RESEARCH REPORT 3251
Development 134, 3251-3257 (2007) doi:10.1242/dev.007112
Analysis of zebrafish sidetracked mutants reveals a novel
role for Plexin A3 in intraspinal motor axon guidance
Kelly A. Palaisa and Michael Granato*
One of the earliest guidance decisions for spinal cord motoneurons occurs when pools of motoneurons orient their growth cones
towards a common, segmental exit point. In contrast to later events, remarkably little is known about the molecular mechanisms
underlying intraspinal motor axon guidance. In zebrafish sidetracked (set) mutants, motor axons exit from the spinal cord at ectopic
positions. By single-cell labeling and time-lapse analysis we show that motoneurons with cell bodies adjacent to the segmental exit
point properly exit from the spinal cord, whereas those farther away display pathfinding errors. Misguided growth cones either
orient away from the endogenous exit point, extend towards the endogenous exit point but bypass it or exit at non-segmental,
ectopic locations. Furthermore, we show that sidetracked acts cell autonomously in motoneurons. Positional cloning reveals that
sidetracked encodes Plexin A3, a semaphorin guidance receptor for repulsive guidance. Finally, we show that sidetracked (plexin
A3) plays an additional role in motor axonal morphogenesis. Together, our data genetically identify the first guidance receptor
required for intraspinal migration of pioneering motor axons and implicate the well-described semaphorin/plexin signaling
pathway in this poorly understood process. We propose that axonal repulsion via Plexin A3 is a major driving force for intraspinal
motor growth cone guidance.
INTRODUCTION
In recent years, many of the basic principles of axonal guidance have
been defined at the molecular and cellular levels (reviewed in
Dickson, 2002). Work in vertebrates and in invertebrates has shown
that a small group of conserved guidance molecules, including
ephrins, netrins, semaphorins and slits, via their cognate receptors,
steer axons towards their synaptic targets. It is also clear now that
axonal trajectories are broken down into segments, and that
individual guidance decisions along these segments are controlled
independently (i.e. using divergent guidance cue/receptor pairs).
Although this enables a relatively small number of guidance
molecules to generate the intrinsic patterns of neural connectivity,
one of the remaining challenges is to ‘reconstruct’ for each neural
cell type all the cue/receptor pairs that control each decision along
its entire axonal trajectory.
For motor axon trajectories, one of the first guidance decisions is
their longitudinal migration towards a segmental spinal cord exit
point. Although the spinal cord is externally unsegmented, motor
axons exit and sensory afferents enter through distinct, segmentally
organized zones along the anteroposterior axis – the motor exit
points (MEPs) and the dorsal root entry zone (DREZ), respectively.
During development, growth cones from motoneurons located
within a spinal segment navigate longitudinally towards a shared
segmental exit point. In rodents and birds, these segmental exit
points are marked by Krox20 (Egr2)-positive cells – the boundary
cap cells (Golding and Cohen, 1997; Niederlander and Lumsden,
1996). Elegant ablation studies have shown that boundary cap cells
are important to confine motoneuron somata to the spinal cord, but
that they appear to be dispensable for guiding motor growth cones
(Vermeren et al., 2003). In contrast to later steps of motor axon
University of Pennsylvania School of Medicine, Department of Cell and
Developmental Biology, Philadelphia, PA 19104-6058, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 10 July 2007
guidance, the cellular and molecular mechanisms by which motor
axons navigate towards the segmental exit point, recognize these
zones and subsequently turn ventrally to exit, are poorly understood.
In the zebrafish, three distinct and identifiable motor axons pioneer
into the periphery through a common, mid-segmental exit point (Fig.
1A) (Eisen et al., 1986). Because Krox20-positive boundary cap cells
have not been identified in zebrafish, caudal primary (CaP)
motoneurons located just dorsal of the exit points can be used as a
landmark for the exit zone (Fig. 1A). CaP growth cones do not
migrate longitudinally, but extend ventrally into the periphery. By
contrast, the growth cones of middle primary (MiP) and rostral
primary (RoP), the somata of which are located further rostrally,
navigate within the spinal cord towards the segmental exit point (Fig.
