 |
 Previous Article | Next Article 
The Journal of Neuroscience, February 1, 2000, 20(3):1030-1035
Spatial Distributions of Guidance Molecules Regulate
Chemorepulsion and Chemoattraction of Growth Cones
Dominique
Bagnard1,
Nicole
Thomasset2,
Marion
Lohrum3,
Andreas W.
Püschel3, and
Jürgen
Bolz4
1 Institut National de la Santé et de la
Recherche Médicale (INSERM) Unité 371, Cerveau et Vision,
69500 Bron, France, 2 INSERM Unité 433, Faculté
de Médecine Laennec, 69372 Lyon cedex 08, France,
3 Max-Planck-Institut für Hirnforschung, Molekulare
Neurogenetik, Abt. Neurochemie, 60528 Frankfurt, Germany, and
4 Universität Jena, Institut für Allgemeine
Zoologie, 07743 Jena, Germany
 |
ABSTRACT |
It is generally assumed that gradients of chemotropic molecules are
instrumental to the wiring of the nervous system. Recently, two members
of the secreted class III semaphorin protein family have been
implicated as repulsive (Sema3A) and attractive (Sema3C) guidance
molecules for cortical axons (Bagnard et al., 1998 ). Here, we
show that stabilized gradients of increasing semaphorin concentrations
elicit stereotyped responses from cortical growth cones, independent of
the absolute concentration and the slope of these gradients. In
contrast, neither repulsive effects of Sema3A nor attractive effects of
Sema3C were observed when axons were growing toward decreasing
semaphorin concentrations. Thus, growth cone guidance by gradients of
chemotropic molecules is robust and reproducible, because it is
primarily independent of the exact dimensions of the gradients.
Key words:
wiring molecules; axonal guidance; gradients; chemotropic
attraction; chemotropic repulsion; semaphorins; cortical development; in vitro assays; time-lapse imaging
| |
INTRODUCTION |
A fundamental issue in developmental
neurobiology is how neurons establish precise connections to distant
target cells. Ramón y Cajal first suggested that the motile tips
of growing axons, which he called growth cones, are guided by
chemotaxis. It took almost 100 years, however, until the existence of
such "chemotropic" molecules was demonstrated experimentally in the
nervous system. In these studies, neurons were cultured next to target
tissue in a gel that stabilized gradients of diffusible molecules and at the same time enabled axons to grow in vitro. Such coculture experiments revealed that specific populations of growing axons are
attracted at a distance by their target cells or by intermediate targets that they encounter on their way toward their final
destinations (Lumsden and Davies, 1983; Tessier-Lavigne et al., 1988).
Moreover, it was also found that cells from regions not innervated by a given axonal population can in some cases release substances that cause
these fibers to grow away from this source of "chemorepulsive" factors (Pini, 1993). Based on such in vitro assays, it was
possible to isolate and clone two families of chemotropic molecules,
the netrins (Tessier-Lavigne and Goodman, 1996) and the
semaphorins (Püschel, 1996), which are expressed in the
developing nervous system. It is now generally accepted that gradients
of long-range guidance cues constitute an important mechanism for the
specification of neuronal connections. In theoretical studies,
different mathematical models have been proposed to describe how growth
cones might be possibly guided by gradients of chemotropic molecules
(Gierer, 1981; Goodhill, 1997). However, despite some notable
exceptions (Baier and Bonhoeffer, 1992; Rosentreter et al., 1998),
there is little experimental work about the mechanisms by which
changing concentrations of chemoattractant and chemorepellent factors
actually influence growing axons. For example, if axons are attracted
by chemotropic factors released from intermediate targets, what does prevent the fibers from stopping in the regions in which the
concentrations of chemoattractive substances are highest, and what
allows them to continue to grow toward their final targets? Similarly,
if chemorepellent substances secreted from nontarget regions define inaccessible territories along specific pathways, do these repulsive factors then also impede axonal growth in the appropriate direction? Axonal pathways are formed in a highly stereotyped manner, and they
appear tightly regulated in space and time. If gradients of chemotropic
molecules are instrumental for the patterning of fiber trajectories,
how finely tuned must the absolute concentration and shape of gradients
be to produce highly reproducible axonal connections?
