonal, rat anti-laminin monoclonal,
or
rat anti-la-
minin polyclonal antibodies, all generously provided by
Dr.
A.
Skubitz, Department of Laboratory Medicine and
Pathology, University of Minnesota.
Previous experiments (Snow et al., 1991) showed
that 15%-40% of the laminin and 3%-5% of the proteo-
glycan present in starting solutions bind to nitrocellu-
lose- and silane-coated glass coverslips. However, these
two substrata were not suitable for the present experi-
ments due to their poor optical properties. Therefore, in
the second method for CS-PG quantitation, the concen-
tration of laminin bound to the glass coverslips and CS-
Figure
2
A
CS-PG
step gradient visualized using epifluorescence microscopy of RITC mixed
with the
CS-PG
prior to making the step gradient. The brightest intensity
(PG,)
represents the
center single stripe of
CS-PG.
the middle intensity (PG,: one on each side of PG,) represents
the intermediate concentration of
CS-PG.
and the outer fluorescent stripe
of
least intensity
represents the lowest concentration of
CS-PG
(PG,: one on each side
of
PG,). Step gradients
visualized
by
labeling with antibodies to
CS-PG
(RITC not used; see Materials and Methods)
resulted in a pattern very similar to that observed with epifluorescence of endogenous RITC
(not shown). The darkest area of thc figure (far right) is the area between stripes containing
laminin alone. Scale bar
=
10 fim.
PG
bound to laminin were determined using 'H-laminin
[made in our laboratory by reductive methylation using
sodium cyanoborohydride according
to
a modification
of the protocols of Jentoft and Dearborn
(
1979)
and
Herbst, McCarthy, Tsilibary, and Furcht
(
19SX)]
and
35S-proteoglycan (kindly provided
by
D.
Carrino and
A.
Caplan. Case Western Reserve IJniversity
).
Step gradients were made
on
glass coverslips as de-
scribed above except that 3H-laminin. and 35S-proteogly-
can were substituted
for
unlabeled laminin and
CS-PG,
respectively. 'H-Laminin (50 or
100
pg/ml startingcon-
centration) was applied to glass coverslips overnight at
4°C.
"S-CS-PG
was added to the substratum either one
time
only
by
soaking a cellulose strip in CS-PG, applying
it to the laminin substrata, and removing it when dry,
or
by making two and three additions
of
"S-CS-PG
to the
first strip as described above. Single stripes of3'S-CS-PG
were made in the same manner but ditfered
by
using
starting concentrations of
100,
1000,
3000,
01-
5000
pg/
ml
'3-CS-PG
in combination with 50 or
100
pg/ml
3H-laminin. The dishes were washed with PBS, then
coated with detergent, and incubated
1
h followed by
scrapping with
a
pipet
tip
to remove the radioactively
labeled molecules. The solutions were counted in
a
Beckman LS-380
I
scintillation counter, and the concen-
trations of each molecule were determined from the
counts.
Dissection and Culture
of
Embryonic
Chicken Retinae and
DRGs,
and Rat
Forebrain Neurons
Chick dorsal root ganglia
(E9)
were dissected in
0.1
M
CMF-PBS by decapitation, evisceration, and removal
of
the vertebi-a1 column and spinal cord. The DRGs were
cleaned
of
surrounding tissue and removed with fine for-
ceps into Dulbecco's Modified Essential Medium
(DMEM) containingo.
1
mg/ml transferrin, 20 nMpro-
gesterone, 5 ng/ml sodium selenite,
0.4
mg/ml
sodium
pyruvate, 5
mM
phosphocreatine. 5
pg/ml
insulin.
10%
fetal bovine serum, glutaniine, and an antibiotic mixture
of penicillin, streptomycin, and fungizone (PSF). With
a
finely drawn glass Pasteur pipette. the DRG explants
were scattered around the center of the dish containing
the CS-PG step gradients in the above media to which
was added 50 ng/ml NGF. DRGs were also dissociated
into single cells by treatment with
0.25%,
bovine trypsin
for
18
min at
37°C.
resuspended in media with serum.
counted, and plated at
a
density of
10,000
cells per cover-
slip.
Retina dissections were performed according to the
protocol of (Halfter, Newgreen, Sauter. and Schwarz,
1983).
Briefly, neural retinae from embryonic day 6
chickens were dissected in Ca2+-Mg2+-free buffer
(CMF). Surrounding tissue, lens, and pigment epithe-
lium were removed. Whole retinae were transferred, vi-
treal side up onto
a
0.2%
concanavalin A-coated black
cellulose acetate membrane filter (Sartorius SM
13006).
The retina and membrane were cut into 350-pm wide
strips, perpendicular to the optic fissure.