1A). Once they exit the spinal cord, CaP, MiP and RoP growth cones
pioneer a common path towards a somitic choice point, at which they
diverge onto cell-type-specific trajectories (Fig. 1A) (Eisen et al.,
1986; Myers et al., 1986). Because each of these primary motoneuron
subtypes can be identified and visualized by their stereotyped axonal
trajectories and cell body position, they present an excellent system
in which to study motor axon guidance, including their intraspinal
navigation, at single-cell resolution (Beattie et al., 2002).
In this manuscript, we identify the guidance receptor Plexin A3
to play a major role in intraspinal motor axon guidance (see below).
Plexins are transmembrane receptors containing a Semaphorin
(Sema) domain followed by a cysteine-rich Met-related domain and
a unique and conserved Sex-Plexin domain (SP) in the cytoplasmatic
region (Tamagnone et al., 1999). Based on sequence similarity,
vertebrate plexins are divided into four subfamilies (A-D), and, in
mammals, plexin A3 has been shown to mediate axonal repulsion
by Sema3A and Sema3F in sympathetic and sensory neurons (Yaron
et al., 2005; Cheng et al., 2001). Most of the secreted class 3
semaphorins, including Sema3A and Sema3F, do not bind plexins
directly, but signal via a receptor complex composed of one of the
four A plexins and the obligate co-receptors neuropilin 1 or
neuropilin 2 (Chen et al., 2000; Giger et al., 2000). While the
neuropilins bind the ligand, the plexins transduce the signal through
their cytoplasmic SP domain. Although members of the plexin A
DEVELOPMENT
KEY WORDS: Zebrafish, Growth cone, Axon, Guidance, Myotome, Plexin A3, Sidetracked, Spinal motoneuron
3252 RESEARCH REPORT
Development 134 (18)
subfamily have been shown to control the development of several
sensory and CNS trajectories, little is known about the specific role
that they play during vertebrate motor axon guidance.
In an antibody-based genetic screen, we had previously isolated
mutants of the sidetracked (set) gene, based on the observation that
motor axons exit from the spinal cord at ectopic positions (Birely et
al., 2005). Here, we show that the sidetracked gene plays a crucial
role early during guidance, when pioneering motor growth cones
navigate towards and then through the segmental exit point. We
positionally cloned the sidetracked gene and showed that it encodes
Plexin A3. Using single-cell labeling, time-lapse microscopy and
chimera analysis we characterized this previously unknown function
of Plexin A3. During intraspinal migration of motor growth cones,
Plexin A3 acts cell autonomously to direct subsets of growth cones,
those further away, towards the segmental exit point. We propose
that this is due to repulsive Plexin A3 signaling, possibly triggered
by semaphorins secreted from the posterior somite. Thus, our results
provide the first molecular genetic insights into the earliest
pathfinding events of spinal motor axons, when they orient and pathfind inside the spinal cord towards the exit point.
Antibody stainings and in situ hybridization
MATERIALS AND METHODS
Labeling and scoring of labeled motoneurons
Fish maintenance and breeding
Embryos were injected with Hb9:GFP plasmid at the one-cell stage and
allowed to develop to 24 hours post fertilization (hpf). Fixed embryos were
stained with anti-GFP and SV2 antibodies, mounted and confocal images
taken. For scoring, we measured the distance between outlying segmental
exit points (a), and between the soma and the endogenous exit point (b). We
then calculated the ratio (a:b) and found that, for wild-type MiP (0.19-0.45)
Chimeric embryos
Chimeric embryos were generated and analyzed as previously reported
(Zeller and Granato, 1999). Donor cells were from Tg(Hb9:GFP) transgenic
embryos.
Time-lapse microscopy
Wild-type and mutant embryos bearing the Tg(Hb9:GFP) transgene were
anesthetized in 0.02% tricaine and embedded in 1.5% low-melting-point
agarose. Time-lapse analysis was carried out on a confocal microscope. At
3-minute intervals, z-stacks of ~15 ␮m were captured and then flattened by
maximum projection.
