Download Common Mechanisms Underlying Growth Cone Guidance and Axon

Common Mechanisms Underlying Growth Cone
Guidance and Axon Branching
Katherine Kalil,1,2 Gyorgyi Szebenyi,2* and Erik W. Dent1
1
2
Neuroscience Training Program and
Department of Anatomy, University of Wisconsin, Madison, Wisconsin 53706
Received 20 April 2000; accepted 25 April 2000
ABSTRACT:
During development, growth cones
direct growing axons into appropriate targets. However,
in some cortical pathways target innervation occurs
through the development of collateral branches that
extend interstitially from the axon shaft. How do such
branches form? Direct observations of living cortical
brain slices revealed that growth cones of callosal axons
pause for many hours beneath their cortical targets
prior to the development of interstitial branches. High
resolution imaging of dissociated living cortical neurons
for many hours revealed that the growth cone demarcates sites of future axon branching by lengthy pausing
behaviors and enlargement of the growth cone. After a
new growth cone forms and resumes forward advance,
filopodial and lamellipodial remnants of the large
paused growth cone are left behind on the axon shaft
from which interstitial branches later emerge. To investigate how the cytoskeleton reorganizes at axon branch
The development of appropriate connections is essential for the correct functioning of the nervous system.
Thus, intense interest has focused on the nerve growth
cone at the axon tip that guides the growing axon
along appropriate pathways and into targets. However, it has been known for many years that axons can
*Present address: Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, 6000
Harry Hines Boulevard, Dallas, TX 75235-9111.
Correspondence to: K. Kalil ([email protected]).
Contract grant sponsor: National Institutes of Health; contract
grant number: NS14428 and NS34270 (KK).
Contract grant sponsor: National Institutes of Health predoctoral training grant award; contract grant number: GM07507
(EWD).
© 2000 John Wiley & Sons, Inc.
points, we fluorescently labeled microtubules in living
cortical neurons and imaged the behaviors of microtubules during new growth from the axon shaft and the
growth cone. In both regions microtubules reorganize
into a more plastic form by splaying apart and fragmenting. These shorter microtubules then invade newly
developing branches with anterograde and retrograde
movements. Although axon branching of dissociated
cortical neurons occurs in the absence of targets, application of a target-derived growth factor, FGF-2, greatly
enhances branching. Taken together, these results demonstrate that growth cone pausing is closely related to
axon branching and suggest that common mechanisms
underlie directed axon growth from the terminal growth
cone and the axon shaft. © 2000 John Wiley & Sons, Inc. J
Neurobiol 44: 145–158, 2000
Keywords: axon branching; growth cone behaviors; microtubules; fibroblast growth factor; time-lapse imaging
also establish connections by extending collateral
branches from the shaft of the primary axon into the
target. Although growth cones are able to bifurcate,
careful observations by O’Leary and colleagues (reviewed in O’Leary et al., 1990) showed that in vivo
layer 5 cerebral cortical axons innervate their targets
by developing interstitial branches from the axon
shaft and not through bifurcation of the terminal
growth cone. It was also shown that collateral
branches form by a process of delayed budding from
the primary axon many millimeters behind the growth
cone. These findings led to the view that for efferent
cortical axons the role of the growth cone at the tip of
the primary axon is limited to guidance and elongation along appropriate pathways. Accordingly, growth
cones of layer 5 cortical axons were thought to bring
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the axons into the vicinity of their targets but fail to
respond to target cues and thus have no overt role in
target selection (O’Leary and Koester, 1993). However, recent studies from our laboratory and others
have shown that growth cone behaviors may serve to
demarcate axon branch points (Halloran and Kalil,
1994; Yamamoto et al., 1997; Szebenyi et al., 1998;
Davenport et al., 1999), that new growth from growth
cones and axon branches may involve similar reorganization of the cytoskeleton (Dent et al., 1999a,b), and
that multifunctional cues can influence axon guidance
as well as collateral branching (Wang et al., 1999).
Taken together these studies are beginning to identify
common mechanisms that directly link growth cone
guidance and axon branching.
ROLE OF INTERSTITIAL AXON
BRANCHING IN TARGET INNERVATION
Studies in vivo have shown that interstitial collateral
branches form at right angles from the axon shaft at
considerable distances behind the growth cone and up
to several days after the growth cone has advanced
beyond the target innervated by the branch (Kuang
and Kalil, 1994; O’Leary and Terashima, 1988).
These delays in branching are thought to account for
waiting periods between the arrival of axons in their
target regions and development of innervation. Delayed interstitial axon branching has been demonstrated in two major efferent cortical pathways: the
corpus callosum (Hogan and Berman, 1990; Norris
and Kalil, 1991), which interconnects the two cerebral
hemispheres, and the corticospinal tract, which arises
from layer 5 of the sensorimotor cortex and innervates
the pontine nuclei (O’Leary and Terashima, 1988;
O’Leary et al., 1990) and the spinal cord (Kuang and
Kalil, 1994). It is known that cortical axons make
transient errors in their caudal trajectories, such that
visual cortical axons extend past their targets in the
pontine nuclei and grow inappropriately into the pyramidal tract in the spinal cord (O’Leary et al., 1990).
