Download Axon Guidance by Growth Cones and Branches: Common

REVIEW ■
Axon Guidance by Growth Cones and Branches:
Common Cytoskeletal and Signaling Mechanisms
ERIK W. DENT, FANGJUN TANG, and KATHERINE KALIL
Growing axons are guided to appropriate targets by responses of their motile growth cones to environmental cues. Interstitial axon branching is also an important form of axon guidance in the mammalian CNS.
Visualization of growing axons in cortical slices and in dissociated cortical cultures showed that growth
cone pausing behaviors demarcate sites of future axon branching. Studies of vertebrate and invertebrate
growth cones suggest common mechanisms that regulate growth cone behaviors and axon branching.
These include reorganization of the actin and microtubule cytoskeleton, dynamic interactions between
microtubules and actin filaments, effects of axon guidance molecules, actions of actin regulatory proteins,
and dynamic changes in intracellular calcium signaling. Future challenges will be to extend high-resolution
imaging of single neurons to studies of intracellular events in the intact nervous system and to apply knowledge of developmental mechanisms to the promotion of axon sprouting after injury in the adult CNS. NEUROSCIENTIST 9(5):343–353, 2003. DOI: 10.1177/1073858403252683
KEY WORDS Cortical development, Growth cone, Cytoskeleton, Axon branching, Calcium
Growth cones are the expanded motile tips of growing
axons. To reach their synaptic partners in the developing
nervous system, axonal growth cones must navigate
along specific pathways in response to molecular guidance cues. Thus, a major focus in axon guidance
research has been to understand how extracellular cues
influence the growth cone to select the appropriate pathway toward the target (Song and Poo 2001; Dickson
2002). However, in many CNS pathways such as those
extending from the cerebral cortex, interstitial axon
branches rather than primary axonal growth cones innervate target neurons (O’Leary and others, 1990). How are
the locations of axon branches determined? Direct visualization of fluorescently labeled axons in living early
postnatal cortical slices suggested that the primary
growth cones of cortical axons demarcate branch points
by developing large complex morphologies and undergoing lengthy pausing behaviors (Halloran and Kalil
1994). In the corpus callosum, these behaviors were
observed only beneath the contralateral cortex, where
axons give rise to interstitial branches tipped by growth
cones (Fig. 1). In contrast, growth cones extending in
Supported by NIH grants NS 14428 and NS 34270 and predoctoral
training grant GMO7507.
Department of Anatomy, University of Wisconsin, Madison (EWD,
KK). Neuroscience Training Program, University of Wisconsin,
Madison (FT, KK).
Address correspondence to: Dr. Katherine Kalil, University of
Wisconsin–Madison, Department of Anatomy, 1300 University
Avenue, Madison WI 53706 (e-mail: [email protected]). Dr.
Dent’s current address: Department of Biology, Massachusetts
Institute of Technology, 68-270, 77 Massuchusetts Avenue,
Cambridge, MA 02139.
Volume 9, Number 5, 2003
Copyright © 2003 Sage Publications
ISSN 1073-8584
other regions of the callosal pathway had smaller, simpler morphologies and grew rapidly without pausing.
These observations suggested a relationship between
pausing behaviors by the growth cone and subsequent
axon branching. In cultures of dissociated cortical neurons, growth cones also developed large complex morphologies during pausing behaviors lasting for many
hours or even days. After the paused growth cones began
to reextend, they left behind remnants from which axon
branches later developed (Szebenyi and others 1998).
These events occurred in the absence of specific targets,
but pausing and branching were enhanced after application of fibroblast growth factor-2 (FGF-2) (Szebenyi and
others 2001), a target-derived factor present in the developing cortex. Because branching mechanisms have
implications for sprouting after injury, this review
emphasizes interstitial axon branching in the developing
mammalian CNS. We describe recent research suggesting that guidance at the growth cone and from axon
branch points shares common mechanisms of cytoskeletal reorganization, extracellular guidance cues, actinassociated regulatory proteins, and intracellular calcium
signaling.