To address these issues, we examined the reaction of developing
cortical axons to defined gradients of two members of the semaphorin
gene family, Sema3A (previously called SemD) and Sema3C (previously
called SemE). Earlier work indicated that Sema3A acts as a repellent or
inhibitory signal for a variety of axonal populations, including
cortical fibers (Messersmith et al., 1995; Püschel et al., 1995;
Püschel, 1996; Bagnard et al., 1998, Polleux et al.,
1998), whereas Sema3C is an attractant signal for cortical axons
(Bagnard et al., 1998). The present study indicates that the exact
dimensions of gradients, such as the magnitude of the concentration or
the slope, are not the essential parameters of axon attraction or
repulsion by semaphorins. However, growth cones are very sensitive to
the direction or "sign" of semaphorin gradients, and they strongly
respond to increasing or uniform concentrations of Sema3A and Sema3C,
but they are not affected by these guidance molecules when the
concentration decreases by <1% over the width of a growth cone.
| |
MATERIALS AND METHODS |
Membrane preparations. Stable human embryonic kidney
293 (HEK 293) cells expressing functional recombinant Sema3A or
Sema3C (pBKFlagSema3AP1b, pBKFlagSema3CP1b) (Adams et al., 1997;
Bagnard et al., 1998), or untransfected (control) cells were used to
prepare cell membranes. Because cortical axons do not grow well on HEK 293 cell membranes, we added membranes from postnatal cortex that contain (unknown) growth-promoting molecules for cortical axons. Postnatal day 3 cortical tissues served to prepare postnatal membranes. Membranes were prepared as described by Götz et al. (1992).
Membranes concentrations were determined by measuring optical densities of solutions after 15-fold dilution in 2% SDS. To quantify semaphorin concentrations, membrane preparations were transferred on
nitrocellulose membrane (D3354; Schleider & Schuell, Dassel, Germany).
Dot blot (protein detection with anti-Flag M2; Sigma) were scanned and digitalized for fluorimaging analysis (FluorImager SI, Image Quant; Molecular Dynamics, Sunnyvale, CA). Concentrations of semaphorin were
determined as a function of a standard Flag protein (N-terminal flag-BAP protein; Sigma) using Excel software analysis. We found that
membrane solutions with optical density 0.1 contain 1 µg/ml semaphorin.
Production of gradients. Membranes gradients were prepared
on capillary pore filters as described by Baier and Bonhoeffer (1992).
Briefly, a drop of membrane solution was pipetted onto a capillary pore
filter placed on a nylon matrix that was connected to a vacuum pump. An
inclined glass coverslip was positioned over the membrane suspension,
so that only the lower edge of the coverslip touched the filter. The
drop adhered to the coverslip and, after application of a vacuum,
contracted toward the lower edge of the coverslip. This resulted in a
graded distribution of membrane particles, with the highest
concentration at the side on which the coverslip was placed on the
filter. Membrane gradients of different slopes were produced by varying
the inclination angle of the coverslip. A detailed description of this
technique is provided by Baier and Klostermann (1994).
The membrane gradients on the capillary pore filters were transferred
to laminin-poly-L-lysine (1 mg/ml; Sigma) -coated glass coverslips (Hübener et al., 1995). It was therefore possible to
obtain a clear phase-contrast image of the axons and the membrane particles. In addition, membrane gradients were also visualized by
supplementing fluorescent beads [3 µl of Covaspheres (Duke Scientific, Palo Alto, CA) per 100 µl of membrane solution] and examined with the appropriate fluorescent filter set. The density of
membrane particles and the density of the fluorescent beads were
quantified with a digital image analysis system (Metamorph; Universal
Imaging, West Chester, PA); there was a linear relationship between
these two measurements. To determine the slope of the gradients, for
each cortical explant the membrane densities were measured in a narrow
stripe (50 × 400 µm) at the beginning ("downhill," lowest
membrane concentration) and the end ("uphill," highest membrane
concentration) of a 600-µm-wide segment centered around the explant.
The slope of the gradient was defined as the change (in percent) of the
mean concentration from the uphill to the downhill side per 25 µm,
the average width of the growth cone.
Tissue culture. Embryonic day 16 embryos were
obtained by cesarean section of pregnant Lewis rats. Brains were
removed and dissected in 4°C Gey's balanced salt solution.
Blocks of neocortical tissue were cut as 200 µm3 using a MacIlwain Tissue Chopper.
Explants were grown on laminin-poly-L-lysine (1 mg/ml; Sigma) -coated coverslips covered with homogeneous carpet of
membranes (uniform substrate) or membranes gradients. Culture medium
was composed of 50% Eagle's basal medium, 25% horse serum, 25%
HBSS supplemented with 0.1 mM glutamin,
and 6.5 mg/ml glucose (all from Life Technologies, Gaithersburg,
MD). Explants were grown for 24 hr before fixation and analysis.
Explants located in the linear part of the gradients were selected for
analysis, and fiber length was determined with an ocular grid of an
inverted microscope equipped with 20× phase-contrast objective. Fiber
outgrowth from cortical explants usually occurs in all directions. We
analyzed axons growing in the quadrant toward increasing and in the
quadrant toward decreasing membrane concentrations. Axons running
oblique to the membrane gradients (deviation angle of 45-90°) were
excluded, because the change in membrane concentrations encountered by
these fibers is different from fibers growing directly uphill or
downhill the membrane gradients.