Axons from forelimb areas of the sensorimotor cortex
also make projection errors, often growing past appropriate targets in the cervical spinal cord and extending into the lumbar cord. Despite these projection
errors, corticospinal connections are topographically
appropriate from the earliest stages of development
(Kuang and Kalil, 1994). These connections form by
interstitial axon collaterals that branch into spinal
targets where they develop elaborate terminal arbors.
Similarly, retinal growth cones also overshoot their
tectal targets but develop topographic tectal connections by extension of interstitial branches at appropri-
ate target sites (Simon and O’Leary, 1992a,b). These
results demonstrate a relative lack of precision in axon
tracts versus specificity of target innervation by axon
collaterals and suggest that in such systems the primary growth cone selects the appropriate pathway but
grows past and ignores target regions. Subsequently,
according to this view, the axon shaft responds to
secreted target-derived factors and extends an interstitial branch into a specific target. Thus, pathfinding
by growth cones and target selection by axon collaterals had been regarded by some authors as separate
phenomena (O’Leary and Koester, 1993; Kennedy
and Tessier-Lavigne, 1995; Bolz and Castellani,
1997; Joosten, 1997).
GROWTH CONE BEHAVIORS IN
TARGET REGIONS
A series of studies on cocultures of explanted cerebral
cortex and their targets in three-dimensional collagen
gels have supported in vivo models of delayed interstitial branching by showing that collateral branches
can be induced to extend toward targets by the release
of a target-derived, diffusible chemoattractant. Observations of cocultured explants from the cortex and
basilar pons, for example, showed that a diffusible
activity from the pons specifically attracts layer 5
cortical axons at a distance. Importantly, the use of
retrograde dye labeling also revealed that many of the
axons attracted to the pontine target are actually collateral branches (Heffner et al., 1990), consistent with
the mode of establishment of corticopontine connections in vivo (O’Leary et al., 1990). Time-lapse imaging of these cocultures confirmed that the collateral
branches stimulated by the pons-derived activity form
interstitially from the axon shaft and not by growth
cone bifurcation (Sato et al., 1994). Although most of
the branches are transient, more branches become
stabilized in the presence of the pontine explant, suggesting that the target-derived activity promotes the
initiation as well as stabilization of interstitial collaterals. In living brain slices of the corticopontine pathway (Bastmeyer and O’Leary, 1996) similar axon
activity was described in the form of dynamic varicosities and filopodia-like extensions that sometimes
become branches. Observations of developing retinotectal arbors in living tadpoles (Harris et al., 1987;
O’Rourke et al., 1994; Witte et al., 1996) and zebrafish (Kaethner and Stuermer, 1992, 1994) also
illustrated the importance of interstitial branching in
the formation of axon arbors.
Although none of these studies of interstitial
branching addressed the role of the growth cone in
Mechanisms of Axon Branch Formation
target selection, other observations suggested that
changes in growth cone morphology and behavior
may be manifested at decision regions related to target
recognition or branch points. Growth cones in fixed
tissue were shown to exhibit dramatic differences in
their morphologies, depending on their locations. In
tracts and pathways simple forms predominate, but
growth cones have more complex morphologies in
decision regions where they change direction and
approach or enter targets (Tosney and Landmesser,
1985; Caudy and Bentley, 1986; Bovolenta and Mason, 1987; Holt, 1989; Norris and Kalil, 1991, 1992).
Consistent with these morphological observations,
video microscopy of growth cone behaviors in semiintact preparations of the vertebrate brain have correlated elongated streamlined forms with growth cone
advance and large complex forms with growth cone
pausing, particularly at decision regions where growth
cones need to recognize targets or make decisions
about crossing the CNS midline (Harris et al., 1987;
Kaethner and Stuermer, 1992; Sretavan and Reichhardt, 1993; Godement et al., 1994; Halloran and
Kalil, 1994; Mason and Wang, 1997).
We have used time-lapse video microscopy in slice
preparations of early postnatal cortex to show that
over many hours of development, growth cones in
different regions of the callosal pathway have strikingly different behaviors (Halloran and Kalil, 1994).