Growth Cone Behaviors
in Decision Regions
Large complex growth cones have been found consistently at choice points in the nervous system where
axons make decisions about growth in new directions,
such as crossing the midline turning or branching
(Mason and Erskine 2000). In many cases, growth cones
stall or slow their forward advance at such choice points.
During the past decade, families of guidance cues have
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Fig. 1. Schematic representation of stages of axon branching in the developing corpus callosum. Cortical neurons and their callosal
axons are drawn in red on images of fixed coronal sections to show extension of an efferent cortical axon through the callosum (1),
pausing and enlarging of the primary growth cone beneath the contralateral cortical target (2), reextension of the primary axon leaving behind remnants of the primary growth cone (3), growth of an interstitial axon branch from the remnant of the primary growth cone
(4), development of an axon arbor from the interstitial axon branch within the cortical target and eventual regression of the primary
axon (5), and completion of the callosal connection (6). These schematics are based on results from direct visualization of callosal
axon outgrowth in living cortical slices and anatomy of callosal development in vivo.
been discovered that either attract or repel axonal growth
cones (or sometimes both, depending on the internal
state of the growth cone), and these molecules are highly
conserved across species. At decision regions, repulsive
axon guidance molecules have been shown to inhibit
growth cone advance. For example, the highly conserved
Slit proteins repel axons at the midline in Drosophila
(Kidd and others 1999) and regulate midline crossing in
regions of the mammalian CNS such as the optic chiasm
(Plump and others 2002) and corpus callosum (Shu and
Richards 2001; Bagri and others 2002). Interestingly,
Slit proteins not only repel growth cones but also induce
branching on sensory axons (Brose and Tessier Lavigne
2000), providing further evidence that axon branching
and growth cone guidance are mechanistically related.
The semaphorins are another family of guidance cues
that can act as growth cone repellents. Both the Slit proteins and the semaphorins, particularly Sema 3A, are
expressed at high levels in the developing mammalian
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cortex and act as chemorepellants for cortical axons
in vitro (Bagnard and others 1998) and in situ
(Polleux and others 1998). However, the mechanisms
by which repulsive guidance molecules inhibit growth
cone advance or regulate axon branching are not well
understood.
Visualizing Cytoskeletal
Dynamics in Cortical Growth Cones
Ultimately, changes in growth cone morphology, motility, and direction of growth depend on changes in the
organization of the actin and microtubule cytoskeleton
(Sabry and others 1991; O’Conner and Bentley 1993;
Lin and Forscher 1993; Tanaka and Sabry 1995).
Microtubules are long hollow polymers of tubulin that
form dense bundles in axons and dendrites. They not
only lend structural support to neuronal processes but
also serve as railways along which materials are trans-
Axon Guidance and Branching Mechanisms
ported from the cell body into the neurites. Actin filaments play an essential role in driving motility and guidance of the growth cone (Luo 2002). In veil-like lamellipodia, actin filaments form a meshwork, but within
filopodia, the spiky fingerlike protrusions from the
growth cone periphery that are important for exploring
the environment, actin filaments form straight bundles.
High-resolution imaging of living growth cones has
provided an unparalleled opportunity to visualize directly the dynamic cytoskeletal changes that underlie growth
cone behaviors. The growth cones of cultured bag cell
neurons from the snail Aplysia (Lin and Forscher 1993;
Schaefer and others 2002) are large and flat and have
been highly advantageous for peering into the inner
machinery of the growth cone. However, to study how
guidance cues such as the semaphorins and Slits influence the organization of the cytoskeleton of mammalian
CNS neurons, it is important to study growth cones of
CNS neurons. Unfortunately, such growth cones are typically too small (5–10 µm in diameter) to achieve the
spatial and temporal resolution to measure local changes
in the cytoskeleton. However, during prolonged pausing
states leading to development of axon branches, the
growth cones of cortical neurons develop large flat morphologies up to 50 µm in diameter (Fig. 2), which
approaches the size of Aplysia growth cones. Branches
also tend to emanate from large, flattened regions along
the axon shaft. Thus, cultured cortical neurons provide a
unique mammalian CNS model for imaging intracellular
events underlying growth cone behaviors and axon
branching.