Time-lapse recordings was similar to the method described previously
(Hübener et al., 1995). In brief, Petri dishes containing a
coverslip with cortical explants were transferred to a chamber on the
stage of an inverted microscope (Axiovert 135; Zeiss, Oberkochen, Germany) in which the temperature (37°) and CO2
concentration (5%) were kept constant. Images were taken every 5 min
during 4 hr periods. To minimize photodynamic damage, a
computer-controlled electronic shutter closed the light path after an
image had been taken.
| |
RESULTS |
Sema3A and Sema3C belong to the class III semaphorins and are
secreted proteins that bind to cell membranes and/or extracellular matrix components. Indeed, Sema3A-collapsin was originally purified from brain membrane extracts (Luo et al., 1995). Therefore, we prepared
membranes from Sema3A- and Sema3C-expressing HEK 293 cells and
untransfected HEK 293 cells as a control to examine the response of
cortical axons to stabilized gradients of semaphorins. As illustrated
in Figure 1, gradients of sigmoid shapes
were formed over a distance of 6-10 mm. The analysis was restricted to
0.6-mm-wide segments centered around the cortical explants (Fig.
1A). The gradients prepared by this method were
generally linear within the analyzed segments, but they differed in
slope and concentration (Fig. 1B).
View larger version (90K):
[in this window]
[in a new window]
|
Figure 1.
Production of semaphorin gradients.
A, Example of a gradient with sigmoid shape over a
distance of >6 mm. The dashed box corresponds to the
linear part of the gradient that was used for the analysis.
B, The linear segments of the analyzed gradients had
various slopes [shallow gradient of 0.8% (filled
triangles) compared with steep gradient of 6.8%
(filled circles)]. C-E, Axons
extending from cortical explants placed on membrane gradients prepared
from untransfected control cells (C),
Sema3A-expressing cells (D), and
Sema3C-expressing cells (E). Phase-contrast
photomicrographs are shown on the left, and the
corresponding fluorescent micrographs visualizing the graded
distribution of membrane particles labeled with fluorescent beads are
on the right. Scale bar, 150 µm.
|
|
Figure 1C-E depicts representative examples of cortical
explants placed on gradients prepared with native cortical membranes mixed 1:1 with untransfected HEK 293 cell membranes (Fig.
1C, control condition) and with membranes from Sema3A- and
Sema3C-producing HEK 293 cells (Fig.
1D,E, respectively). Axonal
outgrowth from explants placed on untransfected cell membranes is
radial, and fibers are equally distributed around the explant and they
reach similar length. In contrast, axonal outgrowth on membrane
gradients from transfected HEK 293 cells is not uniform. As illustrated in Figure 1D, on Sema3A gradients, less axons are
growing toward the side of higher membrane concentration (uphill the
gradient), and they are shorter than axons growing toward the side of
lower membrane concentration (downhill the gradient). In contrast, more fibers are growing uphill Sema3C gradients, and they are longer than
fibers growing downhill Sema3C gradients (Fig. 1E).
To quantify these effects, we measured the length of all axons in the
quadrant facing the uphill side of the gradients and in the quadrant
facing the downhill side of the gradients. Fibers growing oblique to the direction of the gradients were excluded from the analysis, because
these fibers encounter different slopes of the gradients than fibers
growing parallel to the direction of the gradients. As illustrated in
Figure 2A, axons
extending uphill Sema3A gradients were 27% shorter than fibers
extending downhill Sema3A gradients (p < 0.001;
Student's unpaired t test). When membranes from
Sema3C-expressing cells were used, however, cortical fibers extending
on increasing concentration gradients were 49% longer than fibers
extending on decreasing concentration gradients
(p < 0.001; Student's unpaired t
test) (Fig. 2A). On gradients with membranes prepared
from untransfected cells, there was no difference in the length of
fibers extending uphill or downhill gradients (p > 0.1). Thus, increasing concentrations of membrane-bound semaphorins
(haptotactic effects) and diffusible semaphorin gradients in a plasma
gel (chemotactic effects) (Bagnard et al., 1998) evoke similar
responses from cortical axons. On the other hand, cortical axons
growing toward decreasing concentrations of the chemorepellent Sema3A
appeared not to be affected in their growth, and they reached the same
length as fibers extending on control membranes
(p > 0.1). Fibers growing on decreasing
chemoattractive Sema3C gradients were 19% shorter than on
control substrates (p < 0.05) (Fig.
2A).
View larger version (22K):
[in this window]
[in a new window]
|
Figure 2.