In the callosal tract growth cones advance rapidly and
steadily, displaying continual shape changes. These
primary growth cones do not make turns into cortical
targets but extend well beyond them. Subsequently,
axon branches tipped by small growth cones develop
interstitially from the axon shaft and grow dorsally
toward the overlying sensorimotor cortex. Growth
cones of these axon collateral branches have uniformly small compact shapes and extend relatively
slowly within the cortex in straight radial trajectories,
in keeping with earlier electron microscopic evidence
that callosal axons are guided into cortical targets by
extension along radial glial processes (Norris and
Kalil, 1992). The most dramatic behaviors were observed in regions of the callosum beneath cortical
targets, where growth cones have elaborate morphologies and complex behaviors characterized by long
pauses, extension of transitory branches, and repeated
cycles of collapse, withdrawal, and resurgence. These
behaviors are likely to reflect recognition of cortical
target signals by the growth cone. Importantly, interstitial branches to cortical targets develop at those
points where complex growth cone pausing behaviors
previously occurred, suggesting a link between
growth cone pausing and axon branching. A timelapse study of behaviors of growing thalamic axons in
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organotypic cocultures of the lateral geniculate nucleus (LGN) and visual cortex (Yamamoto et al.,
1997) supported this view by demonstrating that regions of growth cone pausing are well correlated with
axon branching. When axons from the LGN encounter layer 4 of the visual cortex, the normal target of
LGN axons, they stop growing for several hours and
often retract, with concomitant collapse of the growth
cone. The emergence of a branch behind the growth
cone several hours later suggested that target-derived
signals may induce growth cone pausing and elicit
growth of interstitial axon branches (Yamamoto et al.,
1997).
ROLE OF THE PRIMARY GROWTH
CONE IN AXON BRANCHING
Studies in brain slices and organotypic cultures have
the advantage of preserving in situ the cellular and
molecular cues present during CNS development.
However, typically only a small region of the axon
can be imaged at any given time and for periods of
only a few hours. Moreover, the resolution of individual axons in these preparations is not sufficient to
demonstrate precisely how growth cone behaviors are
related to axon branching. Therefore, to study in detail
the development of interstitial axon branches in relation to growth cone behaviors, we have used highresolution imaging of dissociated neurons from the
cerebral cortex (Szebenyi et al., 1998). We chose for
analysis pyramidal neurons from early postnatal sensorimotor cortex because in vivo layer 5 pyramidal
neurons give rise to efferent axons that branch interstitially to cortical and subcortical targets (O’Leary
and Terashima, 1988; Norris and Kalil, 1992; Kuang
and Kalil, 1994). A disadvantage of this approach is
that behaviors of dissociated neurons occur in the
absence of normal target cues. However, an advantage
of studying neurons in relative isolation is the ability
to carry out imaging of single neurons at high resolution without the confounding influence of cell– cell
interactions. By continuously imaging cortical neurons at frequent intervals for periods up to 5 days, we
were able to observe the entire length of growing
axons and follow the development of interstitial
branches from the initial behaviors of the primary
growth cone through the elongation of stable
branches. Thus, lengthy periods of observation encompassed the history of the primary growth cone in
relation to subsequent development of branching
along the axon shaft.
We found that cultured cortical neurons develop
axon branches in a manner similar to cortical neurons
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in vivo (Szebenyi et al., 1998). Cortical neurons initially extend numerous minor processes from the cell
body that are approximately equal in length and
tipped by growth cones. One of the processes then
continues to elongate and becomes the single axon.
This scenario is also consistent with development of
hippocampal neurons in culture (Dotti et al., 1988).
About 20 – 40 h after plating, branches begin to extend
interstitially from the axon shaft (Fig. 1) and form
growth cones at their tips. In contrast to numerous
transient short filopodia, branches persist for several
days and grow to lengths averaging ⬎130 ␮m. The
numbers (4 –5), clustering (2–5), positions (up to 1
mm behind the primary growth cone), and delays
(several hours to several days) in extension of axon
branches correspond to features of delayed interstitial
branches observed on developing cortical axons in
vivo (O’Leary and Terashima, 1988; Kuang and Kalil,
1994) and in situ (Bastmeyer and O’Leary, 1996).
Interestingly, as happens in vivo (O’Leary et al.,
1990) the primary axon distal to the interstitial branch
often ceases to grow or degenerates.
Importantly, we found that pausing behaviors by
the primary axonal growth cone are highly correlated
with interstitial axon branching (Szebenyi et al.,
1998), as we had previously observed in situ in target
regions of the corpus callosum (Halloran and Kalil,
1994). Growth cone pausing behaviors are characterized by repeated cycles of collapse, retraction, and
extension without net forward advance. During pausing behaviors, which could last from 1 to 30 h in
dissociated cultures, growth cones become on average
six times larger than those that are advancing and
have large spread out lamellipodia. The greatly expanded lamellipodium then reorganizes by forming a
new growth cone at its tip (Fig. 2). By the end of the
pausing period, this new primary growth cone
emerges from the tip of the lamellipodium to lead the
growing axon, whereas the rest of the large paused
growth cone remains behind on the axon in the form
of filopodial and lamellar activity. After delays of
several hours to several days after the primary growth
cone resumes forward advance, interstitial axon
branches tipped by growth cones subsequently
emerge from filopodial and lamellar protrusions on
these active regions of the axon. These results are
consistent with recent findings in cultures of retinal
ganglion cell axons encountering repellent cues.