Microtubule Reorganization during Growth
Cone Behaviors and Axon Branching
Microtubules are bundled together in the axon shaft and
also extend into the central region of the growth cone.
They are splayed apart in growth cones that are extending, but when growth cones stall, microtubules form
prominent loops in the central region (Tanaka and
Kirschner 1991). Microinjection of cortical neurons
with fluorescent tubulin and live-cell imaging revealed
movements of individual microtubules and reorganization of microtubule arrays in cortical axons and their
growth cones (Dent and others 1999). Microtubule reorganization at the growth cone and at axon branch points
was strikingly similar (Kalil and others 2000). Along the
axon shaft, microtubules splayed apart and locally fragmented prior to development of branches. During prolonged growth cone pausing behaviors, microtubules were
maintained in a tightly bundled loop. During transitions
of the growth cones from pausing to growth states,
microtubules reorganized from looped to splayed configurations followed by fragmentation. Reorganization
and fragmentation permitted short microtubules to
explore new directions of growth with forward and backward movements within the growth cone periphery and
in branches extending from the axon shaft (Fig. 3).
Further observations of long-term branching events
showed that microtubules invaded and remained in
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Fig. 2. An example of a large paused cortical growth cone
imaged in differential interference contrast microscopy. The
central, transition, and peripheral regions of the growth cone
are indicated in green, yellow, and pink, respectively. Note the
presence of organelles in the dense central region and the thin
lamellipodial veil and spiky filopodia in the peripheral region.
branches favored for further growth but withdrew from
branches that regressed.
These findings suggest that the cytoskeletal mechanisms underlying axon branching involve the reorganization of the microtubule array into a more labile form
whereby microtubules can become debundled and fragmented. This plasticity would permit microtubules to
explore the growth cone and newly forming branches.
The phenomenon of retrograde microtubule movements
suggests a mechanism whereby some axonal processes
can regress while others grow. Similarities in microtubule reorganization during growth and guidance at the
growth cone and at axon branch points is consistent with
recent findings showing that molecules such as the Slits,
which influence growth cone guidance, can also promote
axonal branching (Wang and others 1999). At present,
the role of microtuble loops in regulating growth cone
advance and the mechanisms governing their formation
and disassembly are unknown. Does the formation of
loops retard growth cone extension? Or is this rearrangement a concomitant of growth cone arrest that causes
growing microtubules to bend into loops? Interestingly,
in stable synapses at the Drosophila neuromuscular
junction microtubules also form loops that disassemble
during growth and sprouting (Roos and others 2000)
suggesting important parallels for microtubule reorganization as a regulatory mechanism for growth and stability in growth cones as well as developing synapses.
F-Actin—Microtuble Interactions
Microtubules in growth cones predominate in the central
region, and actin filaments predominate in the growth
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Fig. 3. Schematic illustration of a model for microtubule reorganization during axon outgrowth and branching. A,
Microtubules (blue) in the pausing growth cone form a loop in
the central region. As microtubules splay apart in one region of
the loop, short microtubules fragment from longer microtubules
and explore the lamellipodium. B, A branch is emerging from
the axon shaft in a region where local splaying and fragmentation of microtubules occurs. Short microtubule fragments
invade the developing branch. C, The axon branch is extending
as microtubules elongate within it. Arrows indicate directions of
microtubule movement. P and D refer to proximal and distal
segments of the axon. Adapted from Kalil and others (2000).
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cone periphery. However, there is also extensive overlap
between these two cytoskeletal elements in the growth
cone transition region between the central domain and
the periphery (Fig. 4). Recent live-cell imaging studies
have shown that microtubules do not remain confined to
the central region but penetrate into the growth cone
periphery and even invade filopodia where they align
with actin filament bundles (Dent and Kalil 2001;
Schaefer and others 2002). During changes in the direction of axon growth, microtubules reorient toward sites
of focal F-actin accumulation (O’Connor and Bentley
1993; Lin and Forscher 1993). Directed growth of
microtubules toward regions of attenuated F-actin flow
during growth cone target interactions (Lin and others
1994) or capture of dynamic microtubule ends by actin
filaments during growth cone turning (Bentley and
O’Connor 1994; Tanaka and Sabry 1995) suggested the
importance of F-actin–microtubule interactions in
growth cone behaviors, but the exact nature of this interaction had not been demonstrated directly.