Tissue culture dishes containing the step gradients
of
CS-PG and laminin were removed from the incubator
and washed one time in media. Retina strips were trans-
ferred individually to the tissue culture surface, vitreal
side down. The strips were positioned on the laminin
substrata along side and parallel to the strips of CS-PG,
with their orientation of outgrowth facing the proteogly-
can stripes. Metal bars were placed at the ends
of
the
membrane strips
(a
region of the filter to which retinal
explants
do
not extend) to anchor them, and the surface
was covered with Dulbecco's Modified Essential Media/
Ham's
FI
2
Nutrient
(
1:
1
)
to which was added
0.1
mg/
ml transferrin,
20
nM progesterone,
5
ng/ml sodium
selenite,
0.4
mg/ml sodium pyruvate,
5
mM
phospho-
creatine,
5
pg/ml insulin,
500
chick serum.
5%
fetal
bo-
vine serum. glutamine, and an antibiotic mixture ofpen-
icillin, streptomycin and fungizone. Experiments were
repeated
in
serum-free media
to
which
10%
chick
em-
bryo extract was added for retinal cell survival.
Rat forebrain neurons
(
E
17)
were dissected in
0.
I
M
CMF-PBS, with the meninges completely removed and
dissociated to single cells using the same procedure, and
the Same media,
as
for
DRGs
above.
FB
neurons
were
plated
in
NGF containing media described above at a
density
of
10,000
cells per coverslip.
Rate
of
Retinal Ganglion Cell Outgrowth
Neurites and growth cones were analyzed using an in-
verted microscope
(
IM-35; Carl Zeiss, Thornwood,
NY)
with
a
Dage-MTI Model 65 Newvicon videocanma
at-
tached to
a
Panasonic TQ-2026F optical disc time-lapse
video recorder. Phase-contrast images were processed
with
a
Quantex QX-7 Image Processor using the soft-
ware program
ICOS.
The rate
of
neurite outgrowth was
analyzed by direct measurements from a video screen of
the distance traveled by each growth cone over
a
given
time interval. For each of the three neuronal types, neu-
nte outgrowth rates were compared on laminin alone,
the outer tier of the CS-PG step gradient (PG,). the mid-
dle tier
of
the CS-PG step gradient (PG,). and the inner
tier of the CS-PG step gradient (PG,).
RESULTS
Video microscopy was used to analyze the rate
of
outgrowth
of
embryonic chick dorsal root ganglia
neurons, chick retinal ganglion cell neurons, and
rat basal forebrain neurons on a step gradient of
chondroitin sulfate proteoglycan (CS-PG), along
which growth cones encounter stepwise increases
in
CS-PG concentration bound to a laminin-
coated substratum.
In
previous studies
of
neurite
outgrowth in response to CS-PG (Snow et al.,
1990b,
199
1
),
neurites grew from laminin toward
a stripe
of
high concentration of CS-PG. In that
paradigm, CS-PG was attached to nitrocellulose or
glass first, then the entire dish was overlaid with
laminin, resulting in regions with laminin alone
and stripes
of
CS-PG mixed with a growth-promot-
ing concentration of laminin.
In
controls for the
present study, we reversed this application in order
to parallel the procedure used to make the step gra-
dient, that is, we applied laminin to the glass cover-
slip first, to which single stripes of high CS-PG con-
centration
(1
mg/ml) were bound. Importantly,
with either order
of
application of CS-PG and
la-
minin in the single stripe experiment, the response
of the neurites was complete inhibition
of
out-
growth.
To
clarify,
throiighout
the
remainder
of
this
rqmrt,
the
purudigms
where
CS-
PG
strips
weve
overlaid
tiith
laminin
or
\there
C'S-YG
stripes
Mvre
hound
to
u
lutninin
szihstrutu
(ivhich
result
in
un inhibition ofneiirite oiitgrm'th)
tiill
hc
ref2rred
to
as
the
single stripe
to
d(@rentiule
it
,from
[he
purudigrn
discxrsed prcsmtlj>, i.rh ich is
rcftwed
to
as
the
CS-PG step gradient.
Characterization
of
the CS-PG Step
Gradient
Repeated addition of CS-PG to a laminin-coated
substrata generated a step gradient
of
increasing
concentrations of CS-PG visible by epifluores-
cence microscopy (Fig.
2).
The CS-PG step gra-
dient consisted
of
a stripe
of
five tiers: PG, (outer
tiers on each side of the stripe),
PG,
(between
outer tier and center of stripe on each side), and
PG, (single center stripe approximately 400 pm
wide). Figure
2
shows one side
of
the step gradient,
that
is,
from the center
of
PG, to the outer-most
tier, PC,, as well as the region outside the step gra-
dient that consists of laminin alone. The widths
of
PG, and PG, were variable, ranging between
30
and
60
pm, while PG, spanned about 400
pm.