When retinal growth cones are induced to collapse by
contact with inhibitory target cells from the tectum,
with cells transfected with repulsive ephrin molecules
or by mechanical manipulations, lateral extensions in
the form of filopodia and lamellipodia develop rapidly
from the axon shaft behind the growth cone (Daven-
port et al., 1999). Lateral extensions from the axon of
the collapsed growth cone (or even from trailing fasciculated axons) can then continue growing to form
branches tipped by growth cones extending in new
orthogonal directions.
Our results (Szebenyi et al., 1998) suggest a novel
mechanism whereby the primary growth cone demarcates future branch points by pausing, reorganizing
and leaving behind active remnants on the axon from
which interstitial branches later emerge. This result
essentially eliminates the distinction often made between delayed interstitial branching and growth cone
bifurcation because the division of the original primary growth cone into a new growth cone and an
active remnant on the axon shaft is actually a bifurcation. Although this work was carried out on isolated
neurons in the absence of targets, the results are
consistent with the notion that in vivo it is the growth
cone that recognizes targets that will be innervated by
delayed interstitial branches. According to this model
(Fig. 3) interstitial branching results directly from
target recognition by the growth cone, suggesting that
guidance of the primary growth cone and target selection by axon collaterals are not separate events but
closely related phenomena.
CYTOSKELETAL REORGANIZATION
UNDERLYING AXON BRANCHING
The actin and microtubule cytoskeleton of neuronal
growth cones plays a central role in shape changes
and reorienting behaviors underlying axon guidance.
Microtubules (MTs) in growth cones provide structural support and act as tracks for transport of vesicles
as they do in the axon. During growth and navigation
of the axon, the MT array within the growth cone
reorganizes and reorients toward the future direction
of axon outgrowth (Sabry et al., 1991; Tanaka and
Kirschner, 1991; Lin and Forscher, 1993; Tanaka et
al., 1995; Tanaka and Sabry, 1995; Suter et al., 1998).
Filamentous actin (F-actin), which occupies peripheral regions of the growth cone (Forscher and Smith,
1988; Bridgman and Dailey, 1989; Lewis and Bridgman, 1992), drives motility of the veil-like lamellipodia and occupies filopodia, the finger-like microspikes
that protrude from the leading edge of the growth
cone. A major unanswered question is how the actin
and microtubule cytoskeleton interact within the
growth cone during guidance behaviors (O’Connor
and Bentley, 1993; Letourneau, 1996; Suter and Forscher, 1998). For example, local accumulation of Factin could result in tension from actin–microtubule
interactions that would pull the central microtubule
Mechanisms of Axon Branch Formation
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Figure 2 Enlargement and reorganization of a growth
cone during prolonged pausing. During the 18-h pausing
period, the lamella of the growth cone gradually enlarges.
At 6 h a prominent microtubule loop in the growth cone is
apparent under phase optics. At 12 h the new primary
growth cone is emerging from the tip of the reorganized
growth cone. By 18 h the primary growth cone resumes
elongation, and a large lamellar expansion remains behind
on the axon shaft. Etchings on the coverslips serve as
landmarks. Scale bar ⫽ 30 ␮m. Reprinted from Szebenyi et
al. (1998) with permission.
domain toward a target site. On the other hand, depletion of F-actin, through the attenuation of retrograde flow, could result in microtubule advance to-
Figure 1 Extension of interstitial branches from regions of
the axon where prolonged growth cone pausing occurs. Representative images were taken from a series of images acquired
at 3-min intervals during the 3-day observation period. Between 5 and 20 h, the growth cones on the branched axon
pause in the region indicated by the arrows. Later, between 35
and 53 h, a cluster of axon branches extends interstitially from
the axon in this pausing region. Scale bar ⫽ 100 ␮m. Reprinted from Szebenyi et al. (1998) with permission.
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Figure 3 Schematic representation of different stages in
axon branching. (A) A growth cone of an efferent cortical
axon is advancing toward its target, indicated by the circle.
(B) The primary growth cone pauses for extended time
periods in the vicinity of the target and enlarges. (C) After
the primary growth cone resumes forward advance, remnants of the reorganized growth cone are left behind as
filopodial or lamellar activity along the axon shaft. (D) After
a time delay, an interstitial axon branch emerges from a
region of lamellar activity at some distance behind the
primary growth cone and extends toward the target. Reprinted from Szebenyi et al. (1998) with permission.
ward positive extracellular cues (reviewed in
Letourneau, 1996; Suter and Forscher, 1998). Local
regulation of axonal morphogenesis by neurotrophins
(Berninger and Poo, 1996) implies that they induce
cytoskeletal rearrangements (Gallo et al., 1997), leading to directed extension of the growth cone. The role
that MTs play in axon growth is not limited to their
continuous rearrangement at the terminal growth
cone. Studies on cultured hippocampal neurons suggest that MTs fragment within the region of the axon
where interstitial branches form (Yu et al., 1994).
