1 Department of Biological Sciences, Stanford University, 371 Serra Mall,
Stanford, California 94305, USA
2 Genentech Incorporated, 1 DNA Way, South San Francisco, California 94080,
USA
Author for correspondence (e-mail:
marctl{at}stanford.edu)
SUMMARY
During embryonic development, morphogens act as graded positional cues to dictate cell fate specification and tissue patterning. Recent findings indicate that morphogen gradients also serve to guide axonal pathfinding during development of the nervous system. These findings challenge our previous notions about morphogens and axon guidance molecules, and suggest that these proteins, rather than having sharply divergent functions, act more globally to provide graded positional information that can be interpreted by responding cells either to specify cell fate or to direct axonal pathfinding. This review presents the roles identified for members of three prominent morphogen families the Hedgehog, Wnt and TGFß/BMP families in axon guidance, and discusses potential implications for the molecular mechanisms underlying their guidance functions.
Introduction
Neuronal connections form during embryonic development when neurons send
out axons, tipped at their leading edge by the growth cone, which migrate
through the embryonic environment to their synaptic targets. Studies of
developing axonal projections have revealed that axons extend to the vicinity
of their appropriate target regions in a highly stereotyped and directed
manner by detecting a variety of attractive and repulsive molecular guidance
cues presented by cells in the environment
(Dickson, 2002;
Tessier-Lavigne and Goodman,
1996
).
In the 1990s, genetic, biochemical and molecular approaches together
identified four major conserved families of guidance cues with prominent
developmental effects: the Netrins, Slits, Semaphorins and Ephrins
(Dickson, 2002;
Tessier-Lavigne and Goodman,
1996
). Netrins, Slits and some Semaphorins are secreted molecules
that associate with cells or the extracellular matrix, whereas Ephrins and
other Semaphorins are anchored to the cell surface. Netrins can act as
attractants or repellents; Slits, Semaphorins and Ephrins act primarily as
repellents but can be attractive in some contexts. For each of these sets of
cues, one or more families of transmembrane receptors have been identified:
DCC and UNC-5 receptors for Netrins, Roundabout (Robo) receptors for Slits,
Neuropilin and Plexin receptors for Semaphorins, and Eph receptors for
Ephrins. In addition to these `classic' axon guidance molecules, some growth
factors, including Neurotrophins and Scatter Factor/Hepatocyte Growth Factor
(SF/HGF), have been implicated in axon guidance
(Ebens et al., 1996
;
O'Connor and Tessier-Lavigne,
1999
; Tucker et al.,
2001
).
Although the identification of these major guidance cues has increased our understanding of how the nervous system is wired, many guidance events observed during development do not appear to be accounted for by these molecules. Moreover, the number of guidance cues and receptors identified seem small relative to the immense complexity of nervous system wiring; thus, additional guidance cues and receptors probably remain to be discovered. Remarkably, over the last few years, members from three other families of secreted signaling molecules have been shown to act as guidance cues: the Wingless/Wnt, Hedgehog (Hh) and Decapentaplegic/Bone Morphogenic Protein/Transforming Growth Factor ß (Dpp/BMP/TGFß) families. In addition to their axon guidance properties, these molecules share a common characteristic of having been previously identified as morphogens controlling cell fate and tissue patterning. This discovery has facilitated the study of an entirely new set of axon guidance cues and changed our current notions about morphogenic and axon guidance molecules. Additionally, it suggests that these proteins can be thought of more generally as providing graded positional information, which can be interpreted by responding cells as either a cell-fate specification signal or one for axonal pathfinding.
Here, we focus on the emerging evidence that these three morphogen families are reused later in development to guide axons, and compare the similarities and differences in how they provide positional information that can be interpreted for axon guidance versus cell fate specification. Additionally, we also briefly discuss the increasingly appreciated role for morphogens in cell migration (Box 1).
Morphogens, cell fate specification and tissue patterning
Morphogens are signaling molecules produced in a restricted region of a
tissue that provide positional information by diffusing from their source to
form a long-range concentration gradient. A cell's program of differentiation
in response to a morphogen is dictated by its position within the gradient and
thus on its distance from the morphogen source. Two criteria determine whether
a secreted signaling protein acts as a morphogen: it must have a
concentration-dependent effect on its target cells and it must exert a direct
action at a distance. To date, only three protein families have members that
fulfill these criteria: the Wingless/Wnt, Hh and Dpp/BMP/TGFß families
(Teleman et al., 2001).
Although there is abundant evidence for concentration-dependent activity of
signaling proteins during development (reviewed by
Gurdon et al., 1998
), evidence
for direct action at a distance has only been provided recently in some
vertebrate systems (Chen and Schier,
2001
; Briscoe et al.,
2001
). In the following section, we summarize briefly some of the
biological processes that involve members from each morphogen family, with a
special emphasis on vertebrate neural tube development, which provides a
convenient system in which to compare and contrast roles of classic guidance
molecules and morphogens in axon guidance.