The
characteristics of the CS-PG step gradient differ
from the single stripe assay described previously
(Snow
et
al.,
1990b,
1991).
The two assays are
compared schematically in Figure
3.
Two methods were used to determine the con-
centration
of
CS-PG in the step gradient. The first
method used quantitative fluorescence microscopy
328
Snow
and
Letourneau
CS-PG
Step Gradient
%RGC
%DRG
[CS-PGI
[LNI
*
Tier
A
Single Stripe
[I
Substratum
2
0.006ug/rnrn
LN
Figure
3
Schematic representation
of
the characteristics
of
the CS-PG step gradient.
(A) A
schematic diagram of Figure
2.
The bottom
of
the figure indicates the tier
of
the step gradient
(
PGi, PG,, PG,,
or
laminin; LN). The row above the tier designation indicates the concentra-
tion
of
laminin applied to the coverglass, washed, and air dried, prior to CS-PG binding.
(*Note: This concentration is assumed to be constant, although the possibility exists that an
increase in CS-PG results in a decrease in laminin binding.)
Also
shown is the concentration
of
CS-PG bound
to
the laminin substratum determined by quantitative immunofluorescence
and expressed per unit area. The row
of
cartoon drawings depict how growth cones can grow on
CS-PG depending upon neuronal type with the top-most row giving the percentage of either
RGC
or
DRG neurons that can elongate onto that tier.
(B)
A
schematic representation
of
a
single stripe
of
CS-PG for comparison with view
(A).
Growth cones growing first on laminin
stop or turn at a border
of
large CS-PG concentration (this study and Snow et al., 1990b). Note
that the concentration
of
CS-PG in the single stripe lies between that
for
PG, and PG,,
demonstrating that once “adapted”
to
CS-PG from growing on low concentrations
of
the PG,
growth cones can
grow
onto and above the concentrations that are inhibitory when presented
as a single stripe
of
large CS-PG concentration (bound to laminin).
Neurite Outgrowth
on
a
CS-PG
Step
Grcldient
329
to estimate the concentration of
CS-PG
bound in
the tiers of the step gradient by comparison to the
binding
of
known concentrations of
CS-PG.
The
results of this assay determined that the level of
fluorescence observed in
PG,
could be produced
by
a
single application
of
CS-PG
at a concentration
of 200-600 pg/ml. The level of fluorescence ob-
served in
PG,
could be produced by a single appli-
cation of 2.5-3.0 mg/ml
CS-PG,
and the level of
fluorescence observed in
PGi
could be produced by
a single application
of
5.0-5.5
mg/ml. Since the
concentration of
CS-PG
used to initially make the
step gradients was 1.0 mg/ml., clearly
CS-PG
binds to the same areas in repeated applications in
order to create the larger concentrations observed
in tiers
PG,
and
PG,.
The second method used to determine the con-
centration of
CS-PG
in each tier of the step gra-
dient measured the amount of
CS-PG
actually
bound to the dish using
a
1
.O
mg/ml starting con-
centration of 35S-proteoglycan. The amount of la-
minin bound was also measured using 3H-laminin
and showed that 0.006 pg/mm2 laminin was
bound when
100
pl
of a
100
pg/ml solution of
laminin was applied to the coverglass. The result-
ing values for the concentration of
CS-PG
bound
in each tier
of
the step gradient are expressed in
Figure
3(
A)
as
amount
CS-PG
per mm2. The val-
ues ranged from a mean
of
0.2
pg/mm2
in
PGi,
to
0.06 pg/mm2 in
PG,
to 0.006 pg/mm2 in
PG,.
These values stand in comparison to 0.020-0.025
pg/mm2
CS-PG
bound to laminin in the single
stripe assay.
Comparison
of
Neuronal Behavior on the
CS-PG Step Gradient for Three Neuron
Types
The rate of neurite outgrowth on the
CS-PG
step gradient was measured using video micros-
copy for three embryonic neuronal cell types:
chicken retinal ganglion cells, chicken dorsal root
ganglion neurons, and rat forebrain neurons. Sin-
gle neurites of each neuronal type were followed
and their rate
of
outgrowth assessed as they grew
from laminin onto the
CS-PG
step gradient. Each
cell type displayed some responses that were com-
mon to all cell types tested, while some cell types
displayed unique responses to the various tiers of
the
CS-PG
step gradient.
Chick DRG Neurons
Chick
E9 DRGs
were explanted onto a laminin
substratum adjacent to the
CS-PG
step gradient.