The response of cortical axons toward semaphorins
depends on their spatial distribution. a, Growth on
gradients. The length of cortical axons growing on gradients of control
membranes or membranes containing Sema3A or Sema3C was determined
separately for those growing uphill (filled bars)
or downhill (open bars) gradients (n is
number of explants; Student's unpaired t test;
**p < 0.001; ns, not significant).
b, Growth on homogeneous substrates. Each
dot corresponds to the mean axonal length measured for
different explants on uniform substrates of control membranes
(open squares), Sema3C-containing membranes
(filled triangles), or Sema3A-containing
membranes (filled circles). The reduction of
axonal length was strongly correlated with the concentration of Sema3A,
whereas decrease of fiber length induced by Sema3C was independent of
its concentration.
|
|
The observation that fibers extending on downhill Sema3A gradients were
not affected by the presence of this molecule might be simply because
Sema3A concentrations on the downhill side are not sufficient to evoke
an axonal response. We therefore also examined axonal growth on
homogeneous membrane carpets, using similar membrane densities as axons
encounter on the downhill side of the prepared gradients. Under these
conditions, the length of cortical axons on uniform Sema3A membranes
was markedly reduced (34% reduction; p < 0.001). A
reduction in fiber length was also observed on homogeneous substrates
containing Sema3C (17% reduction; p < 0.05).
Strikingly, the reduction of axonal length induced by Sema3A was
proportional to the concentration of the molecule (r2= 0.614; p < 0.005), whereas Sema3C elicited a constant decrease in length that
was independent of the membrane density
(r2= 0.04; p > 0.1). These results indicate that the function of axonal guidance cues
depends critically on the spatial context in which they are presented.
Increasing and uniform concentrations of Sema3A evoke a strong
repulsive response in cortical axons, whereas decreasing amounts of
Sema3A do not influence the elongation of these fibers. Likewise,
chemoattractive effects by Sema3C are only elicited when cortical axons
encounter increasing concentrations of the molecule, but not if they
are exposed to uniform or decreasing amounts of Sema3C (Fig.
2B).
Given that cortical axons are attracted by increasing concentrations of
Sema3C (Bagnard et al., 1998; present results), it was surprising that,
compared with control membranes, cortical axons were shorter when
exposed to uniform or decreasing concentrations of Sema3C. Previous
work indicated that the repulsive effects of Sema3A are caused
by the growth cone collapse activity of this molecule (Luo et
al., 1993). Does Sema3C, depending on its spatial distribution, also
induce growth cone collapse of cortical axons? To address this issue,
we used time-lapse imaging to examine axonal growth on Sema3A, Sema3C,
and control substrates. The average growth speed of cortical axons on
the control substrates was 18.6 µm/hr, and growth cones collapsed
only rarely (0.34 collapses/hr). Consistent with previous work, on
Sema3A membranes, there was a fourfold increase in the rate of growth
cone collapses, and the average growth speed decreased to 8.6 µm/hr
(p < 0.001; Student's t test)
(Table 1). Strikingly, the net growth
speed, i.e., the mean growth speed excluding the periods of growth cone
collapse, was not different on control (19.5 µm/hr) and on Sema3A
substrates (17.2 µm/hr). In contrast, as depicted in Table 1, on
substrates with uniform or decreasing Sema3C concentrations the rate of
growth cone collapse was low (0.22 and 0.24 collapses/hr,
respectively), but the growth speed was reduced to almost half of the
speed observed on control substrates. Thus, the reduction of axonal
length on Sema3A substrates is caused by the collapse of growth cones,
whereas on uniform Sema3C substrates and downhill Sema3C gradients by a
decrease of the growth speed of the fibers. For uphill Sema3C gradients, when this molecule acts as a chemoattractant signal for
cortical axons, growth speed was significantly increased compared with
control conditions (Table 1).
The response of cortical axons to Sema3A and Sema3C gradients was not
dependent on the semaphorin concentrations used in the present
experiments. The membrane particle density reached by axons on the
uphill side in the different experiments varied 8.8-fold, and it ranged
from 1.1-9.7 relative units. On the downhill side of the gradients,
axons encountered a membrane concentration between 0.8-4.4 units
(almost sixfold variation). As illustrated in Figure 3, there was no correlation between fiber
length and the density of membranes containing Sema3A and Sema3C, on
neither the downhill side nor the uphill side of the gradients
(downhill: r2 = 0.26, p > 0.1 for Sema3C;
r2 = 0.01, p > 0.1 for Sema3A; uphill: r2 = 0.06, p > 0.1 for Sema3C;
r2 = 0.25, p > 0.1 for Sema3A). Thus, although for some gradients the relative
semaphorin concentration on the uphill side was lower than the
concentration on the downhill side of other gradients, in each case
effects of semaphorins were only observed for fibers growing uphill but
not downhill. For example, in four explants with Sema3A gradients, the
relative concentration encountered by axons on the uphill side was
<2.9 units, but their length was reduced by >30%. On uniform
substrate, such concentrations induced only a reduction by <10%. In
contrast, the highest concentration on the downhill side of Sema3A was
3.9, but there was no effect on axonal length. When presented as a
uniform substrate, at this concentration Sem3A produced a greater than
30% reduction in axonal length.