In vertebrate embryos, one of the first steps in nervous system development is the specification of the diverse neural cell fates. Members of each of the three morphogen families are expressed in the developing neural tube and are implicated in its patterning, as summarized below.
The Hedgehog family
Hedgehog proteins are found in insects and vertebrates, but not nematodes.
There is a single Hedgehog gene in flies, and three in mammals: Sonic hedgehog
(Shh), Indian hedgehog (Ihh) and Desert hedgehog (Dhh). Shh is secreted by the
notochord and by floor-plate cells at the ventral midline of the neural tube,
and functions as a graded signal for the generation of distinct classes of
ventral neurons along the dorsoventral (DV) axis of the neural tube
(Fig. 1A) (reviewed by
Jessell, 2000;
Ingham and McMahon, 2001
;
Marti and Bovolenta, 2002
). In
agreement with its role as a morphogen, Shh is able to induce a range of
ventral spinal cord cell fates in a concentration-dependent manner
(Roelink et al., 1995
) and has
been shown to exert a direct action at a distance to specify neural tube cell
fate (Briscoe et al.,
2001
).
|
|
Members of the Dpp/BMP/TGFß family regulate cell fate by inducing the dimerization of type I and type II TGFß receptors, resulting in phosphorylation and activation of the intracellular kinase domain of the type I receptor (Fig. 2B). Targets of the type I receptor are the receptor-regulated Smads (R-Smads) which, upon phosphorylation, associate with co-Smads and translocate to the nucleus where they activate transcription.
The Wingless/Wnt family
Roof-plate cells also express several members of the Wnt family (reviewed
by Lee and Jessell, 1999).
Although Wnt1 and Wnt3a are required for normal specification of dorsal
neurons (Muroyama et al.,
2002
), it also remains an open question whether they function
specifically as morphogens in this system.
Wnt ligands can activate several different signal transduction pathways.
The most extensively studied is the canonical Wnt pathway, which controls gene
expression by stabilizing ß-Catenin
(Fig. 2C). This pathway
involves evolutionarily conserved cellular components (reviewed by
Nelson and Nusse, 2004;
Strutt, 2003
). Frizzled (Fz)
proteins are seven-transmembrane-domain molecules that function as Wnt
receptors. When Wnts are absent, ß-Catenin is phosphorylated by
GSK3ß, leading to its degradation. Binding of Wnts to their receptors
results in Dishevelled (Dsh) activation and suppression of GSK3ß
activity, thus stabilizing ß-Catenin. Accumulated ß-Catenin converts
the lymphoid enhancer factor (Lef)/Tcf from a transcriptional repressor to an
activator.
Recently, much attention has been given to two ß-Catenin-independent non-canonical Wnt pathways, the Wnt/Ca2+ pathway and the planar cell polarity (PCP) pathway (Fig. 2C). The PCP pathway involves a non-canonical, ß-Catenin-independent, Wnt/Fz pathway that requires Dsh. The Wnt/Ca2+ pathway is thought to signal via heterotrimeric G-proteins to mobilize intracellular Ca2+ and, in some contexts, to stimulate protein kinase C (PKC).
Below, we discuss the functions of these morphogen families in axon guidance.
Morphogens in axon guidance
The Hedgehog family
Shh is a chemoattractant for commissural axons
During spinal cord development, commissural neurons, which differentiate in
the dorsal neural tube, send axons that project toward and subsequently across
the floor plate, forming axon commissures
(Fig. 1B) (see also
Colamarino and Tessier-Lavigne,
1995). These axons project toward the midline in part because they
are attracted by Netrin 1 (Ntn1), a long-range chemoattractant secreted by the
floor plate (Kennedy et al.,
1994
; Placzek et al.,
1990
; Serafini et al.,
1996
; Serafini et al.,
1994
; Tessier-Lavigne et al.,
1988
). In mice mutant for Ntn1 or its receptor
Dcc, many commissural axon trajectories are foreshortened, fail to
invade the ventral spinal cord, and are misguided
(Fazeli et al., 1997
;
Serafini et al., 1996
).
However, some of them do reach the midline, indicating that other guidance
cues cooperate with Ntn1 to guide these axons. Further analyses of
Ntn1 knock-out mice have suggested that the floor plate might
actually express an additional diffusible attractant(s) for commissural axons
(Serafini et al., 1996
;
Charron et al., 2003
).
Given its expression by the floor plate and its long-range effects in the
spinal cord, Shh was a candidate for a midline-derived axonal guidance cue.
Shh was indeed shown to function as an axonal chemoattractant that can mimic
the Ntn1-independent chemoattractant activity of the floor plate in in vitro
assays (Charron et al., 2003).