Moreover, in a recent study demonstrating induction
of filopodia on regions of axons in contact with nerve
growth factor (NGF)-coupled beads, it was found that
local debundling of microtubules occurs in the axon
shaft along with actin accumulation (Gallo and Letourneau, 1998).
To begin to understand the cytoskeletal mechanisms underlying directed axon growth, we investigated specific changes that occur in the MT array
within the terminal growth cone and at sites of interstitial axon branch formation. Some authors have argued that the reorganization of the MT array is based
solely on the assembly and disassembly of MTs and
not on their movement through the cytoplasm (reviewed in Hirokawa et al., 1997), whereas other authors have argued that individual MTs can interact
with motor proteins that actively transport them to
new locations (reviewed in Baas, 1997; 1999; Baas
and Brown, 1997). To date, this issue remains controversial, in large part because of results based on
relatively low-resolution fluorescence analyses. Nevertheless, there is compelling evidence from indirect
studies that individual MTs are capable of movement
(Yu et al., 1996; Slaughter et al., 1997; Gallo and
Letourneau, 1999).
We took a direct approach by visualizing the
movements of individual MTs with high-resolution
time-lapse fluorescence digital imaging of dissociated
cortical neurons microinjected with fluorescently labeled tubulin (Dent et al., 1999b). We hypothesized
that in order for new growth to occur at the growth
cone and from interstitial axon branches, MTs must
rearrange from a bundled array to a more plastic
configuration. We therefore investigated the reorganization of the bundled MT array during transitions
from quiescent to growth states and determined
whether individual MTs in these regions are capable
of independent movement. During pausing, growth
cones are typically large and flat (Szebenyi et al.,
1998) and in their central regions MTs form prominent loops (Fig. 4) characteristic of slowly growing
axons (Tsui et al., 1984; Lankford and Klein, 1990;
Sabry et al., 1991; Tanaka and Kirschner, 1991). We
found that during transitions of the growth cones from
quiescent to growth states, MTs undergo a dramatic
reorganization that involves an initial breaking away
of short MTs from the MT loop, followed by a splaying apart of the looped MTs and their entry into
branches forming from the growth cone (Fig. 5). MTs
explore growth cone lamellipodia with forward, backward, and lateral movements, and individual MTs are
able to move independently of one another. To observe MT reorganization and movements at axon
Mechanisms of Axon Branch Formation
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Figure 4 Cortical neurons fixed and stained with fluorescent phalloidin, which binds actin
filaments and antibodies to ␣-tubulin, which label microtubules. (A) MTs (pseudocolored green) in
the axon splay apart at branch points, as shown in the inset. Actin filaments (pseudocolored red) are
concentrated in distal regions of the growth cone and developing axon branches. (B) MTs form a
prominent loop in the central region of the growth cone and are closely apposed to actin filaments
in the distal region of the growth cone, as shown in the inset. In both images regions of overlap
between MTs and actin filaments appear yellow. Scale bar ⫽ 10 ␮m; inset ⫽ 5 ␮m.
branch points, we focused on the expanded regions of
the axon shaft that resemble flat lamellipodia from
which axon branches develop (Szebenyi et al., 1998).
Observations at early stages of branch formation revealed disruptions in the bundled MT array where
MTs splay apart (Figs. 4 and 6). MTs explore filopodial processes extending from the axon [Fig. 6(A)]
and continue to invade elongating branches [Fig.
6(B)]. Only those filopodia that contain MTs develop
into branches, whereas those lacking MTs either disappear or remain as filopodia. However, even longer
branches tipped by growth cones and heavily invested
with MTs are capable of regressing, suggesting that
although MT invasion is necessary for development
of branches, the presence of MTs does not guarantee
the survival of a branch. In cases where filopodia
develop along the axon shaft but MTs within the axon
remain bundled, branches never form, even though
MTs frequently penetrate into these transient filopodia. These results suggest that development of
branches from the axon shaft involves local splaying
apart of the MT bundle accompanied by breakdown of
longer MTs and invasion of shorter MTs into nascent
branches. This reorganization of MTs is similar to that
observed during formation of branches from paused
growth cones.
MTs invade developing branches from the axon
shaft and move forward and backward within them,
sometimes growing and shrinking at the same time.
Even longer MTs were seen to retreat from growing
axon branches. Imaging of branch formation along the
axon shaft over extended time periods revealed that
MTs undergo continual redistribution during the simultaneous extension of some processes, accompa-
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nied by invasion of MTs and the regression of others
concomitant with a loss of MTs. This suggests that
anterograde and retrograde MT transport, in addition
to MT polymerization and depolymerization, play a
role in directing MTs toward branches favored for
growth and away from processes that retract. The
retrograde movement of MTs was unexpected, because MT movement in intact cells has thus far only
been documented in the anterograde direction, when
MTs move parallel to their own long axis (Terasaki et
al., 1995; Keating et al., 1997).