View larger version (21K):
[in this window]
[in a new window]
|
Figure 3.
Influence of semaphorin concentration on axonal
length. a, Axonal length at the uphill side of the
gradient. On the uphill side of gradients, the average length of
cortical axons was higher compared with control gradients when they
were exposed to increasing concentrations of Sema3C, whereas it was
lower in the case of Sema3A. Under all conditions, the fiber length was
independent of the relative semaphorin concentration on the uphill
side. b, Axonal length at the downhill side of the
gradient. On the downhill side, the average length of cortical axons
was shorter in the presence of decreasing concentrations of Sema3C. The
magnitude of this effect was identical for all concentrations tested.
Strikingly, the inhibition observed on uniform substrate and on the
uphill side of Sema3A gradients was not detected when axons were
growing toward decreasing Sema3A concentration. Concentrations are
indicated as membrane particle × 103/mm2. Open
squares, Control membranes; filled circles,
Sema3A; filled triangles, Sema3C-containing
membranes.
|
|
The different membrane concentrations used here also lead to different
slopes of the gradients, ranging from 0.3 to 4.1% (14-fold variation),
when expressed as percent difference of membrane concentration over 25 µm. As was observed for the variation of the relative concentrations
of semaphorins, there was no correlation between slopes of the
gradients and the effects on axonal length (downhill: r2 = 0.02 for Sema3C;
r2 = 0.01 for Sema3A,
p > 0.05; uphill:
r2 = 0.36 for Sema3C;
r2 = 0.14 for Sema3A,
p > 0.05). As already mentioned, we initially restricted our analysis of fiber length to axons that were growing approximately parallel to the directions of the gradients (deviation angle of <45°), because axons emerging from the explants at angles >45° encounter different slopes of the gradients. The results presented here, however, indicate that the slopes of Sema3A and Sema3C
gradients does not affect the behavior of cortical growth cones. We
therefore reanalyzed several explants and measured the length of axons
extending at angles >45° toward the gradient direction. We found
that there was no significant difference in axonal length of axons
extending parallel or oblique to the gradient direction (length uphill
Sema3A gradients: 104.8 ± 6.1 µm for n = 8 "oblique" axons; 97.1 ± 6.3 µm for n = 10 "parallel" axons, p > 0.04; length uphill Sema3C
gradients: 309.3 ± 9.1 for n = 12 oblique axons; 296.5 ± 5.2 for n = 10 parallel axons,
p > 0.2). Thus, cortical axons are able to read the
direction or "sign" of the slope of semaphorin gradients, but their
response is over a wide range, independent of the shape and the
absolute concentration of these gradients (Fig.
4).
View larger version (20K):
[in this window]
[in a new window]
|
Figure 4.
Influence of the slope of concentration gradients
on axonal length. The slope of gradients prepared from control
membranes (open squares), or membranes containing Sema3A
(filled circles) or Sema3C (filled
triangles) did not influence the average axonal length both
when axons that grow uphill (a) or downhill
(b) the gradients were analyzed. The slope of the
concentration gradients are shown as percent difference of membrane
density over 25 µm, the average size of a cortical growth cone.
|
|
| |
DISCUSSION |
We demonstrated in a previous study that Sema3A can act as a
chemorepellent and Sema3C as a chemoattractant guidance molecule for
cortical axons. Fibers extending toward Sema3A-secreting cell aggregates are short, and they are deflected away from these
aggregates. When confronted with alternating stripes of membranes
containing Sema3A and control membranes, cortical axons avoid the
Sema3A substrate. Sema3C elicits opposite responses; in the stripe
assay, fibers grow preferentially in membrane lanes containing Sema3C, and in a gradient of Sema3C produced by aggregates of transfected HEK
293 cells, fibers are long and they turn toward the source of Sema3C
(Bagnard et al., 1998). The present findings with membrane gradients
indicate that repellent effects of Sema3A and the attractant effects of
Sema3C are not observed when axons encounter decreasing concentrations
of these molecules. Cortical axons are very sensitive to the direction
of concentration gradients, but their response is highly stereotypic
and primarily independent of how much and the range over which these
concentration vary. As reported here, cortical axons growing down a
very shallow decreasing concentration gradient (slope of 0.3%) are not
affected by Sema3A, whereas on a uniform substrate (slope of 0%) their
growth is strongly reduced. Using our estimates of the relative
concentrations on the uphill and downhill side on Sema3A gradients, we
demonstrate that a concentration change from 1.15 to 1.22 units over
the width of the growth cone exerts a strong effect on fiber growth.