The chemoattractant activity of Shh, like the chemoattractant activity of
floor plate derived from Ntn1 mutants, can be blocked by cyclopamine,
which blocks the actions of Shh in cell fate determination by inhibiting the
Shh signaling mediator Smo. This shows that Smo is required for Shh-mediated
axon attraction and, importantly, that the Netrin 1-independent
chemoattractant activity of the floor plate also requires Hh signaling. As Shh
is the only Hh family member expressed in the spinal cord at this stage, these
results suggest that Shh is functioning as a floor plate-derived
chemoattractant for commissural axons.
While the reorienting effect of Shh could be due to a direct
chemoattractant effect, an alternative explanation is suggested by the fact
that Shh is a potent morphogen. Because in these assays commissural axon
turning occurs within the spinal cord tissue explant, it seemed possible that
Shh was not acting directly on the axons but was rather repatterning and
altering the expression of guidance cues by cells within the explant, which
then secondarily guided the axons to the Shh source. Arguing against this
possibility is the finding that the spinal cord explants used to assess
chemoattractant activity are at a developmental stage at which they have
apparently lost the competence to be repatterned by Shh, as assessed using a
battery of markers of DV patterning
(Charron et al., 2003).
A direct action of Shh in attracting the axons was supported further by two
sets of experiments (Charron et al.,
2003). First, Shh was shown to attract the growth cones of
isolated Xenopus spinal axons in dispersed cell culture in a
cyclopamine-dependent manner, proving that Shh, acting via Smo, can function
as a chemoattractant at least for these Xenopus axons. A direct
demonstration that it can attract rodent commissural axons remains, however,
to be obtained. A second way of providing evidence that Shh can act directly
on commissural axons to guide them relied on blocking Shh signaling
selectively in commissural neurons without blocking it in the terrain through
which their axons course. This was achieved by the conditional inactivation of
a floxed allele of Smo using Cre recombinase expressed under the
control of the Wnt1 promoter, which drives Cre expression in the dorsal spinal
cord (and in neural crest progenitors). When Cre, driven by this promoter, was
used to delete a floxed Smo allele in the dorsal spinal cord,
commissural axon trajectories were defective in the ventral spinal cord, where
Cre is not expressed (Table 1). This result strongly implies that the axonal misrouting is not due to
repatterning of the ventral spinal cord, and must instead reflect a guidance
defect arising from loss of Smo function in commissural neurons. The most
parsimonious explanation, in light of the finding that Shh can attract
isolated Xenopus spinal axons in a Smo-dependent fashion, is that the
defects in commissural axon growth in the ventral spinal cord in the
conditional Smo knock-out mouse reflect loss of chemoattraction of
those axons by Shh. Taken together, these results therefore imply that Shh
functions to guide commissural axons both in vitro and in vivo by acting
directly as a chemoattractant on these axons through a Smo-dependent signaling
mechanism.
|
Shh is a negative regulator of retinal ganglion cell axon growth
Retinal ganglion cell (RGC) axons growing towards the diencephalic ventral
midline are faced with the decision to project either contralaterally or
ipsilaterally in response to guidance cues at the optic chiasm
(Fig. 3). Homozygous
inactivation of the mouse Pax2 gene alters the development of the
optic chiasm and RGC axons never cross the midline in these mice.
Interestingly, whereas in wild-type mice Shh expression is downregulated in
the chiasm as RGC axons are migrating towards this region, Shh expression is
ectopically maintained along the ventral midline in
Pax2-/- mice (Torres
et al., 1996). These observations raised the possibility that the
continuous expression of Shh at the ventral midline might contribute to
preventing RGC axon crossing. In agreement with this idea, Trousse et al.
(Trousse et al., 2001
) found
that ectopic expression of Shh in the midline region interferes with RGC axon
growth and prevents them from crossing the midline
(Fig. 3) (Trousse et al., 2001
).
Consistent with the idea that Shh might be acting directly on RGC axons, it
was shown that these manipulations do not affect patterning and neural
differentiation in the eye. Further experiments will be required to determine
whether the chiasm region is repatterned in these experiments, but results
from in vitro experiments support the idea that Shh acts directly to control
RGC axon migration. These studies show that the addition of exogenous
recombinant Shh to retinal explants decreases the number and length of growing
axons, without interfering with the rate of proliferation and differentiation
of cells in the explant, and time-lapse analysis shows that addition of Shh to
retinal explants rapidly causes growth cone arrest and subsequent retraction
of RGC axons (Trousse et al.,
2001
). As the response of the growth cone to many extracellular
guidance cues appears to be modulated, and in some cases perhaps even
mediated, by intracellular cyclic nucleotide levels (cAMP and cGMP)
(Song et al., 1997
;
Song et al., 1998
), the
possibility was explored that the effect of Shh on retinal axons in vitro
might be due to a change in cAMP levels. In agreement with this, the addition
of Shh to retinal growth cones was shown to decrease intracellular levels of
cAMP, a finding consistent with the observation that lowering cAMP levels
favors growth inhibition (Song and Poo,
1999
).
|
The opposite effects of Shh on commissural and retinal axons (attraction
and repulsion) might be due to an intrinsic or extrinsic factor that modulates
cyclic nucleotide levels, much as extrinsic factors can convert Netrin
attraction to repulsion by modulating cyclic nucleotide levels
(Hopker et al., 1999).