Figure
4A
shows that as the growth cones elon-
gated from laminin onto the
CS-PG
step gradient,
their rates of elongation decreased from a mean of
36.5 pm/h on laminin (S.E.M.
=
4.506)
to
19.8
pm/ h on
PG,
(S.E.M.
=
4.346).
A
cumulative fre-
quency distribution plot for the percentage of
DRG
neurons with a rate of neurite outgrowth
greater than a given rate, x, on laminin and the
CS-PG
step gradient, demonstrates
a
shift toward
lower rates of neurite outgrowth
on
CS-PG
substrata in comparison to growth on laminin
[Figure
4(B)].
The majority of
DRG
neurites did not proceed
past the first tier of the
CS-PG
step gradient.
Rather, growth cones lined up along the border be-
tween
PG,
and
PG,
. Using epifluorescence com-
bined with phase microscopy with a
10X
objective,
the position of the neurites with respect to the step
gradient could be determined [Fig.
4(C
and
D)]
and was verified at high magnification (not
shown
)
.
Dissociated
DRG
neurons attached to and ex-
tended neurites on laminin alone and
on
PG,
but
not on higher levels of the
CS-PG
step gradient.
This behavior is consistent with the results of ex-
plant outgrowth in these assays and further sup-
ports that
DRGs
respond to
CS-PG
in a concentra-
tion-dependent manner.
Chick RGC Neurons
Chick E5-6
RGC
stripe explants were grown on
laminin with their preferred orientation of out-
growth (i.e., toward the optic fissure
in
vivo)
facing
the step gradient. In contrast to
DRG
neurons,
many
RGC
growth cones could elongate up the
CS-PG
step gradient, some growing onto the high-
est concentration of
CS-PG
without turning
[
Fig.
5(C)].
Of
RGC
neurites, 32% grew onto the first
tier of
CS-PG, 18%
grew onto
CS-PG,,
and
8%
elongated onto the highest concentration of
CS-PG
[Fig. 3
(A)].
This result was surprising in light of
our finding from previous studies (Snow et al.,
199
1
)
that
RGC
growth cones are more sensitive
than
DRG
growth cones when presented with the
single stripe
of
CS-PG
(see Discussion). The
RGC
growth cones appear to behave differently when
presented with a step-wise increase in the concen-
tration
of
CS-PG.
As
the
RGC
neurites elongated, their rate of out-
growth decreased from 28.8 pm/h on laminin
(S.E.M.
=
2.915)
to
19.9
pm/h on
PG,
(4.365)
to
8.4
pm/h on
PG,
[S.E.M.
=
1.984)
to 2.0 pm/h
on
PGi
(S.E.M.
=
0.176; Fig.
5(
A)].
The cumula-
tive frequency distribution plot for the percentage
DRG Neurite Outgrowth Rates on
Laminin and a
CS-PG Step
Gradient
40
1
A
LN PGo
Cumulative Frequency Distribution
Plot
for
E9
Chicken DRGs
-lh
-
PG
(outer)
100
80
60
40
20
0 0
20
40 60 80
100
120
Elongation
rate
x
(pmihr)
Figure
4
DRG growth on the CS-PG step gradient.
(A)
Growth rates for DRG neurites on the CS-PG step gra-
dient, on
LN:
mean
=
36.5 pm/h.
n
=
27,
S.E.M.
=
4.506;
on
PC,:
mean
=
19.8
Fm/h,
17
=
6,
S.E.M.
=
4.346.
(B)
Cumulative frequency distribution
plot
for
elongation rates of
E9
chicken DRGs on laminin alone
and
on
the
CS-PG
step gradient dcrnonstrating
a
left-
ward shift with growth
up
the
CS-PG
concentration gra-
dient in comparison to growth on laminin alone. (C)
Phase contrast
of
DRG explants on laminin and the
CS-
PG
step gradient. Arrows indicate
PGo/PG,
border.
Scale
bar
=
50 um. (D) Fluorescent image of
(C)
show-
of RGC neurons with a rate of neurite outgrowth
greater than
a
given rate,
x,
is shown in Figure
5
(B)
for growth on laminin, PG,, PG,, and PG,. and
demonstrates a shift toward lower rates of neurite
outgrowth on CS-PG tiers
in
comparison to RGC
axonal growth on laminin.
Dissociated
RGCs
attached
to
laminin alone
and in some cases
to
PG, (data not shown). Cell
bodies on PG, extended neurites both up the con-
centration step gradient of CS-PG and down the
step gradient toward laminin alone, with
no
obvi-
ous preference for direction. Rates of neurite out-
growth were not analyzed for neurites that grew
from
a
high CS-PG concentration down the CS-PG
step gradient toward and/or onto laminin. Future
studies will be necessary
to
address this phenome-
non. The few cells that attached
to
the higher levels
of the CS-PG step gradient did not extend pro-
cesses. This result is consistent with previous data
that showed that RGC explants placed directly
on
high concentrations of CS-PG would not extend
neurites (Snow et al.,
199
1
).