Fig. 1. Motoneuronal defects in sidetracked mutants.
(A) Schematic of primary motoneurons. (B) 24 hpf wildtype embryos stained with SV2. Endogenous exit points
are labeled with asterisks. (C) 24 hpf sidetracked (set)
embryos stained with SV2. Endogenous exit points are
labeled with asterisks, ectopic exit points are marked by
arrows, and branches by an arrowhead. (D-F) Wild-type
embryos injected with Hb9:GFP plasmid and stained with
anti-GFP (green) and SV2 (red) to visualize CaP, MiP and
RoP trajectories. Broken lines indicate the position of
somite boundaries. (G-L) set embryos injected with
Hb9:GFP plasmid and processed as above. Notice that the
labeled somata are located at a distance from the
endogenous exit point (asterisk). (G) Normal projecting
CaP. (H,I) Example of MiP/RoP neurons bypassing the exit
point. (J) Rostrally projecting MiP/RoP motoneurons. Notice
that growth cones exit the spinal cord through the
adjacent, rostral exit point. (K,L) Examples of ectopicexiting RoP/MiP axons. CaP, caudal primary; MiP, middle
primary; RoP, rostral primary.
DEVELOPMENT
All experiments, except where indicated, were performed with the
sidetrackedp55emcf allele (Birely et al., 2005). The Tg(Hb9:GFP) transgenic
line (Flanagan-Steet et al., 2005) primarily labels motoneurons, but also
labels ventral interneurons, presumably ventral longitudinal descending
spinal interneurons (VeLDs).
Antibody stainings were performed as previously described (Zeller et al.,
2002). The following primary antibodies were used: znp-1 [1:200;
(Trevarrow et al., 1990); Antibody Facility, University of Oregon]; and SV2
(1:50, Developmental Studies Hybridoma Bank, University of Iowa).
Antibodies were visualized with corresponding Alexa-Fluor-488, -546 or
-594 conjugated secondary antibodies (1:500; Molecular Probes, Eugene,
OR). Embryos were imaged using confocal microscopy. Colorimetric in situ
hybridizations were performed according to Odenthal and Nusslein-Volhard
(Odenthal and Nusslein-Volhard, 1998). Images were processed using
Adobe Photoshop and Adobe Illustrator.
and RoP (0.22-0.57), the ratio is between 0.19 and 0.57, whereas, for
CaP:VaP, this ratio is always less than 0.19. We then applied this to
sidetracked mutants and considered all soma with a ratio between 0.19 and
0.57 as presumptive MiP/RoP neurons.
Molecular biology
To clone plexin A3, sidetrackedp13umal mutant embryos were first identified
using SV2 antibody staining, their genomic DNA was isolated, and
the entire coding sequence of ENSDARG00000007172 and
ENSDARESTT00000003054, which contains the 5⬘ region including the
translational start, was amplified with gene-specific primers. The amplification products were cloned, sequenced and analyzed using Sequencher
software.
RESULTS AND DISCUSSION
sidetracked guides motor growth cones towards
the segmental exit points
We had previously shown that, in sidetracked mutants, motor axons
displayed two prominent phenotypes: branching at various points
along the axon and exiting from the spinal cord at ectopic locations
(Fig. 1B,C) (Birely et al., 2005). To identify which of the three
subpopulations (CaP, MiP, RoP) exit ectopically, we labeled
individual motoneurons by injecting the motoneuronal Hb9:GFP
construct into one-cell-stage embryos (Flanagan-Steet et al., 2005).
RESEARCH REPORT 3253
This results in embryos with a number of stochastically labeled
motoneurons. Analysis based on cell body position and axonal
trajectory of wild-type siblings at 24 hours post fertilization (hpf)
revealed the stereotypic somata position and axonal trajectories
for RoP, MiP and CaP, demonstrating that all three primary
motoneurons are labeled by this method (Fig. 1D-F; see Materials
and methods for scoring of individual motoneurons). In addition,
this also labeled a fourth, variable primary motoneuron, VaP, which
is present in less than 50% of the hemisegments, and which is also
located dorsal to the exit zone, adjacent to CaP (Eisen et al., 1990).