Our overall impression was that shorter MTs have
more complex and rapid movements. Individual MTs
sometimes move at constant rates but can exhibit
saltatory movements, changing speed and direction
within seconds and accelerating rapidly to velocities
up to 30 ␮m/min. MTs longer than 10 ␮m tend to
move at rates significantly slower than those shorter
than 10 ␮m. The inverse relationship of MT length
and transport rates may be due to more drag (Willard
and Simon, 1983) on longer MTs. If this were correct,
increasing the length of a MT by polymerization and
further stabilizing it through interactions with MTassociated proteins (Desai and Mitchison, 1997)
would result in slower MT movements. In contrast, in
regions of new growth, short MTs undergoing more
active movements may be required for rapid exploration of growth cones and developing branches. As
MTs in these processes invade regions favored for
growth, some of the MTs could then become stabilized in preferred directions by elongating and slowing down. Recent studies indicate that cytoplasmic
dynein is a key motor protein that drives MT movement in cellular extracts (Heald et al., 1996) and in
neurons (Ahmad et al., 1998). Similarities in the rates
of anterograde and retrograde movement observed in
the present study suggest that the same motor might
Figure 5 Movement and fragmentation of individual MTs
in a paused growth cone. (A) A large paused growth cone
has a prominent MT loop in the central region. MT movements shown in image sequences (B) and (C) occur in
regions indicated by the boxes. In sequence (B) a MT
elongates while moving rapidly into the peripheral lamellipodium and then shortens while moving laterally (40 – 60 s).
In sequence (C) a MT elongates (0 –30 s) and then fragments into two shorter MTs (40 s). The shorter MT segment
remains stationary without elongating or shortening (50 –70
s), whereas the longer MT segment grows slightly (50 s) and
then fragments a second time (60 –70 s). Matching images
in (B⬘) and (C⬘) highlight in white the MTs shown in (B)
and (C). Scale bar ⫽ 5 ␮m. Reprinted from Dent et al.
(1999) with permission.
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Figure 6 Splaying apart of the MT array within the axon shaft during interstitial branching. The
sequence of black and white with matching pseudocolor images shows changes in the MT array
before (A, A⬘) and during (B, B⬘) development of an interstitial branch. Pseudocolor images indicate
fluorescence intensity from low to high as shown in the scale. Arrows in the first image in the
sequence in (A) point to two regions where MTs are splayed apart in contrast to the bundled array
in the nonbranching region of the axon shaft (arrowhead). P and D refer to proximal and distal
segments of the axon, respectively. Six minutes later the MTs in the upper region (arrow) have
formed a bundle, whereas those in the lower region (arrow ) remain splayed. At 28 min MTs have
invade a filopodial process (arrow). In (B) the same region of the axon is shown 5 h later when MTs
are invading an interstitial branch developing in the position of the filopodium shown at 28 min in
(A). Arrows in (B) indicate distal ends of the MTs. Times are shown in hours and minutes. Scale
bar ⫽ 5 ␮m. Reprinted from Dent et al. (1999) with permission.
be responsible for both types of movement. In theory,
the forces generated by cytoplasmic dynein could
result in the transport of MTs with their plus (Ahmad
et al., 1998) or minus (Heald et al., 1996) ends leading
(reviewed in Baas, 1999).
Previous ultrastructural studies on cultured hippocampal neurons suggested that the presence of short
MTs at branch points result from the fragmentation of
longer microtubules (Yu et al., 1994). Our direct
observations confirmed that indeed MTs do undergo
fragmentation (Fig. 5). During transitions from quiescent to growth states, fragmentation of longer MTs
would result in a higher number of shorter MTs ideally suited for rapid exploratory movements within
growth cones and developing branches. After invasion into appropriate regions, these short MTs could
then elongate and become stabilized, allowing for
further growth of the axon to occur. Fragmentation of
MTs in neurons may be locally regulated. For example, focal application of a calcium ionophore to axons
of Aplysia neurons was shown to elicit new growth
cones that develop into branched neuritic processes
(Ziv and Spira, 1997). In these branching regions the
MT array appears to be discontinuous, suggesting that
locally induced MT fragmentation is correlated with
growth of new axonal processes. What mechanisms
might account for MT fragmentation in the axon?
Several recent studies suggest that the protein katanin,
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known to have MT-severing properties in vitro (McNally and Vale, 1993; Hartman et al., 1998), is
present in a variety of cell types including neurons
(McNally and Thomas, 1998; Ahmad et al., 1999).
One interesting possibility is that MT fragmentation
may be regulated by intrinsic and/or extrinsic factors
that locally activate katanin.