However. in the pres-
ent study, the examination
of
explants misplaced
onto
CS-PG stripes showed that
RGC
cell bodies
extend processes that circulate within the explant
itself and do not exit the explant onto the CS-PG
containing substrata (data now shown).
Rat
Forebrain Neurons
Growth
of
neurites from embryonic day
17
rat fore-
brain neurons was analyzed on the CS-PG step gra-
dient.
FB
neurites grew
on
laminin at a rate
of
53.7
pm/h (S.E.M.
=
7.8),
on
PG, at 41.3 pm/h
(S.E.M. =7.6),andonPGmat 15.7pm/h[S.E.M.
=
5.0;
Fig. 6(A)]. Figure
6(
B)
shows the cumula-
tive frequency distribution plot for FBs indicating
the same trend
as
for Figures 4(A) and
5(A).
In
single cell cultures, FB neuronal cell bodies at-
tached
to
laminin and PG,,
but
did not routinely
attach
to
PG, or PG, regions (not shown). Neu-
rites were found to extend onto PG, from PG,, but
did not grow onto PG,. In contrast to
DRG
neu-
rons, FB neurites were able to attach to and grow
directly on strips created
by
a
single application of
1
mg/ml CS-PG [Fig.
6(C)].
In previous studies
(Snow
et
al., 1990b). DRGs rarely attached or ex-
tended neurites directly onto a substrata treated
ing the borders of the step gradient with respect to
DRG
neurite outgrowth. The growth cones characteristically
stop/turn
at
the
PG,/
PG, border. indicated by arrows.
Dotted line delincates
PG,/
PG,
border: scale sainc
as
in
Neuritr
0irtgroiz.th
on
u
C’S-PG
Stcp
Gmdicnt
331
RGC Neurite Outgrowth Rates
on
Laminin and a CS-PG Step Gradient
LN
PGo PGm PGI
A
Cumulative Frequency Distribution
Plot for
E6
Chicken RGCs
---(r-
LN
-t
PG
(outer)
-t-
PG
(middle)
.-b
PG
(Inner)
B Elongation
rate
x
(pmihr)
Figure
5
RGC
growth on
the
CS-PG
step
gradient.
(A)
Growth
rates
for
RGC neurites
on
laminin
and
the
CS-
PG
step
gradient.
on
LN:
mean
=
28.8
pm/h,
n
=
38,
S.E.M.
=
2.915;
on
PG,: mean
=
19.9
pm/h,
n
=
12,
S.E.M.
=
4.365;
on PC,: mean
=
8.4
pm/h,
n
=
7,
S.E.M.
=
1.984;
and
on
PG,:
mean
=
2.0
pm/h,
n
=
3,
S.E.M.
=
0.176.
(B)
Cumulative
frequency
distribution
plot
for
elongation
rates
of
E6
chicken
RGCs
on
laminin
alone and on
the
CS-PG step
gradient
demonstrating
the
same
trend
as
in
4B.
(C)
A
single
RGC
neurite
labeled
with
antibody
RA4
(kindly
provided
by
Dr.
Steve
McLoon)
elongating
from
right
to left. Arrow heads
point
to
borders,
from
right
to
left:
LN/PG,,
PGJPG,,
PG,,/PG,.
Small
arrows
point
to
RA4
labeling
that
is
fainter
than
middle
portion
(fluorescence
observed
as
a
result
of
labeling
RGC
neurites
with
antibody
RA4
is
sometimes
intermittent), but
present,
showing
that
the
neurite
extends
across laminin
and
all
levels
of
the
CS-
PG
gradient.
Scale
bar
=
10
pm.
with
a
single application
of
1
mg/ml CS-PG and
RGCs never elongated
a
neurite directly onto this
concentration of CS-PG (Snow et al.,
1991
).
DISCUSSION
Although often inhibitory to neurite outgrowth
in
vivo
and
zy1
vitro,
the presence of chondroitin sul-
fate proteoglycan in regions where neurons extend
processes indicates that factors other than the mere
presence of CS-PG are important in the response of
growing neurites. For this reason, a study of the
response of growth cones to intermediate ratios of
CS-PG to laminin was conducted by analyzing neu-
rite outgrowth on a step gradient of increasing con-
centrations
of
CS-PG bound to
a
laminin-coated
substratum. The paradigm also tested whether
growth cones would respond differently to
a
step-
wise distribution of CS-PG superimposed on a
constant concentration of laminin, than to
a
single
stripe of
a
large concentration of CS-PG bound to
laminin (single stripe assay). Using video micros-
copy, neurite outgrowth on the CS-PG step gra-
dient was compared for three neuronal types: chick
dorsal root ganglia neurons, retinal ganglion cell
neurons, and rat forebrain neurons.