However, axons growing down a gradient with more than five times higher
Sema3A concentrations, changing from 6.2 to 5.8 units, are not
influenced by this repulsive molecule. This property would facilitate
process outgrowth by enabling axons to grow without being inhibited by
the presence of repulsive guidance cues released from inappropriate
territories, as long as they grow away from these regions.
The finding that the length of cortical axons is reduced when they grow
downhill Sema3C gradients appears paradoxical, because this molecule
exerts a strong attractive effect when axons grow in the opposite
direction, uphill the gradient. We noted previously that, on uniform
Sema3C substrates, axonal outgrowth and axonal length is reduced
compared with control substrates (Bagnard et al., 1998). Based on this
assay alone, Sema3C could be classified as a repulsive or repellent cue
for cortical axons. The present data with time-lapse imaging of growing
axons indicated that the reduction of axonal length on Sema3A and
Sema3C substrates is caused by different cellular mechanisms. Sema3A
increases the rate of growth cone collapses but has no effect on the
net growth speed. Thus, Sema3A is a potent collapse inducer, whereas
Sema3C has no significant effect on growth cone retractions. Instead, on substrates with uniform and decreasing concentrations of Sema3C, there is a reduction of the average growth speed compared with neutral
substrates. Because the known families of repulsive guidance receptors
show almost no sequence similarity in their cytoplasmatic domain, it
has been proposed that different classes of repulsive receptors mediate
different types of repellent responses (Bashaw and Goodman,
1999). Moreover, in vitro studies with
Xenopus spinal axons have demonstrated that attractive
actions of guidance molecules can in some cases be converted to
repulsion by changing the levels of cAMP or cGMP (Ming et al., 1997;
Song et al., 1998). It will therefore be interesting to monitor and
manipulate the activity of cyclic nucleotide signaling pathways when
axons grow uphill and downhill semaphorin gradients.
Recently, there has been much discussion of how axons attracted by
long-range guidance to intermediate targets do not stall, but rather
continue to grow to their ultimate target. For example, netrin-1
released by cells in the floor plate in the ventral midline of the
spinal cord and hindbrain attracts commissural axons, which then grow
past the midline and extend contralaterally (Kennedy et al., 1994;
Colamarino and Tessier-Lavigne, 1995). Given that netrin-1
concentration is highest in the floor plate, how then can axons grow
away from the midline? Several mechanisms have been suggested to
explain how axons can "ignore" attractive signals once they crossed
the midline (Klambt et al., 1991; Goodman, 1996; Kidd et al.,
1998, 1999; Brose et al., 1999). The capacity of growth cones to ignore
decreasing concentration of chemoattractants may represent one of the
strategies allowing axons to grow past intermediate targets. An
alternative explanation would be that Sema3C exhibits a repulsive
activity when axons encounter decreasing concentration of the factor.
Nevertheless, monitoring of axons growing downhill show that, rather
than being inhibited, these fibers behave as they would do in
vivo in decision regions, slowing down and enlarging growth cones
(Stirling and Dunlop, 1995). Thus, decreasing concentration of the
attractant signal might represent the loss of the driving force of the signal.
The findings presented here provide new insights into the basic
mechanisms of chemotropic guidance. In many studies using functional
assays to investigate target-derived chemotropic activities, it was
often assumed that the effects would depend on the distance from the
growth cone to the target. The closer a growth cone is to a source of a
diffusible guidance signal, the higher its response should be to this
signal. In a trivial sense, this is always the case; if the
concentration of a factor is below a certain threshold, then it has no
influence on growth cone behavior. However, as shown here for gradients
with semaphorins, there might be a wide range of distances from the
target at which growth cones exhibit similar physiological responses.
Within certain limits, the only critical parameter of gradients that
determine how fibers grow is whether there is an increase or a decrease
in the concentration of these guidance molecules. Growth cone guidance
by gradients is therefore robust and stereotyped, because it is
primarily independent of the exact dimensions of the gradients. It is
now an intriguing question to examine how this mechanism is implemented
at the level of the growth cone (or perhaps along the axon). Evolution
might have selected such a mechanism because, in nervous tissue in
which complex patterns of connections are being formed, it may be
difficult to produce highly reproducible gradients of wiring signals
with well defined slopes and with well adjusted absolute
concentrations. The results presented here suggest that, even if
gradients of wiring molecules vary to some extent from one brain to
another, brains are still wired the same way.
| |
FOOTNOTES |
Received June 14, 1999; revised Oct. 11, 1999; accepted Nov. 5, 1999.
This work was supported by the Human Frontiers Science Program and
Deutsche Forschungsgemeinschaft Grant Pu102/4-2. We thank Susan Amara
for helpful comments on this manuscript.