By contrast, analysis of sidetracked mutants revealed that
motoneuron somata located immediately above the endogenous exit
point (i.e. presumptive CaP/VaP neurons) were unaffected (14/15;
Fig. 1G), whereas approximately 50% (17/32) of motoneurons with
cell bodies located rostrally to the exit point (i.e. presumptive MiPs
and RoPs) displayed dramatic pathfinding defects. In mutants,
presumptive RoP/MiP axons projected towards the exit point but
bypassed it (6/32, Fig. 1H,I), or, instead of projecting caudally
towards the exit point, projected rostrally (4/32, Fig. 1J), or exited
ectopically at the position of their cell body (7/32, Fig. 1K,L). Thus,
single-cell labeling demonstrates that the sidetracked phenotype is
not simply caused by axonal overgrowth, but rather that sidetracked
plays a pivotal role in intraspinal guidance.
Fig. 2. Time-lapse analysis and branching.
(A) Individual movie frames (see Movie 1 in the
supplementary material) showing wild-type CaP/VaP
migration. Notice the growth cone from an interneuron
extending along the ventral aspect of the spinal cord
(arrowhead). (B) Individual movie frames (see Movie 2 in
the supplementary material) showing the ectopic exit of a
sidetracked MiP/RoP axon (asterisk). The soma is located
between two CaP/VaP pairs. Mutant growth cones
pioneer an ectopic exit zone into the periphery, where
they often branch (yellow arrow) and sometimes join
endogenous motor axons (white arrow). Notice the
growth cone from an interneuron extending along the
ventral aspect of the spinal cord (arrowhead). sidetracked
mutants display excessive branching. (C,D) 24 hpf
embryos injected with Hb9:GFP plasmid, and stained with
anti-GFP (green) and SV2 (red) to visualize CaP, MiP and
RoP trajectories, respectively. (C) Wild-type (wt) embryo.
(D) sidetracked embryo. Notice the small branches along
the axonal shaft. (E,F) 48 hpf wild-type and sidetracked
embryos stained with znp1 and SV2. Notice the persisting
ectopic exit points (arrows) and the long branches
(arrowheads) present in the mutants. CaP, caudal primary;
MiP, middle primary; RoP, rostral primary.
DEVELOPMENT
Plexin A3 in motor axon guidance
We next used time-lapse microscopy to examine the behavior of
sidetracked growth cones in intact, live embryos. In Tg(Hb9:GFP);
sidetracked siblings, motor axons exited at segmental exit points and
navigated into the periphery (Fig. 2A and see Movie 1 in the
supplementary material). In Tg(Hb9:GFP); sidetracked mutants
CaP and VaP axons navigate like their wild-type counterparts
through segmental exit points, whereas presumptive RoP/MiP axons
exit at ectopic positions, taking novel paths into the periphery (Fig.
2B and see Movie 2 in the supplementary material). By contrast,
presumptive VeLD interneurons, axons of which extend caudally,
navigate properly in sidetracked mutants (Fig. 1A,B, arrowheads),
suggesting that intraspinal guidance in general is unaffected.
Although we show that, in sidetracked mutants, presumptive
VeLD and primary commissural ascending (CoPA) interneuron axon
projections are indistinguishable from those in wild type (Fig. 2 and
data not shown), we cannot exclude the possibility that sidetracked
also plays a role in the guidance of other spinal cord neurons.
However, it is unlikely that the observed phenotypes are caused by
mis-specified or misplaced motoneurons. We previously showed
that, in sidetracked mutants, anteroposterior polarity of the overlying
somites, and the number and position of each of the three primary
motoneuron populations – CaP, MiP and RoP – are unaffected [Fig.