From the present study, we have learned that
(1)
growth cones that do not cross onto CS-PG
bound to laminin (single stripe assay)
nil/
elongate
onto CS-PG presented in the form of a step gra-
dient. Once
a
cell has extended
a
process on a low
concentration
of
CS-PG bound to laminin, it can
sometimes grow onto concentrations of CS-PG
that are inhibitory in the single stripe assay;
(2)
neurites growing up
a
step gradient of CS-PG slow
their rate of outgrowth with each step of the gra-
dient, that is, with increasing concentrations of
CS-
PG; and
(3)
different neuronal cell types show dif-
ferent responses to the CS-PG step gradient. These
results offer
a
possible explanation for how CS-PG
can inhibit certain populations of neurites while
allowing the passage of others.
Comparison
of
Growth Cone Behavior on
a Step Gradient
of
CS-PG Versus Growth
on the CS-PG Single Stripe
When chick DRG or RGC growth cones were con-
fronted with a single stripe of CS-PG bound to la-
minin after growing on laminin alone, they
stopped or turned, and grew in an alternate direc-
tion. However, given the step gradient of bound
CS-PG spanning a concentration range of
0.006-
332
Snow
and
Letaiirneuu
FB Neurite Outgrowth
Laminin and a CS-PG Rates on
Step Gradient
LN
PGo PG
m
a
Cumulative Frequency Distribution
Plot for
El7
Rat
FBs
IN
PGO
Ern
--
-~
7-7
7.t
0
20
40
60
80
100
120
Elongation rate
x
(pm/hr)
Figure
6
FB
growth
rates
on
the
CS-PG step gradient.
(A)
Rates
of neurite outgrowth
for
E
17
rat
FB neurons
on
laminin
and the CS-PG step gradient:
on
LN:
mean
=
53.7
pm/h.
n
=
10,
S.E.M.
=
7.8:
PG,:
mean
=
41.3
pm/h,n=5,S.E.M.=7.6;onPGm
mean=
15.7,n=4.
S.E.M.
=
5.0.
(B)
Cumulative
frequency
distribution
plot
for FB neurite outgrowth on
laminin
and
the
CS-PG
step
gradient.
(C) FB neurons
growing
on
single
stripe
of
CS-PG
bound
to laminin
(concentration
CS-PG bound
=
0.02
pg/mm2).
Scale
bar
=
10
pm.
0.2
pg/ mm', the same cell types extended neurites,
in some cases, to the highest concentration of
CS-
PG without turning, concomitant with a decrease
in growth rate at each tier of the step gradient.
What might be the difference between these two
forms of presentation of CS-PG to growth cones?
In the single stripe assay, CS-PG is inhibitory to
the advance of all neuronal types tested (Snow et
al., 1990b and this study). Hypotheses for this inhi-
bition include: the influence
of
negative charge due
to high sulfation, and steric hindrance due to the
molecular size of proteoglycans and their extensive
degree
of
hydration. The proteoglycans may bind
to active sites on growth-promoting molecules
(such as laminin) to render them ineffective for the
support of neurite outgrowth-and/or a specific
receptor-mediated response.
Given these hypotheses for the mechanisms
of
neurite outgrowth inhibition by sulfated PGs, it is
difficult to understand how some growth cones can
grow on CS-PG when it is presented in the form of
a step gradient, since presumably similar mecha-
nisms would be, in effect, regardless of the size of
the step increase of CS-PG. Although the precise
mechanism(
s)
that enable growth cones to elon-
gate on the CS-PG step gradient are unknown, we
have considered a number of possible explana-
tions.
Growth cones may undergo adaptation to
CS-
PG as they traverse the CS-PG step gradient. When
a growth cone contacts the outer tier of the step
gradient, it encounters a low concentration of CS-
PG bound to laminin. There may be enough la-
minin present in this outer tier to support contin-
ued outgrowth. While exposed to low levels of
CS-
PG, a growth cone may either up-regulate its
ability or number
of
laminin receptors to provide
greater sensitivity to laminin or may down-regu-
late a CS-PG receptor, making it less sensitive to
the inhibitory effects of CS-PG. Both adaptations
may occur simultaneously. Such adaptations
might allow a growth cone to progress on the
CS-
PG step gradient to points beyond the concentra-
tions that inhibited outgrowth in the single stripe
assay.