Correspondence should be addressed to Jürgen Bolz,
Universität Jena, Institut für Allgemeine Zoologie,
Erberstrasse 1, 07743 Jena, Germany. E-mail:
bolz{at}pan.zoo.uni-jena.de.
| |
REFERENCES |
-
Adams RH,
Lohrum M,
Klostermann A,
Betz H,
Püschel AW
(1997)
The chemorepulsive activity of secreted semaphorins is regulated by furin-dependent proteolytic processing.
EMBO J
16:6077-6086[ISI][Medline].
-
Bagnard D,
Lohrum M,
Uziel D,
Püschel AW,
Bolz J
(1998)
Semaphorins act as attractive and repulsive guidance signals during the development of cortical projections.
Development
125:5043-5053[Abstract].
-
Baier H,
Bonhoeffer F
(1992)
Axon guidance by gradients of a target-derived component.
Science
255:472-475[Abstract/Free Full Text].
-
Baier H,
Klostermann S
(1994)
Axon guidance and growth cone collapse in vitro. Neuroprotocols: Companion Methods
Neurosci
4:96-105.
-
Bashaw GJ,
Goodman CS
(1999)
Chimeric axon guidance receptors: the cytoplasmic domains of slit and netrin receptors specify attraction versus repulsion.
Cell
97:917-926[ISI][Medline].
-
Brose K,
Bland KS,
Wang KH,
Arnott D,
Henzel W,
Goodman CS,
Tessier-Lavigne M,
Kidd T
(1999)
Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance.
Cell
96:795-806[ISI][Medline].
-
Colamarino SA,
Tessier-Lavigne M
(1995)
The role of the floor plate in axon guidance.
Annu Rev Neurosci
18:497-529[ISI][Medline].
-
Gierer A
(1981)
Development of projections between areas of the nervous system.
Biol Cybern
42:69-78[ISI][Medline].
-
Goodhill G
(1997)
Diffusion in axon guidance.
Eur J Neurosci
9:1414-1421[ISI][Medline].
-
Goodman CS
(1996)
Mechanisms and molecules that control growth cone guidance.
Annu Rev Neurosci
19:341-377[ISI][Medline].
-
Götz M,
Novak N,
Bastmeyer M,
Bolz J
(1992)
Membrane bound molecules in rat cerebral cortex regulate thalamic innervation.
Development
116:507-519[Abstract].
-
Hübener M,
Götz M,
Klostermann S,
Bolz J
(1995)
Guidance of thalamocortical axons by growth-promoting molecules in the developing rat cerebral cortex.
Eur J Neurosci
7:1963-1972[ISI][Medline].
-
Kennedy TE,
Serafini T,
De la Torre JR,
Tessier-Lavigne M
(1994)
Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.
Cell
78:425-435[ISI][Medline].
-
Kidd T,
Russell C,
Goodman CS,
Tear G
(1998)
Dosage-sensitive and complementary functions of roundabout and commissureless control axon crossing of the CNS midline.
Neuron
20:25-33[ISI][Medline].
-
Kidd T,
Bland KS,
Goodman CS
(1999)
Slit is the midline repellent for the robo receptor in Drosophila.
Cell
96:785-794[ISI][Medline].
-
Klambt C,
Jacobs JR,
Goodman CS
(1991)
The midline of the Drosophila central nervous system: a model for the genetic analysis of cell fate, cell migration, and growth cone guidance.
Cell
64:801-815[ISI][Medline].
-
Lumsden AGS,
Davies AM
(1983)
Earliest sensory nerve fibres are guided to peripheral targets by attractants other than nerve growth factor.
Nature
306:786-788[Medline].
-
Luo Y,
Raible D,
Raper JA
(1993)
Collapsin: a protein in brain that induces the collapse and paralysis of neuronal growth cones.
Cell
75:217-227[ISI][Medline].
-
Luo Y,
Shepherd I,
Li J,
Renzi MJ,
Chang S,
Raper JA
(1995)
A family of molecules related to collapsin in the embryonic chick nervous system.
Neuron
14:1131-1140[ISI][Medline].
-
Messersmith EK,
Leonardo ED,
Shatz CJ,
Tessier-Lavigne M,
Goodman CS,
Kolodkin AL
(1995)
Semaphorin III can function as a selective chemorepellent to pattern sensory projections in the spinal cord.
Neuron
14:949-959[ISI][Medline].
-
Ming G-L,
Song H-J,
Berninger B,
Holt CE,
Tessier-Lavigne M,
Poo MM
(1997)
cAMP-dependent growth cone guidance by netrin-1.
Neuron
19:1225-1235[ISI][Medline].
-
Pini A
(1993)
Chemorepulsion of axons in the developing mammalian central nervous system.
Science
261:95-98[Abstract/Free Full Text].
-
Polleux F,
Giger RJ,
Ginty D,
Kolodkin A,
Ghosh A
(1998)
Patterning of cortical efferent projections by Semaphorin-Neuropilin interactions.