8 in Birely et al. (Birely et al., 2005)]. Moreover, cell
transplantations have shown that, when placed in ectopic locations,
motoneurons either develop axonal trajectories appropriate for their
original soma positions or appropriate for their new soma position,
but never exit the spinal cord at ectopic positions (Eisen, 1991).
Thus, we conclude that sidetracked primarily guides growth cones
of the distantly located RoP and MiP motoneurons towards the
segmental exit point.
Finally, we found that sidetracked is also crucial for axonal
morphogenesis once motor growth cones navigate the periphery. In
contrast to Hb9:GFP-labeled wild-type axons, sidetracked motor
axons already displayed excessive branching along the entire shaft
at 24 hpf. This is unlikely to be a mere consequence of inappropriate
axonal pathfinding, because sidetracked motor axons that exited
Development 134 (18)
through the endogenous exit point also displayed this phenotype
(Fig. 2C,D). In sidetracked mutants, exuberant branching became
even more pronounced at 48 hpf (Fig. 2E,F). In wild-type embryos,
such extensive side branches have only developed by around 72 hpf,
and invade the myotome to form distributed and myoseptal
neuromuscular synapses (Downes and Granato, 2004; Liu and
Westerfield, 1990), suggesting that sidetracked functions to restrict
motor axons from extending into non-synaptic muscle territories
precociously. Thus, sidetracked motor axons exit at ectopic spinal
cord locations and also form excessive branches that invade
inappropriate territories, consistent with the idea that both
phenotypes are caused by defective axonal repulsion.
sidetracked encodes the zebrafish Plexin A3
guidance receptor
We first used bulk segregant analysis to locate the sidetracked
mutation on chromosome 8 (Fig. 3A). We then refined the map
position by meiotic recombination mapping, defining a crucial
interval between marker fb82f05.x1 (1/420 recombinants) and
marker Z43564b (2/598 recombinants). On the ENSEMBL genome
assembly, these two markers delineate a 480 kb interval, in
which many predicted and three known protein coding genes are
located: sodium and chloride dependent creatine transporter 1
(ENSDARG00000043646), calcium/calmodulin-dependent protein
kinase type 1b (ENSDARG00000043648) and plexin A3
(ENSDARG00000007172) (Fig. 3A). The conceptually translated
protein (XM_690717) is 61% identical to zebrafish Plexin A4
(NP_001004495) but 73% identical to human and mouse Plexin A3,
suggesting that XM_690717 encodes zebrafish Plexin A3.
Sequencing of the plexin A3 coding region from sidetracked mutants
revealed a single base-pair change (T2312A), resulting in a nonsense mutation, truncating the protein at position 662 in the second
PSI (Plexin-Semaphorin-Integrin) domain (Fig. 3B,C). Because this
truncates the protein before the transmembrane domain and the
signal transducing Sex-Plexin domain, the sidetrackedp13umal allele
probably represents a null allele. Although it is possible that the
Fig. 3. Molecular cloning and plexin A3 expression.
(A) Molecular genetic map of the sidetracked (plexin A3)
region. (B) Domain organization of the wild-type Plexin
A3 protein. (C) The sidetrackedp13umal allele is a
presumptive null. For domains, see main text. (D) Lateral
view of a wild-type embryo processed for plexin A3 in
situ hybridization. Low-level expression is detectable
throughout the spinal cord and appears enriched in
neurons close to the somite boundary. (E) Highmagnification view. Expression of plexin A3 is enriched in
two somata adjacent to the somite boundary, consistent
with the positions of middle primary (MiP) and rostral
primary (RoP). Square brackets indicate motoneuron cell
bodies; broken lines indicate the position of somite
boundaries. G-P/IPT, glycine-proline-rich/
immunoglobulin-like fold shared by plexins; MRS/PSI,
Met-related sequence/plexin-semaphorin-integrin
domain; SP, Sex-Plexin; TM, transmembrane.