Further, the degree of change of bound CS-PG
interpreted by a growth cone may be more impor-
tant than the absolute concentration of the mole-
cule, since growth cones on the CS-PG step gra-
dient can grow onto concentrations of CS-PG that
are inhibitory in the single stripe assay. Experi-
ments designed to determine whether inhibition of
neurite outgrowth by CS-PG is due to relative
changes in CS-PG concentration or to absolute
concentrations are in progress in our laboratory.
At some point for each neuronal type or subset
of neurons, the concentration of CS-PG may be-
come too large. At this point, some direct or indi-
rect signalling event
(
Stnttmatter and Fishman,
Ncurite
Outgrowth
on
a
CS-PG
Step
Gradient
333
199
1;
Walter, Allsopp, and Bonhoeffer, 1990) may
alter the cytoskeleton, causing the growth cone to
stop or turn.
Differences in the Response
of
Each Cell
Type to the CS-PG Step Gradient
A
significant difference in neuronal behavior is evi-
denced by the fact that RGC neurites are more ca-
pable of growing up the CS-PG concentration gra-
dient than are the DRGs or FBs. This may suggest
that RGC neurons are more capable of adapting to
the CS-PG than the other cell types. With regard to
the observation that 32% of RGC neurites grow
onto PG,, 18% onto PG,, and
8%
onto PGi, it is
unknown whether there are subpopulations of neu-
rons possessing different capabilities for response
to CS-PG or whether each neuron has an equal
probability of elongating onto the subsequent tier
of CS-PG, with various degrees of success.
In the case of DRG neurons, where 22% grow
onto PG, [Fig. 3 (B)]
,
the result is not surprising,
based on previous results (Snow et al.,
1990b).
Fur-
ther, chick DRG neurites do not progress past the
border between PG, and PG,. For DRGs, then,
the response to CS-PG appears to be strictly con-
centration dependent under the present conditions
(i.e., a CS-PG step gradient with slopes different
from the ones we tested may produce different re-
sults), without evidence of adaptation.
FB
neurons also appear to have
a
concentration-
dependent response to CS-PG, stopping
at
the
border between PG, and PG,
,
possibly suggesting
that they are somewhat more resistant to the inhibi-
tory effects of CS-PG, or that they may have a re-
duced requirement for laminin in comparison to
DRGs, or both. However, unlike DRGs,
FB
neu-
rons will attach to and grow abundantly on a stripe
treated with
a
single application of
1
mg/ml CS-
PG. This difference between DRGs and FBs sug-
gests different mechanisms of interaction between
cell surface molecules and the CS-PG and/or
la-
minin.
Relevance
of
the
in
vifro
Data to
Development
For some neurons, the response to CS-PG is con-
centration dependent. This phenomenon
in
vitro
reflects the behavior of DRG neurons
in
vivo,
for
example, DRG neurons encounter CS-PG as well
as KS-PG (Snow et al.,
1990a)
at the dorsal mid-
line of the developing embryo. One possibility is
that the concentration of sulfated PG in the roof
plate of the spinal cord approximates 0.02
pg/
mm2, since DRGs turn away from CS-PG at that
concentration
in
vitro,
whether presented as a sin-
gle stripe or in a step gradient. However, the turn-
ing response may be a result of the combined ef-
fects of CS-PG and KS-PG and perhaps other mole-
cules present in the roof plate.
In
vivo,
a graded distribution of CS-PG has been
shown in the rodent retina (Brittis et al., 1992).
RGC cell bodies reside in a low concentration of
CS-PG prior to differentiation (Snow et al., 199
I;
Brittis et al., 1992), and higher concentrations of
CS-PG flank the territory of RGCs. CS-PG immu-
nofluorescence moves peripherally with the periph-
eral wave of RGC differentiation. The ability to
extend an axon into areas with low concentrations
of CS-PG would be advantageous in the retina.
The concentrations of CS-PG in the retina are un-
known, but they could potentially be high enough
in the peripheral retina to induce turning toward
the optic fissure. These data indicate that CS-PG
may play a role in the guidance of RGC axons.
Further, assymmetrical distribution of other mole-
cules have been described in the literature in such
regions as the retina and tectum (Gottlieb, Rock,
and Glaser, 1976; Marchase, 1977; Trisler,
Schneider, and Nirenberg, 198
1;
Bonhoeffer and
Huf,
1985; Irwin, Bremer, Irwin, and McCluer,
1985; Rabacchi, Neve, and Drager, 1990;
McLoon, 199
I
)
.
Interesting are the forebrain neurons that grow
through the subplate, since CS-PG is expressed in
this region (Palmert et al., 1986; Sheppard et al.,
1990, 199
1
)
.