Alternatively, as the molecular mechanisms underlying the effects of Shh on
commissural and retinal axons are poorly understood, it is also possible that
these two effects are mediated by distinct signaling pathways that result in
opposite guidance effects a possibility that also has a precedent in
the case of Netrins, which can attract axons by activating DCC family
receptors, and repel them by activating UNC5 family receptors
(Tessier-Lavigne and Goodman,
1996
; Dickson,
2002
).
The Dpp/BMP/TGF-ß family
BMPs are chemorepellents for commissural axons
In Ntn1 and Dcc mutants, commissural axons initially
migrate ventrally for approximately the first third of their normal trajectory
before becoming misrouted (Serafini et
al., 1996; Fazeli et al.,
1997
), suggesting that an additional guidance cue might be acting
to control their dorsal migration.
The proximity of commissural neurons to the roof plate and their initial
growth away from the dorsal midline indicated that the roof plate might repel
commissural axons away. A direct test of this possibility showed that the roof
plate expresses a diffusible activity that repels commissural axons in vitro
(Fig. 1B)
(Augsburger et al., 1999). By
testing a battery of candidate diffusible molecules that might act as
repellent signals, it was found that BMP7 and BMP6, two BMP family members
expressed by the roof plate, can each mimic the chemorepellent activity of the
roof plate in vitro without causing changes in spinal cord cell fate at the
doses used for chemorepulsion. The inhibition of BMP7 activity using soluble
inhibitors of BMP activity, BMP7-blocking antibodies and genetic inactivation
of Bmp7 showed that BMP7 contributes to the chemorepellent activity
of the roof plate for commissural axons
(Augsburger et al., 1999
).
Moreover, BMP7 was shown to induce the collapse of commissural axon growth
cones, providing evidence that it can act directly on growth cones to elicit a
rapid change in cytoskeletal organization
(Augsburger et al., 1999
).
Further evidence indicated that the roof plate chemorepellent BMP complex
consists of a BMP7 and GDF7 heterodimer, as genetic inactivation studies
showed that expression of both Bmp7 and Gdf7 by roof-plate
cells is required for the fidelity of commissural axon growth in vivo
(Butler and Dodd, 2003
).
Together, these results support a model in which a GDF7:BMP7 heterodimer
mediates the roof plate chemorepellent activity that guides the initial
trajectory of commissural axons in the developing spinal cord.
Although GDF7 and BMP7 are essential for the roof plate chemorepellent activity that guides the initial trajectory of commissural axons, it is interesting to note that the initial high incidence of mispolarization of commissural axons in the mutants is later restored and they recover their correct projection pattern for their entire trajectory. Although the mechanisms underlying this `rescue' are not known, it is possible that the chemoattractant activity of Netrin 1 and/or Shh contributes to the correction of the ventral trajectory.
The molecular mechanisms underlying the effect of BMPs on growth cones are
not known. Although BMPs typically activate signaling through type I and II
receptors, it is not known whether these receptors also transduce the BMP
chemorepellent activity. The activation of BMP receptors normally leads to the
transcriptional activation of BMP target genes by Smads, but the timecourse of
commissural growth cone collapse is difficult to reconcile with the idea that
a transcriptional effect mediates the chemorepellent effects of BMPs. Thus,
the guidance effect of BMPs could be mediated independently of Smads or may
involve non-transcriptional effects. Indeed, some of the cytoplasmic
components implicated in guidance responses to other pathfinding cues are
activated in response to TGFß/BMP family members. For example, BMPs
activate PKA and LIM kinase (Lee and
Chuong, 1997; Foletta et al.,
2003
), and other TGFß family members stimulate phospholipase
C, PKC and Rho GTPase (Halstead et al.,
1995
; Choi et al.,
1999
).
It is interesting to note that gradients of BMPs and Shh appear to cooperate at least twice during neural tube development: first to specify cell fate, and later to guide commissural axons to the ventral midline (see Fig. 1). In the case of Shh, the same molecule plays both roles. In the case of BMPs, it remains to be determined whether the same family member can play both roles or whether different BMPs independently perform each role.