Microtubules and actin filaments are highly dynamic,
undergoing continual cycles of growth and shortening
through polymerization and depolymerization, respectively. At the same time, actin filaments and microtubules can move and undergo dramatic changes in their
organization and location within the growth cone. A
study of actin–microtubule interactions was carried out
in living cortical neurons (Dent and Kalil 2001) by coinjecting fluorescently labeled tubulin, which is incorporated into microtubules, and phalloidin, which binds to
actin filaments. Time-lapse imaging revealed that
branching from the growth cone and the axon shaft was
always preceded by splaying apart of looped or bundled
microtubules accompanied by localized accumulation of
F-actin. Dynamic microtubules co-localized with Factin in transition regions of growth cones and axon
branch points. The dynamic actin filaments observed
closely apposed to microtubules in the growth cone transition region resembled actin-based protrusive structures
(termed intrapodia) previously documented in growth
cones of sympathetic neurons (Rochlin and others
1999). The use of high-resolution time-lapse fluorescence microscopy made it possible to analyze the
dynamic growth and shortening of microtubules and
actin filaments in cortical growth cones and to image the
interactions between individual microtubules and the
actin filaments associated with them (Fig. 5).
Interactions between microtubules and actin filaments
involved their coordinated polymerization and depolymerization (Dent and Kalil 2001). Drugs that attenuated
either microtubule or actin dynamics, such as nocodazole
and latrunculin, respectively, abolished F-actin–microtubule interactions at the growth cone and at axon branch
points. Consequently, these drug treatments inhibited
axon branching and caused undirected axon outgrowth
without affecting axon elongation. These findings
demonstrated for the first time that actin–microtubule
interactions are essential for initiating axon growth in
new directions. Subsequent studies of invertebrate neu-
Axon Guidance and Branching Mechanisms
Fig. 4. Locations of microtubules and actin filaments in a large paused cortical growth cone and developing axon branch. A, A fixed
growth cone has been stained with phalloidin and antibodies to tubulin in order to fluorescently label F-actin (pseudocolored red) and
microtubules (pseudocolored green), respectively. Microtubules are bundled together in the axon shaft and form a prominent loop in
the central region of the growth cone. They also extend out into the periphery where they overlap (pseudocolored yellow) with F-actin.
B, A schematic illustrates locations of different populations of actin filaments and microtubules. Microtubules (green) are located in
the shaft of the axon and its branch and in the central region of the growth cone but also extend into the lamellipodium and filopodia.
F-actin forms dot-like structures in the axon shaft and central region of the growth cone, a meshwork in the lamellipodium, and straight
bundles in filopodia. Intrapodial F-actin is shown in yellow in the growth cone transition region where they colocalize with microtubules
extending away from the central microtubule loop. Adapted from Dent and Kalil (2001).
ronal growth cones also showed that actin filaments are
important for the guidance of microtubules into the
periphery (Zhou and others 2002), particularly in filopodia where microtubules were found to extend along actin
filament bundles that served to guide the anterograde
and retrograde transport of microtubules (Schaefer and
others 2002). Similarly, dynamic interactions between
actin filaments and microtubules were visualized in
migrating epithelial cells (Salmon and others 2002),
suggesting that coupling of F-actin and microtubule
movements may be a common feature of migrating cells.
Taken together, these results imply that factors, such as
extracellular cues, that influence the dynamics of either
F-actin or microtubules may affect both cytoskeletal elements presumably through bidirectional signaling pathways that have not yet been identified.