Our results demonstrate a similar reorganization of
the microtubule cytoskeleton at the terminal growth
cone and at sites of branch formation (Fig. 7). This
should perhaps not be surprising, given that interstitial
branches form from regions of activity on the axon
shaft at sites where the terminal growth cone has
previously paused (Szebenyi et al., 1998). The similar
MT behaviors in these two regions are probably important for enabling individual MTs to move more
effectively into the lamellipodia and filopodia of
growth cones and developing interstitial branches. It
has long been recognized in vivo that after a developing cortical axon extends an interstitial branch toward a callosal or spinal target the region of the
cortical axon distal to the branch degenerates
(O’Leary et al., 1990). This process is probably accompanied by a redistribution of MTs. At present the
mechanisms that regulate the long-term redistribution
of MTs are unknown, but it is compelling to speculate
that the kinds of anterograde and retrograde MT
movements that we observed might be a key factor in
determining whether an axon branch degenerates or
continues to grow and stabilize.
Thus far we have considered only the reorganization and movement of MTs during new axonal
growth. Although actin is known to play an important
role in regulating changes in the distribution of MTs
at the growth cone, the precise nature of actin–microtubule interactions in the growth cone remains to be
elucidated (reviewed in Suter and Forscher, 1998) and
has not been well characterized in any motile cell
(reviewed in Waterman-Storer and Salmon, 1999).
We have used the direct approach of visualizing
movements of MTs and actin filaments in living cortical neurons during branching from the growth cone
and the axon shaft (Dent et al., 1999a). By microinjecting fluorescent tubulin and phalloidin (which selectively binds filamentous actin) into the same neuron. we were able to image simultaneous changes in
both cytoskeletal elements. Preliminary results show
that actin filaments in peripheral regions of the growth
cone and on the axon shaft are concentrated where
new growth is occurring. Further, actin filaments appear to accumulate prior to the invasion of MTs
toward sites of axon branching. In fact, the first overt
sign of branching along the axon shaft or at the
growth cone is a focal accumulation of actin filaments
Figure 7 Schematic illustration of a model for reorganization of the MT array during directed axon outgrowth.
According to the model, longer bundled MTs in terminal
growth cones and developing axon branches locally fragment into shorter MTs. Individual short MTs then explore
these developing processes with rapid movements. New
growth occurs in regions where MTs become stabilized and
elongate. In (A) MTs in the pausing growth cone have
formed a looped array. Short MTs move away from the MT
loop to explore the growth cone lamellipodium. In (B) a
branch is emerging from the axon shaft accompanied by
local fragmentation of the MT array and invasion of the
nascent branch by short MTs. In (C) the axon branch is
extending as individual MTs elongate and move within it.
Arrows indicate directions of movement. P and D refer to
proximal and distal segments of the axon, respectively.
Mechanisms of Axon Branch Formation
accompanied by splaying of MTs (Fig. 4). Subsequently, MTs invade regions of high filamentous actin
concentration where the two cytoskeletal elements
become closely apposed. These results suggest that
MTs and actin filaments may be directly coupled.
EFFECTS OF TARGET-DERIVED
FACTORS ON AXON BRANCHING
Although much of our work has been carried out on
isolated neurons in the absence of their normal targets, it is known that target-derived factors influence
growth cone behaviors and elicit development of axon
collaterals. During the past decade different cues have
been identified that regulate the guidance of the
growth cone (reviewed in Tessier-Lavigne and Goodman, 1996; Cook et al., 1998; Mueller, 1999; Song
and Poo, 1999). These cues include neurotrophins and
other highly conserved families of guidance molecules. NGF (Letourneau, 1978; Gundersen and Barrett, 1979) and BDNF (Song et al., 1997), for example, attract growth cones of sensory neurons. Basic
fibroblast growth factor (FGF-2) has been shown to
play a role in targeting retinal axons to the frog optic
tectum in vivo (McFarlane et al., 1995, 1996). Families of molecules such as the netrins and semaphorins
and most recently the slit proteins are known to exert
attractive or repulsive effects on specific growth cones
at specific locations in the vertebrate and invertebrate
nervous systems (Mueller, 1999). There is increasing
evidence that many of these cues affect not only the
behavior of the growth cone and the guidance of the
primary axon but also the initiation and extension of
collateral branches from the axon shaft. For example,
in vivo applications of neurotrophin 3 (NT-3) in the
spinal cord (Schnell et al., 1994) and brain-derived
neutrophic factor (BDNF) in the optic tectum (CohenCorey and Fraser, 1995) promote sprouting of corticospinal axons and arborization of optic axons, respectively. Guidance cues such as the netrins and
semaphorins have been characterized primarily for
their role in attracting or repelling various growth
cones. However, one such molecule, the mammalian
Slit 2N protein, which is homologous to slit proteins
that repel axons at the midline in Drosophila, was
recently found to stimulate formation of collateral
axon branches on rat dorsal root ganglion neurons in
collagen gels (Wang et al., 1999; reviewed in Van
Vactor and Flanagan, 1999).