The TGFß family member unc-129 is required for motor axon guidance
Intriguing evidence for a role of TGFß family members in axon guidance
has come from studies on the C. elegans gene unc-129, a
TGFß family member that is required for proper guidance of pioneer motor
axons along the DV axis (Colavita and
Culotti, 1998; Colavita et
al., 1998
). Mutations in unc-129 cause defects in the
dorsally oriented trajectories of motor axons that resemble those present in
unc-5, unc-6/Netrin and unc-40/Dcc mutants, without causing
other overt patterning or morphological defects
(Colavita and Culotti, 1998
;
Colavita et al., 1998
;
Hedgecock et al., 1990
). The
dorsal expression of unc-129 suggests that it might be acting as a
chemoattractant for motor axons; however, whether UNC-129 acts directly on
growth cones remains to be established. Interestingly, UNC-129 function does
not require DAF-4, the only known type II TGFß receptor in C.
elegans, suggesting that UNC-129 may act through a novel receptor
mechanism. Identification of the UNC-129 receptor will help determine whether
UNC-129 functions directly to guide motor axons, and will help identify the
mechanisms of UNC-129 signaling.
Interestingly, unc-129 appears to act in parallel to the
unc-6/Netrin pathway (Colavita et
al., 1998; Nash et al.,
2000
). Thus, if UNC-129 acts directly as a guidance cue, it would
provide another example of a BMP collaborating with a Netrin to guide axons;
in this case, however, the sign of the guidance would be inverted compared
with the spinal cord, with the BMP attracting and the Netrin repelling. As in
the spinal cord, the presence of the two cooperative pathways providing `a
push from behind' and `a pull from afar' would then help to ensure the
necessary fidelity in axon guidance required for invariant and robust
development.
The Wg/Wnt family and commissural axon guidance
Wnt5 repels commissural axons from the posterior commissure
The ability of Wnt proteins to stimulate a reorganization of the
cytoskeleton during axonal growth and growth cone extension
(Hall et al., 2000) suggested
that Wnt proteins might also be involved in guiding axons to their targets.
The first direct demonstration of a guidance role was obtained in studies of
commissural neurons in the fly central nervous system (CNS). During
Drosophila development, the array of axons composing the CNS has a
ladder-like structure: each body segment comprises an anterior and a posterior
commissural tract that cross the midline and join one of the two lateral
longitudinal tracts that extend the length of the embryo
(Fig. 4). The attractive and
repulsive signals regulating the decision of commissural axons to cross or not
have been well characterized; however, how axons choose between the two major
subdivisions of the crossing pathways the anterior or posterior
commissure was only recently elucidated.
|
To explore the mechanism underlying Drl function, a soluble, labeled
version of the extracellular domain of Drl was used to detect potential
cell-surface ligands for Drl in the fly ventral nerve cord
(Bonkowsky et al., 1999;
Yoshikawa et al., 2003
).
Drl-binding sites were observed specifically in the posterior commissure,
suggesting that Drl functions to guide axons into the anterior commissure by
repelling them away from the region of ligand expression in the posterior
commissure. The fact that Drl, like other Ryk family members, possesses a
so-called Wnt inhibitory factor (WIF) domain, which in other proteins
functions to bind Wnt proteins (Patthy,
2000
), suggested that a Wnt might be the repellent in the
posterior commissure that repels the axons by binding Drl. Indeed loss of
wnt5 function resulted in commissural axon defects similar to those
in drl mutants, and decreased the ability of misexpressed Drl to
force axons into the anterior commissure
(Fig. 4)
(Yoshikawa et al., 2003
).
Moreover, overexpressing Wnt5 throughout the midline prevented the anterior
commissure from forming, whereas the overexpression of Wnt5 in drl
mutants did not. Taken together, these results imply that Wnt5 repels
Drl-expressing axons and suggest that Drl might function as a receptor for
Wnt5. In a direct test of this possibility, the soluble Drl extracellular
domain was shown to bind to the endogenous Wnt5 protein from fly extracts, and
its binding to the fly ventral nerve cord was found to disappear in
wnt5 mutants (Yoshikawa et al.,
2003
). Thus, biochemical and genetic data indicate that Wnt5 is a
Drl ligand responsible for repelling axons from the posterior commissure.
Importantly, this work is the first to identify a ligand for the Drl family
of receptors and suggests that the other member of the family, Drl2, might
also act as a Wnt receptor. This receptor-ligand interaction appears to be
specific for Wnt5 as Drl does not interact with the two other Wnt family
members tested, Wingless and Wnt4, a finding consistent with the lack of
genetic interaction between drl and either wg or
wnt4 (Yoshikawa et al.,
2003). It remains to be determined whether Drl acts to transduce
the repulsive Wnt signal directly, or functions to prevent, or to reverse,
attraction through an alternative receptor, possibly of the Fz family.
Wnt4 controls the anteroposterior guidance of commissural axons
After commissural axons have reached and crossed the floor plate, they make
a sharp anterior turn toward the brain
(Fig. 1C). The molecules
involved in the DV projection of commissural axons to and at the floor plate
have been well described, but it is only recently that a cue controlling
anteroposterior (AP) guidance has been identified. Using a novel in vitro
assay, evidence was obtained that the activity responsible for the anterior
guidance of post-crossing commissural axons is an increasing posterior to
anterior gradient of a diffusible attractant
(Lyuksyutova et al., 2003).