Effects of Guidance Molecules
on the Axonal Cytoskeleton
To understand how guidance molecules regulate directed growth cone extension and axon branching, it will be
necessary to understand their effects on the organization
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of the actin and microtubule cytoskeleton. This will
require a more complete understanding of the signal
transduction pathways that link activation of guidance
molecule receptors to reorganization of the cytoskeleton
(Song and Poo 2001). Previous studies, for example,
have shown that Sema 3A–induced collapse of DRG
growth cones was accompanied by actin depolymerization at the leading edge of the growth cone (Fan and others 1993; Fritsche and others 1999; Fournier and others
2000), which is mediated by the Rho family of GTPases
(Liu and Strittmatter 2001). Semaphorin 3A (Bagnard
and others 1998; Polleaux and others 1998) and several
of the Slit proteins repel cortical axons and can reduce
their branching (Bagnard and others 1998). One possibility is that guidance factors may affect microtubule
organization through changes in the actin cytoskeleton.
This hypothesis was tested by applying Sema 3A to cortical cultures (Dent and others forthcoming). Exposure
of cortical neurons to Sema 3A reduced branching by
more than 50% without affecting axon length in contrast
to FGF-2, which increased axon branching by 2 to 3
times (Szebenyi and others 2001). Within an hour of
exposure to Sema 3A, approximately 60% of cortical
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Fig. 5. Tandem polymerization and depolymerization of microtubules and F-actin in growth cones. A, Images of the same living growth
cone at a single time point showing location of actin filaments at left, microtubules in the middle panel, and the merged image at right.
In the merged image, F-actin is pseudocolored red, microtubules are pseudocolored green, and their colocalization in the transition
region is shown in yellow. B, Higher power images taken from the boxed region in A (at right) showing that actin filaments and microtubules polymerize and depolymerize together. From 5 to 55 sec, microtubules and actin filaments polymerize outward and reach the
periphery of the growth cone. From 80 to 105 sec, one microtubule and associated bundle of F-actin (1) depolymerizes and moves
rearward with the retrograde actin flow while the other microtubule and associated bundle of F-actin (2) remains extended but turns
perpendicular to the retrograde actin flow. C, Positions of the tips of microtubules and actin filaments in B are plotted with respect to
each other. The distance between the tips of the microtubules and F-actin are also plotted. All points below the horizontal line at 0.9
µm indicate that microtubule and F-actin tips are coextensive at that time point. D, Higher power images taken from the boxed region
in A (at left) show co-polymerization of F-actin and microtubules along F-actin bundles (arrows) in the growth cone periphery.
Polymerization versus movement of the microtubule was determined by measuring the length of the microtubule (yellow arrowheads)
relative to a dark speckle (white arrowheads) in merged images at 35 and 45 sec. The increasing distance between the two arrowheads from 35 to 45 sec shows that the microtubule is polymerizing rather than moving. E, The positions of the tips of the microtubules and actin filaments in D are plotted with respect to each other. Scale bars, (A) 10 µm, (B) and (D) 3 µm. Data from Dent and
Kalil (2001).
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Axon Guidance and Branching Mechanisms
axonal growth cones had collapsed. Interestingly, however, large paused growth cones did not completely
collapse but showed some retraction of actin-based
filopodia and lamellipodia. Rapid sequential imaging of
fluorescently labeled microtubules and actin filaments
in growth cones after bath application of Sema 3A and
FGF-2 revealed that within a few minutes, Sema 3A
depolymerized bundled actin in filopodia, decreased
actin dynamics in intrapodia, and attenuated motility and
protrusion of lamellipodia. Microtubules decreased their
exploratory behaviors in the transition region and collapsed into the microtubule loop. These changes resembled the attenuation of cytoskeletal dynamics after application of nocodazole and latrunculin (Dent and Kalil
2001) but were less severe. These results are consistent
with the finding that cultures treated with Sema 3A over
several hours had almost no large paused growth cones
and suggest that Sema 3A reduced actin dynamics,
which in turn attenuated microtubule dynamics. Thus,
the reduction in axon branching may have resulted from
the failure of microtubules to invade the growth cone
periphery and delineate new directions of growth at axon
branch points. In contrast, FGF-2, which increased cortical axon branching and enlarged growth cones over
time (Szebenyi and others 2001), promoted actin polymerization and the formation of microtubule loops. A
recent study of embryonic Xenopus spinal neurons also
demonstrated the importance of microtubule dynamics
for growth cone responses to axon guidance cues (Buck
and Zheng 2002). Attraction and repulsion of growth
cones by gradients of glutamate and netrin, respectively,
were completely blocked when microtubule dynamics
were inhibited by nocodazole or taxol. Local stabilization of microtubules in one side of the growth cone
induced attractive steering, whereas local microtubule
depolymerization caused growth cones to steer away,
demonstrating an instructive role for microtubules in
directional steering of the growth cone. Although actin
dynamics were not visualized directly, inhibition of actin
polymerization with cytochalasin and Rho GTPase signaling by toxin B both blocked taxol attraction of the
growth cones, supporting the view that steering of the
growth cone in response to guidance cues requires coordinated actin-microtubule interactions.