Science
282:1904-1906[Abstract/Free Full Text].
-
Püschel AW
(1996)
The semaphorins: a family of axonal guidance molecules?
Eur J Neurosci
8:1317-1321[ISI][Medline].
-
Püschel AW,
Adams RH,
Betz H
(1995)
Murine semaphorins D/collapsin is a member of a divers gene family and creates domains inhibitory for axonal extension.
Neuron
14:941-948[ISI][Medline].
-
Rosentreter SM,
Davenport RW,
Löschinger J,
Huf J,
Jung J,
Bonhoeffer F
(1998)
Response of retinal ganglion cell axons to striped linear gradients of repellent guidance molecules.
J Neurobiol
37:541-562[ISI][Medline].
-
Song HJ,
Ming GL,
He Z,
Lehmann M,
Mckerracher L,
Tessier-Lavigne M,
Poo MM
(1998)
Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides.
Science
281:1515-1518[Abstract/Free Full Text].
-
Stirling RV,
Dunlop SA
(1995)
The dance of the growth cones: where to next?
Trends Neurosci
18:111-115[ISI][Medline].
-
Tessier-Lavigne M,
Goodman CS
(1996)
The molecular biology of axon guidance.
Science
274:1123-1133[Abstract/Free Full Text].
-
Tessier-Lavigne M,
Placzek M,
Lumsden AGS,
Dodd J,
Jessell TM
(1988)
Chemotropic guidance of developing axons in the mammalian central nervous system.
Nature
336:775-778[Medline].
Copyright © 2000 Society for Neuroscience 0270-6474/00/2031030-06$05.00/0
This article has been cited by other articles:
|
|
|
|
 
L. Roth, C. Nasarre, S. Dirrig-Grosch, D. Aunis, G. Cremel, P. Hubert, and D. Bagnard
Transmembrane Domain Interactions Control Biological Functions of Neuropilin-1
Mol. Biol. Cell,
February 1, 2008;
19(2):
646 - 654.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B Gonthier, C Nasarre, L Roth, M Perraut, N Thomasset, G Roussel, D Aunis, and D Bagnard
Functional Interaction between Matrix Metalloproteinase-3 and Semaphorin-3C during Cortical Axonal Growth and Guidance
Cereb Cortex,
July 1, 2007;
17(7):
1712 - 1721.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Banu, J. Teichman, M. Dunlap-Brown, G. Villegas, and A. Tufro
Semaphorin 3C regulates endothelial cell function by increasing integrin activity
FASEB J,
October 1, 2006;
20(12):
2150 - 2152.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. T. Legg and T. P. O'Connor
Gradients and Growth Cone Guidance of Grasshopper Neurons
J. Histochem. Cytochem.,
April 1, 2003;
51(4):
445 - 454.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Mann, C. Peuckert, F. Dehner, R. Zhou, and J. Bolz
Ephrins regulate the formation of terminal axonal arbors during the development of thalamocortical projections
Development,
March 10, 2003;
129(16):
3945 - 3955.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. M. Isbister, P. J. Mackenzie, K. C. W. To, and T. P. O'Connor
Gradient Steepness Influences the Pathfinding Decisions of Neuronal Growth Cones In Vivo
J. Neurosci.,
January 1, 2003;
23(1):
193 - 202.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Ricard, V. Rogemond, E. Charrier, M. Aguera, D. Bagnard, M.-F. Belin, N. Thomasset, and J. Honnorat
Isolation and Expression Pattern of Human Unc-33-Like Phosphoprotein 6/Collapsin Response Mediator Protein 5 (Ulip6/CRMP5): Coexistence with Ulip2/CRMP2 in Sema3A- Sensitive Oligodendrocytes
J. Neurosci.,
September 15, 2001;
21(18):
7203 - 7214.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Bagnard, C. Vaillant, S.-T. Khuth, N. Dufay, M. Lohrum, A. W. Puschel, M.-F. Belin, J. Bolz, and N. Thomasset
Semaphorin 3A-Vascular Endothelial Growth Factor-165 Balance Mediates Migration and Apoptosis of Neural Progenitor Cells by the Recruitment of Shared Receptor
J. Neurosci.,
May 15, 2001;
21(10):
3332 - 3341.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Bagnard, N. Chounlamountri, A. W. Puschel, and J. Bolz
Axonal Surface Molecules Act in Combination with Semaphorin 3A during the Establishment of Corticothalamic Projections
Cereb Cortex,
March 1, 2001;
11(3):
278 - 285.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. E. Spencer, K. Lukowiak, and N. I. Syed
Transmitter-Receptor Interactions between Growth Cones of Identified Lymnaea Neurons Determine Target Cell Selection In Vitro
J. Neurosci.,
November 1, 2000;
20(21):
8077 - 8086.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