Several members of the Wnt family were then shown to be able to affect the
growth of post-crossing commissural axons. Among them, Wnt4 was found to be
expressed in an increasing posterior to anterior gradient, at least at the RNA
level. Importantly, an ectopic posterior source of Wnt4 was found to redirect
post-crossing axons posteriorly in vitro, whereas the Wnt inhibitors sFRP1,
sFRP2, and sFRP3 (secreted frizzled-related proteins; soluble proteins that
block the interaction of Wnts with their receptor) made post-crossing
commissural axons stall and turn randomly along the AP axis. In the presence
of Wnt4, the growth cones of post-crossing commissural axons were enlarged and
more complex; addition of sFRP2 reduced this effect within one hour,
suggesting that Wnt4 might be acting directly on the growth cone. These
results indicate that Wnt activity is essential for the normal guidance of
post-crossing commissural axons, and that Wnt4 can act as an instructive
post-crossing commissural axon attractant
(Fig. 1C).
In agreement with a role for Wnt factors in the control of post-crossing
guidance of commissural axons, it was found that mice lacking the Wnt receptor
Frizzled 3 (Fz3) have normal pre-crossing commissural axon behavior but
display defects in AP guidance of commissural axons after midline crossing
(Lyuksyutova et al., 2003). It
will, of course, be important to determine whether Fz3 is required
specifically in commissural neurons for this effect; however, a lack of
apparent patterning defects in the neural tube of fz3 mutants
(Lyuksyutova et al., 2003
),
combined with the in vitro experiments described above, already provide strong
evidence that Wnt-Frizzled signaling directly guides commissural axons along
the AP axis of the spinal cord.
It is noteworthy that the inactivation of Fz3 in mice also results
in other axonal abnormalities (Wang et
al., 2002). These animals display severe defects in many major
axon tracts within the forebrain, including complete loss of the
thalamocortical, corticothalamic and nigrostriatal tracts and of the anterior
commissure, and a variable loss of the corpus callosum. These results suggest
that, in addition to guiding commissural axons at the spinal cord midline,
Wnt-Frizzled signaling might also play a much broader role in axonal
development, although in each case it will again be important to determine
whether Wnt-Frizzled signaling mediates axon guidance directly, or only
indirectly as a secondary consequence of cell fate changes.
An interesting question is how commissural neurons, which apparently
express Fz3 early before reaching the floor plate
(Lyuksyutova et al., 2003), do
not respond to the Wnt gradient until after they have crossed the floor plate.
A crucial step in our understanding of how guidance cues accomplish their
pathfinding role and then `pass the baton' to subsequent cues is to determine
how axonal responsiveness is regulated during an axon's migratory journey. One
mechanism is to have the receptor or an essential signaling component of the
pathway expressed and/or transported at the right time and right place only
when an axon is required to respond. Such a mechanism has been shown to be
responsible for the induction of Slit responsiveness in post-crossing
commissural axons in Drosophila, which is explained by the
upregulation of Robo protein on the axons after crossing
(Kidd et al., 1998
).
Alternatively, an inhibitory molecule could block the function of a receptor
and/or its signaling components when it is inappropriate for the axon to
respond to a particular cue (Sabatier et
al., 2004
; Stein and
Tessier-Lavigne, 2001
). Thus, mechanisms inhibiting premature (in
this case, pre-crossing) sensing of Wnts (e.g. through active inhibition of
the receptor or a signaling component) or, alternatively, allowing the sensing
of Wnts only post-crossing (e.g. through induction of a required receptor or
signaling component) might also act to confer post-crossing-specific
responsiveness to Wnts.
LRP6 is a Fz co-receptor required for the canonical Wnt/ß-catenin
signaling pathway (He et al.,
2004). The axon guidance signaling pathway downstream of Fz3 has
not been investigated but, interestingly, the pathfinding of commissural axons
is reported to be normal in LRP6 mutant embryos, suggesting that the
canonical Wnt signaling pathway is not required for Wnt-mediated commissural
axon guidance (Lyuksyutova et al.,
2003
).
The requirement of a Fz receptor to guide commissural axons along the AP
axis contrasts with the finding that, in flies, Wnt5 acts through Drl to
mediate axonal repulsion from the posterior commissure
(Yoshikawa et al., 2003). It
is possible that the Wnt/Fz pathway signals attraction and the Wnt/Drl pathway
repulsion. Alternatively, as discussed above, it may be that the presence of
Drl modulates signaling through Fz receptors. In this case, Drl would not
directly transduce a repulsive signal, but would reverse an attractive signal
transduced by Fz receptors, similar to the repellent effect that the
expression of the UNC-5 receptor has on UNC-40/Frazzled/DCC-mediated
attraction by UNC-6/Netrin (Hamelin et
al., 1993
; Hong et al.,
1999
).