Actin-Associated Regulatory Proteins
and Cytoskeletal Reorganization
Actin-associated proteins have been identified that regulate actin filament assembly, and some of these have
been implicated in inducing protrusions at the leading
edge of motile cells (Pantaloni and others 2001; Bear
and others 2001). The Ena/VASP family of actin regulatory proteins, consisting of Ena (Drosophila Enabled),
Mena (the mammalian homolog of Ena), VASP
(vasodilator-stimulated phosphoprotein), and EVL
(Ena/VASP-like), is associated with the Abl (Abelson)
tyrosine kinase and is involved in cytoskeletal dynamics
(Renfranz and Beckerle 2002). In Drosophila, overex-
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pression of Abl and deletion of Ena induced a bypass
phenotype in which the motor axon extended beyond the
normal choice point and failed to stop and branch (Wills
and others 1999). In mice, deletion of Mena caused
defects in axon guidance in the corpus callosum and hippocampal commissure (Lanier and others 1999). Mena
localized to the tips of filopodia in hippocampal growth
cones (Lanier and others 1999), consistent with the role
of Ena/VASP proteins in regulating actin dynamics in
the growth cone. Abl and Ena/VASP proteins are hypothesized to link extracellular signaling pathways to actin
cytoskeletal dynamics that modulate growth cone motility (Lanier and Gertler 2000). Nevertheless, the precise
physiological functions of the Ena/VASP proteins during
axon guidance are not known. In the Listeria pathogen
model, these proteins increased actin polymerization and
enhanced the rate of actin-based motility, but this effect
appears to represent unregulated actin polymerization
(Bear and others 2001). Paradoxically, overexpression of
Mena in fibroblasts reduced the rate of cell motility,
whereas inhibition of Ena/VASP function increased the
rate of fibroblast migration (Bear and others 2000) by
controlling the geometry of actin filament networks in
lamellipodia (Bear and others 2002). Thus, the bypass
phenotype observed in Drosophila Ena mutants could
result from a failure of actin regulatory mechanisms that
normally slow growth cone advance and permit branching at specific choice points. It is therefore significant
that Ena was shown to mediate repulsive axon guidance
at the Drosophila midline by interactions with the cytoplasmic domain of the Robo receptor that responds to
secreted Slit proteins (Bashaw and others 2000).
Staining of cortical growth cones with antibodies to the
Ena/VASP proteins Mena, EVL, and VASP showed that
all three proteins localized to the tips of filopodia and in
the transition region localized selectively to the tips of
actin-based intrapodia (Dent and others, unpublished
observations). Because actin and microtubules interact
in this region, it is tempting to speculate that the
Ena/VASP proteins, by enhancing actin assembly at the
tips of intrapodia, may regulate microtubule advance
into the growth cone periphery.