The presence of CS-PG in the sub-
plate in a region where axons grow appears to con-
tradict the theory that CS-PG is inhibitory to neu-
rite outgrowth. However, it has been shown that
laminin or other growth-promoting molecules, if
present in high enough concentration, can override
the inhibitory effects of CS-PG on neunte out-
growth. Growth-promoting molecules in the sub-
plate may bind CS-PG, reducing its inhibitory ef-
fect. Further, from the present results, it is feasible
that an appropriate combination of CS-PG and la-
minin or other growth-promoting molecules may
regulate the timing of FB neurite outgrowth as
well. It is important to consider that although fore-
brain neurons extend a neurite while passing
through the subplate (Shoukimas and Hinds,
1978), a study has not been done to determine the
position of the FB neuron growth cones with re-
spect to the CS-PG in that region. Whether a gra-
dient of CS-PG exists in the rat forebrain and may
influence the direction of neurite outgrowth re-
mains
to
be determined.
Important to consider in the analysis of these
334
Snobc.
and Lctozimcaii
Tier
Figure
7
IF? iyiw
See
D~~c~i~oon
for details.
Model of
how
different neurons may respond differently
to
a step gradient
of
CS-PG
data is that only cartilage CS-PG was tested in the
CS-PG step gradient. Many other types
of
sulfated
proteoglycans exist
in
vivo
that exhibit different
sulfation patterns, chain length. combinations of
carbohydrate chain types, and protein core compo-
sition. In particular, neurites may respond differ-
ently
to
neural PGs (Snow et
al.,
1990a; Cole and
McCabe. 1991 )than
to
nonneural forms. Such dif-
ferences may be important in the response of a
growth cone to a PG step gradient and will require
future studies for elucidation.
A
Model for Neurite Outgrowth on
a
CS-
PG
Step Gradient
We suggest
a
model to explain how a CS-PG step
gradient might influence the timing and direction
of specific neurons
in
vivo
(
Fig.
7
)
.
From our data,
we know that different neuronal types respond dif-
ferently to the CS-PG step gradient, extending their
axons to a specific level
of
the gradient. Axons
grow at faster rates on laminin than on CS-PG
(areas represented by
“LN”
in Fig.
7
) .
For some
cell types or subpopulations of neurons, complete
avoidance
of
a
territory could be accomplished
by
the expression of a large concentration of sulfated
PGs, such
as
in the
roof
plate of the developing
spinal cord (Snow et
al.,
1990a). Such a barrier
would require growth cones to turn into an alter-
nate direction (not depicted in Fig.
7).
However,
given
a
step gradient of CS-PG, neurons could be
influenced
to
slow their rate
of
outgrowth
as
they
encounter CS-PG (i.e., neurite on PG,), but per-
haps not alter its direction until it reaches
a
con-
centration that provides
a
turn command to the
growth cone (curved portion
of
schematic axons).
Such a turn command may be provided by an ap-
propriate ratio
of
CS-PG to laminin. While some
neuronal types would turn
at
low concentrations of
CS-PG (neurite on PG,), others could grow onto
higher concentrations
of
CS-PG
UY
long
as
the),
first
cncorintered
lowc~v
levclr
of
tho
inolccule
(re-
mainder of neurites). Thus,
a
CS-PG step gradient
could induce certain populations of neurites
to
wait while others could elongate toward their ap-
propriate target, dependent upon their intrinsic sen-
sitivity to CS-PG, their ability
to
adapt to CS-PG
and/or their ability
to
negotiate relative changes in
CS-PG concentration.
CONCLUSIONS
These data indicate that the responses
of
cells to
CS-PG
in
viva
may depend upon other molecules
with which CS-PG is complexed or expressed.
When present in high concentrations and lacking
significant contributions from growth-promoting
molecules, CS-PG may prevent neurites from en-
tering forbidden regions of the developing nervous
system. Alternatively, when present in
a
gradient
and/or modified by growth-promoting molecules,
such
as
laminin, the inhibitory effects
of
CS-PG
may be eliminated
or
reduced. Since growth cones
decrease their rate of outgrowth with increasing
step concentrations of
CS-PC,
such
a
molecule
may
be
capable of influencing the timing and/or
direction of neurite outgrowth
in
viva.
The authors wish
to
thank Kokila
F.
Roche and Ger-
ald Sedgewick
for
excellent technical assistance. Drs.
Lloyd Culp, Arnold Caplan, David Carrino. James
McCarthy.
Amy
Skubitz, and
Leo
Furcht for reagents.
Dr. Virginia Seybold for materials and suggestions for
experimental design, and
Drs.
Maureen Condic and
Jerry Silver for helpful discussions of the manuscript.
This study was supported by
NIH
grants
EY06331-01
(D.M.S.)and HD19950
(P.C.L.).
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