Taken together, previous findings suggest that
axon guidance and interstitial branching may be regulated by common factors affecting the growth cone.
To investigate this possibility, we have used bath and
155
local application of growth factors on cultured cortical
neurons to determine their effects on the development
of collateral branches in relationship to behaviors of
the growth cone (unpublished results). A survey of a
number of growth factors revealed that fibroblast
growth factor (FGF)-2 is particularly effective in promoting branching of cortical axons (Szebenyi et al.,
1999). Application of FGF-2 for as little as 2 h is
sufficient to elicit maximal branching, which is three
times greater than for neurons in untreated control
cultures. As we had previously determined (Szebenyi
et al., 1998) branches had to be at least 30 ␮m long to
persist and become stabilized. Also, branches typically occur in clusters, are often tipped by growth
cones, and frequently rebranch.
Previous observations of growth cone pausing in
relation to branching showed that the larger the primary growth cone becomes during pausing the more
axon branches develop in the pausing region. We
therefore investigated effects of FGF-2 on growth
cone size and rates of extension. We found that because of lengthy pausing periods by the growth cones,
axons of FGF-2–treated neurons extend at only half
the rates of controls and that by 20 h after treatment,
growth cones become twice as large as untreated
controls. Thus, one mechanism of action of FGF-2
could be a direct effect on growth cones by slowing or
arresting their extension and greatly increasing their
size. Both of these effects would promote collateral
branching. Another possibility is that growth factors
can stimulate local regions of the axon shaft to
branch. This was demonstrated in a recent study in
which application of NGF-coupled beads to the axons
of dorsal ganglion neurons elicited filopodial sprouts
in close proximity to the bead (Gallo and Letourneau,
1998).
Using a similar approach, we coupled heparin to
polystyrene beads, coated them with FGF-2, and then
applied them at low densities to cortical cultures. We
found that in comparison with BSA-coated control
beads, which were associated with branches only a
fraction of the time, the FGF-2– coated beads often
promoted interstitial branching in close proximity
(within 10␮m) to the bead but not at greater distances.
Beads contacting or landing close to growth cones
also elicit development of branches. In fact, our results suggest that beads acting on localized regions of
the axon are more likely to elicit branches on distal as
opposed to proximal regions of the axon. In a previous study (Gallo and Letourneau, 1998), application
of NGF-coupled beads elicited filopodia that develop
within minutes of bead application and are often transient. Moreover, in order for a branch to develop, the
growing process had to be in continual contact with
156
Kalil et al.
the NGF-coupled beads. In contrast, we found that
branches in our cultures develop over a much longer
time course (1–3 days), are stable over the entire
observation period, grow to lengths averaging over 90
␮m, and often rebranch. Branches continue to elongate even when the bead moved away from contact
with the axon. This shows that continuous contact
with the FGF-2– coated beads is not necessary for
growth of cortical axon branches. Thus, FGF-2 can
act locally to induce axons to branch, but such
branches occur preferentially on more labile regions
of the axon in the vicinity of the growth cone. Large
increases in growth cone size and slowing of growth
cone extension also suggest that effects of FGF-2 on
axon branching in many cases directly involves the
growth cone. This is consistent with a hypothesis that
FGFs in the optic tectum may slow growth cone
advance and switch axons form a growing mode into
an arborizing or branching mode (McFarlane et al.,
1996). Because FGF-2 has been shown to increase
L-type calcium channels at branch points during
branching of cultured hippocampal neurons (Shitaka
et al., 1996) and calcium transients are known to slow
growth cone advance (Gomez et al., 1995; Gomez and
Spitzer, 1999), it is possible that local changes in
intracellular calcium and other second messengers
may be a common pathway by which growth factors
such as FGF-2 induce growth cone pausing leading to
axon branching.
FUTURE DIRECTIONS
The mechanisms by which environmental cues are
transduced into the cytoskeletal reorganization that
underlies guidance and branching of the axon are
poorly understood. Nevertheless, it is becoming clear
that these two developmental events are interrelated
and may share many of the same cellular and molecular mechanisms. In the future, to understand the
mechanisms by which axon guidance and branching
are orchestrated, it will be important to determine the
effects of extracellular guidance molecules on the
cytoskeleton, the role of intracellular second messengers in transducing these effects, the molecular machinery regulating the local splaying and fragmentation of microtubules, the identity of the motor proteins
driving anterograde and retrograde microtubule
movements, and the nature of the interactions that link
the microtubule and actin cytoskeleton. In all of these
studies high-resolution imaging of living neurons during axon guidance and branching events will be an
essential technique.
Movies of several figures can be viewed at http://
kalil.anatomy.wisc.edu.
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