Regulation of Growth Cone Advance
by Intracellular Calcium Signaling
During axon guidance, localized changes in the organization and dynamics of the cytoskeleton are likely to be
regulated by changes in intracellular signaling events in
restricted regions of the growth cone. Changes in levels
of intracellular calcium are an important signaling
mechanism known to influence various aspects of neuronal development including gene transcription (West
and others 2001), axonal and dendritic outgrowth
(Gomez and Spitzer 1999; Redmond and others 2002;
Lohmann and others 2002), and neuronal migration
(Komuro and Rakic 1996). Dynamic calcium oscillations are particularly important in regulating the rate of
neurite outgrowth (Gu and Spitzer 1995; Gomez and
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others 1995; Gomez and Spitzer 1999). Recent evidence
from work on Xenopus spinal neurons also suggests that
electrical stimulation, which transiently elevates intracellular calcium, modulated the responses of growth
cones to positive and inhibitory guidance factors (Ming
and others 2001). Rapidly propagated calcium spikes
involve influx of calcium through voltage-gated channels, whereas transient calcium waves in growth cones
spread passively by diffusion and involve calcium entry
by an unidentified channel. In Xenopus spinal neurons,
the rate of axon elongation was shown to be inversely
related to the frequency of calcium waves (Gu and
Spitzer 1995). Experiments in vivo (Gomez and Spitzer
1999) in Xenopus spinal cord showed that suppressing
calcium transients accelerated axon outgrowth, whereas
imposing calcium transients slowed the rate of growth
cone advance. Interestingly, at choice points where
growth cones normally stalled for up to 1 h, growth
cones generated higher frequencies of calcium transients
(10–15 per h). However, at present the mechanistic relationship between calcium transients and growth cone
behavior and morphology is unclear.
Although dynamic calcium changes have been documented in the developing cortex (Yuste and others 1992;
Mao and others 2001), it was not known whether calcium transients might also play a role in regulating cortical axon outgrowth. In a recent study (Tang and others
2003), calcium transients were measured in cortical
growth cones during different stages of axon outgrowth
and branching by means of the sensitive calcium indicator Fluo 4 AM. The incidence of calcium transients was
highly correlated with axonal growth cone morphologies
and behaviors. Small, rapidly extending growth cones
showed few calcium transients, whereas more than 80%
of large paused growth cones as well as those with characteristic branching morphologies exhibited frequent
calcium transients (Fig. 6). In one fortuitous case, a
small growth cone that exhibited no calcium transients
was observed approaching another cell. On contact with
the cell, the growth cone paused and began to show highfrequency calcium transients that persisted throughout
the 60-minute imaging period. Calcium transients in cortical neurons were oscillatory and occurred throughout
the entire neuron, in contrast to the low-amplitude, lowfrequency calcium waves previously observed in growth
cones of spinal cord neurons in embryonic Xenopus
(Gomez and Spitzer 1999). The frequency and amplitude
of calcium transients were inversely correlated with
growth cone extension and ranged from 6 spikes per
hour and 40% above baseline for small extending growth
cones to 14 spikes per minute and 1000% above baseline
for large paused growth cones, in marked contrast to calcium waves in Xenopus, which occurred in growth cones
at a mean frequency of 8 to 9 times per h. The addition
of tetrodotoxin to the cortical cultures immediately
silenced all calcium transients demonstrating their relation to electrical activity. Antibody staining revealed the
presence of L-type calcium channels on cortical neurons
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and treatments with the L-type channel blocker nifedipine blocked calcium transients. These results demonstrated that calcium influx occurred primarily through Ltype channels, although a contribution from intracellular
stores was also found. Importantly, silencing these calcium transients with L-type channel blockers increased the
rate of axon outgrowth and prevented growth cone pausing, thereby reducing the axon branching associated
with pausing behaviors. Evidence for an increase in Ltype calcium channels at branch points from FGF2–stimulated hippocampal neurons (Shitaka and others
1996) supports the view that calcium oscillations may
play a role in regulating cortical growth cone pausing
and branching.
These in vitro results demonstrated that calcium transients play an important role in regulating rates of cortical growth cone extension and consequently the development of axon branches. Calcium exerts its effects on
rates of neurite outgrowth through intracellular signaling
pathways that ultimately regulate the cytoskeleton.