Shh guides commissural axons along the longitudinal axis of the spinal cord
In addition to Wnt4, a recent publication has reported that Shh also guides
commissural axons in the rostral direction along the longitudinal axis of the
spinal cord (Bourikas et al.,
2005). Using a subtractive hybridization approach to identify
guidance cues responsible for the rostral turn of post-crossing commissural
axons in chick embryos, Bourikas and colleagues identified differentially
expressed candidates, the function of which they investigated by RNA
interference (RNAi)-mediated in ovo gene silencing. Unexpectedly, one of their
candidates turned out to be Shh. In agreement with these results, silencing of
the Shh gene by a different RNAi construct, or by injecting a
hybridoma producing a function-blocking Shh antibody, led to axon stalling at
the contralateral floor plate border, with some axons turning caudally or
rostrally, apparently in a random manner. Importantly, marker analysis
revealed that the patterning of the spinal cord was not apparently affected by
these manipulations, suggesting that these experiments were done after neural
cell fate specification by Shh had occurred. Finally, post-crossing
commissural axons were shown to avoid ectopic Shh in vivo. Together, these
results provide strong evidence that Shh is essential for the normal guidance
of commissural axons along the longitudinal axis of the spinal cord.
A Shh gradient could be guiding commissural axons directly, or could
alternatively be acting only indirectly by controlling a graded distribution
of a distinct guidance cue. For example, Shh could be involved in repressing
Wnt4 expression along the AP axis (contributing to a Wnt gradient) or inducing
a sFRP gradient. Two lines of evidence, however, were provided for a direct
role of Shh (Bourikas et al.,
2005). The first came from an investigation of the receptor
mechanism for this guidance. Interestingly, neither cyclopamine nor Smo RNAi
interfered with the rostral turn of commissural axons along the longitudinal
axis, suggesting that Smo might not be involved in this process. Instead,
RNAi-mediated silencing of Hip1, a gene encoding a Shh-binding
membrane protein transiently expressed in commissural neurons at the time when
they cross the floor plate (as well as in the peri-ventricular region),
resulted in the same post-crossing phenotype as Shh RNAi. These results, which
contrast with the essential role of Smo in Shh-mediated attraction of
commissural axons to the floor plate
(Charron et al., 2003
), suggest
that Hip1 might be involved in transducing a Shh guidance signal in
post-crossing commissural neurons. The relatively restricted expression of
Hip1 mRNA to commissural neurons would be consistent with a direct
action of Shh on these axons. A second line of evidence that supports a direct
role for Shh was obtained in vitro, in experiments that showed that
post-crossing commissural axons from spinal cord explants could be repelled by
Shh beads in vitro. Together, these results suggest a model in which Shh could
be functioning directly through Hip1 as a chemorepellent for post-crossing
commissural axons (Fig.
1D).
Although it is not yet known whether Shh guides post-crossing commissural axons in rodents nor whether Wnt4 guides post-crossing commissural axons in chicks, it is nonetheless interesting to note that the complementary Wnt4 and Shh gradients might act cooperatively in the rostral guidance of commissural axons.
Interpreting positional information: signaling components in axon guidance and cell fate specification
In the studies summarized above, members of all three morphogen families were shown to act rapidly (in an hour or less) to affect growth cone morphology. Although these results appear to be inconsistent with these proteins mediating their axon guidance effects through their canonical, transcriptional signaling pathways, this needs to be formally proven, as none of the above studies has addressed this issue directly. Nonetheless, even if a transcriptional response is found to be necessary, additional local signaling would still be required in the growth cone to generate a polarized response that leads to growth cone turning in a specific direction. Evidently, a purely transcriptional response consisting of a retrograde signal to the nucleus followed by an anterograde signal back to the growth cone cannot account for the polarized turning effect of a guidance cue. Studies aimed at understanding the molecular mechanisms underlying growth cone turning by morphogens will be necessary to identify the molecules that link morphogen signaling to localized growth cone effects.
In this regard, at least three possible models may account for the effects of morphogens in axon guidance. The first is based on the fact that the Wnt, BMP and Hh signaling pathways are only beginning to be understood; many of their intermediate signaling molecules remain to be identified and characterized. Thus, it might be that the signaling proteins that elicit the growth cone effects are also components of the signaling pathways that are required for cell fate specification.
A second model is that the same cue might be acting through entirely
different signaling pathways, including the use of a different receptor. In
the case of BMP/TGFß family members, no known receptor has so far been
implicated in the commissural axon guidance activity of these proteins in
vertebrates. In worms, UNC-129 does not appear to require the classical
TGFß receptors, suggesting that it may be functioning through an
alternative receptor family; it will be exciting to determine whether the
classical BMP receptors are required for the guidance activity of the BMPs on
vertebrate commissural axons or whether they signal through non-classical BMP
receptors. In the case of Shh, although Smo is required for Shh-mediated
commissural axon guidance to the floor plate, it is not known whether Ptc, the
Shh-binding component of the Shh receptor, is involved. This finding contrasts
with chick post-crossing commissural axon guidance, where Smo does not appear
to be required for the rostral turn away from the Shh gradient
(Bourikas et al., 2005).