Interestingly, the highest frequencies and amplitudes of
calcium transients were associated with the presence of
a stable microtubule loop in the central region of large
paused cortical growth cones. Although it is known that
calcium can regulate neurite elongation in part by influencing the stability of actin filaments (Lankford and
Letourneau 1989), which can in turn influence microtubule stability, it is not well understood how calcium
transients influence the cytoskeleton to cause the growth
cone to stop and branch. Recent studies have shown that
calcium signaling is involved in the turning responses of
growth cones to gradients of diffusible guidance factors
(Hong and others 2000) and that global calcium spikes
can influence axon path finding by modulating responses of the growth cone to guidance cues (Ming and others
2001). Because stimulation of developing Xenopus
spinal neurons at different frequencies elicited different
effects on growth cone behaviors exposed to local gradients of guidance cues, Ming and others (2001) speculated that the signaling mechanism may be sensitive to the
pattern of stimuli. Results from studies of calcium transients in cortical neurons (Tang and others 2003) suggest
that global calcium activity in cortical neurons can regulate axon outgrowth through frequency dependent
mechanisms. Thus, it will be important to determine
whether guidance cues can influence behavioral
responses of the growth cone by modulating the frequencies and amplitudes of these calcium transients.
Conclusions
Growth cone behaviors are mediated by intracellular signaling pathways that link guidance receptors to the
cytoskeleton (Song and Poo 2001; Luo 2002). Although
many elements in the signaling pathways regulating
actin and microtubule dynamics have been identified,
our understanding of their functions in cytoskeletal reorganization driving growth cone behaviors is incomplete.
Axon Guidance and Branching Mechanisms
Fig. 6. Calcium transients occur preferentially in growth cones with large complex morphologies. A-F, Calcium transients in cortical
growth cones with morphologies representative of progressive stages in growth cone extension, pausing, and branching. At top are
differential interference contrast images of each growth cone followed by pseudocolor fluorescence images of intracellular calcium in
the growth cone at 10-sec intervals. A, A small simple growth cone shows no detectable calcium fluctuations. B, A pausing growth
cone without a central microtubule loop exhibits a single calcium transient of relatively low amplitude. C, A large paused growth cone
with a partially formed microtubule loop shows a single high-amplitude calcium transient at 10 sec. D, A large paused growth cone
with a prominent microtubule loop has the highest amplitude calcium transients, which occur at 10 and 30 sec. E, A pausing growth
cone from which an axon is extending exhibits a single transient of moderate amplitude at 10 sec. The growth cone at the tip of the
new axon (arrow) shows a simultaneous calcium transient. F, A branching axon exhibits a single high-amplitude calcium transient at
10 sec. G, Sequence of fluorescence images at 10-sec intervals showing changes in calcium levels in three different cortical neurons
in close proximity. The smaller growth cones at left and center show little fluctuation in calcium levels in contrast to the large paused
growth cone, which shows a very large calcium transient at 10 sec. Scale shows relative fluorescence intensity over baseline in
pseudocolor images. Scale bar (in G), 10 µm, is the same for all figures. H, At left are average areas of growth cones of three different morphologies, exemplified by growth cones in A, B, and D. At right are average growth rates of these growth cones. I, Sequence
of fluorescence images of a growth cone shows increase in calcium levels as the growth cone changes from extending (0 minutes) to
pausing (6, 10, and 16 minutes) in response to contact with another cell in the dish. Data from Tang and others (2003).
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This review has focused on growth cone behaviors and
axon branching as a means of establishing connectivity
in the developing nervous system. However, in response
to injury or denervation, collateral branches can sometimes sprout interstitially from more mature axons that
are no longer growing. Does sprouting involve
cytoskeletal and signaling mechanisms similar to those
documented for developing axon branches? How do
axon collaterals sprout in the absence of a growth cone?
What factors limit sprouting in the adult CNS? Answers
to these questions will be important for stimulation of
directed axon growth after injury. Moreover, this review
has considered only a few subsets of extracellular axon
guidance cues, actin regulatory proteins, and intracellular calcium signaling events that have been implicated in
governing cytoskeletal changes associated with growth
cone advance and axon branching. Clearly, many other
signaling intermediates are currently the subject of
intense research, and many others await discovery.
Finally, most of the assumptions about regulation of the
cytoskeleton are based on results from analysis of
cytoskeletal organization in cells following fixation or in
dissociated living neurons extending growth cones on
artificial substrates. To understand the mechanisms that
orchestrate the cytoskeletal reorganization required for
growth cone behaviors and extension of axon branches
in vivo, future studies will need to test these assumptions
in the context of motile growth cones responding to physiological guidance cues in the intact nervous system.
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