Additional experiments on commissural and retinal axons are required to
determine the receptor components mediating Shh effects on axon guidance.
Finally, for Wnt-mediated axon guidance, where the identity of the receptors
involved has been more thoroughly investigated, an unexpected situation was
uncovered: the non-classical Wnt receptor Drl is required for repulsion from
the posterior commissure in Drosophila, and the classical receptor
Fz3 is required for attraction towards the anterior pole of the spinal cord in
mouse.
The third model for how morphogens guide axons is a combination of the
first two, and postulates that a morphogen might use the upstream part of the
classical cell fate signaling cascade but then diverge and use a non-classical
pathway to elicit its effects on the growth cone. In this model, the
divergence or branching from the classical pathway might occur
directly downstream of the receptor or further down the signaling cascade. AP
commissural axon guidance by Wnt4 through Fz3 might use such a mechanism:
although this effect requires a Fz receptor, the fact that the Fz co-receptor
LRP6 is not required suggests that Wnt4 may regulate commissural axon guidance
through a non-canonical pathway. In this case, the non-canonical and
potentially overlapping Wnt/Ca2+ and PCP pathways are
potential candidate signaling cascades to mediate the axon guidance effects of
Wnt4. Indeed, in Xenopus, Wnt4 and its related family members Wnt5a
and Wnt11 were found to regulate various morphological events by activating
signaling by heterotrimeric G-protein, Ca2+ and PKC pathways
(Strutt, 2003), and Fz3 was
shown to activate PKC (Kuhl et al.,
2000
). Together, these results raise the possibility that axon
guidance mediated by Wnt4 might be operating through a non-canonical,
PKC-dependent Wnt/Fz signaling pathway and, more importantly, that axon
guidance and PCP might overlap not only conceptually by controlling
the polarity of a growth cone or an entire cell, respectively but also
mechanistically. Indirect evidence supporting mechanistic links between PCP
and axon guidance is provided by the recent finding that the receptor tyrosine
kinase-like protein PTK7 regulates planar cell polarity in vertebrates
(Lu et al., 2004
), whereas its
Drosophila homolog Off-track (Otk) participates in Semaphorin
signaling in axon guidance (Winberg et
al., 2001
).
Concluding remarks
The discovery that morphogens can be reused to guide axons has generated considerable excitement in the field. It remains an open question as to how widespread these guidance effects are. At one extreme, the examples of guidance by morphogens may be isolated instances. At the other, morphogens may prove to be as important as the classic axon guidance molecules (Netrins, Slits, Semaphorins, Ephrins and growth factors) in guiding axons. Elucidating the precise contribution of morphogens will, however, continue to be difficult for some time, given the significant difficulty in determining in any particular situation whether a morphogen is functioning directly or indirectly to regulate axonal guidance. In any gain- or loss-of-function experiment in vivo, the morphogen may be altering the expression of guidance cues in the environment where the guidance effects are observed, or the fate of cells (and hence their responses to guidance cues) that are showing guidance responses. Thus, several tests are required to prove that altered guidance in such experiments reflect direct guidance effects of the morphogen: (1) evidence against a change in the fate of cells or expression of other guidance cues in the environment; (2) evidence that cell autonomous manipulation of the morphogen's signal transduction pathway in the responsive neuron results in similar guidance deficits; and (3) evidence that growth cones of responsive axons can respond directly to the morphogen which may most frequently be obtained in vitro in growth cone collapse or turning assays. It can be expected that the level of proof that is obtained will increase over time as the signaling pathways linking the morphogens to the cytoskeleton for growth cone turning are elucidated. These findings should provide entry points with which to interfere selectively with the guidance effects of the morphogens in the responsive neurons, without altering their transcriptional effects either in those neurons or in the environment. Nonetheless, the collective weight of the experiments summarized above, many of which attempted and succeeded, at least partly, in distinguishing between direct and indirect effects of the morphogens, already provide strong evidence that morphogens have widespread roles in axon guidance, no doubt with more to come.
ACKNOWLEDGMENTS
We thank Christelle Sabatier and Avraham Yaron for critical reading of the manuscript, and members of the Tessier-Lavigne Laboratory for helpful discussions. F.C. is an Arnold and Mabel Beckman Foundation Senior Research Fellow. Research carried out in the Tessier-Lavigne Laboratory was supported by the Howard Hughes Medical Institute.
Footnotes
* Present address: Molecular Biology of Neural Development, Institut de
Recherches Cliniques de Montréal (IRCM), 110 Pine Ave West, Montreal,
Quebec H2W 1R7, Canada
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