DEVELOPMENT
3254 RESEARCH REPORT
Plexin A3 in motor axon guidance
RESEARCH REPORT 3255
truncated protein is expressed, it is unlikely that it retains biological
activity because it lacks both the transmembrane and the signal
transducing domains. Thus, given the absence of a second plexin A3
in the zebrafish genome, sidetracked mutants probably lack all
Plexin A3-mediated semaphorin signaling.
sidetracked (plexin A3) acts cell autonomously to
guide motor growth cones
During the time period of intraspinal motoneuron guidance (18-24
hpf), plexin A3 expression is detectable at low levels throughout the
spinal cord, and in each spinal segment is enriched in two ventral
neurons positioned at the level of the somite boundary (Fig. 3D,E).
The dorsoventral and anteroposterior position within each segment
is consistent with the position of MiP and RoP motoneurons (Fig.
1A,E,F) (Feldner et al., 2005). To determine whether sidetracked
indeed functions in these two motoneurons, we generated chimeric
embryos in which labeled wild-type blastula cells were transplanted
into age-matched sidetracked hosts. Analysis of 26 hpf chimeric
embryos revealed that MiP and RoP motoneurons derived from
wild-type donors developed wild-type-like axonal trajectories in
sidetracked mutants (n=6/6 RoPs, 6/6 MiPs, Fig. 4A). Conversely,
mutant-derived motoneurons developed sidetracked-like axonal
trajectories when transplanted into wild-type hosts (n=4/6 RoPs; 4/5
MiPs; Fig. 4B). Thus, Plexin A3 acts cell autonomously in MiP and
RoP, suggesting a role as a receptor that is crucial for intraspinal
guidance.
How does Plexin A3 control intraspinal motor axon guidance? In
most circumstances in which it has been examined, type A plexins
transduce axonal repulsion (reviewed in Negishi et al., 2005;
Tamagnone et al., 1999). One attractive model is that Plexin A3sensitive RoP and MiP growth cones avoid a repellent that diffuses
anteriorly and posteriorly from the posterior part of each somite (Fig.
4E,F). Such a simple model would explain why wild-type RoP and
MiP growth cones migrate caudally to a mid-segmental point, where
repulsion would be minimal. It would also account for the various
pathfinding defects observed in sidetracked mutants: RoP and MiP,
now insensitive to the repellent, bypass the endogenous exit point,
or turn rostrally towards the next anterior exit point (Fig. 1H,J), or
just exit the spinal cord below their soma (Fig. 1K,L).
We next examined the distribution of known plexin ligands at the
posterior somite compartment. Plexin A family members function
in a complex with neuropilins as receptors for semaphorins, in
particular for Sema3 family members (reviewed in Negishi et al.,
2005). In the zebrafish, a number of Sema3 family members have
been identified, including Sema3Aa (Yee et al., 1999), Sema3Ab
(Roos et al., 1999), Sema3C (Yu and Moens, 2005), Sema3D
(Halloran et al., 1999), Sema3Fa, Sema3Fb, Sema3Ga, Sema3Gb
(Yu and Moens, 2005) and Sema3H (Stevens and Halloran, 2005).
Of these, Sema3Aa has been reported to be expressed in the
posterior region of each somite, and knockdown of sema3Aa and
nrp1a expressed in motoneurons affects axon guidance in the
DEVELOPMENT
Fig. 4. Plexin A3 acts cell autonomously, mediating the
chemorepulsion of motor axon growth cones from posterior
somites by semaphorin ligands. (A,B) Chimera analysis. Donor cells
are labeled with rhodamine dextran (red) and donor motoneurons
express GFP (green); hosts were also stained with SV2 antibody (green)
to reveal all motor axonal trajectories. (A) A wild-type (wt) donorderived MiP neuron (red) in an otherwise mutant (mut) host properly
exits the spinal cord, projects to the choice point (arrowhead), and then
forms a dorsal collateral. Arrow indicates normal dorsal trajectory.
(B) sidetracked donor-derived motoneurons (red, asterisks) in an
otherwise wild-type host exit the spinal cord ectopically (arrows).
(C) sema3Fa expression at 21 hpf. Broken lines indicate the position of
somite boundaries. (D) sema3Ab expression at 21 hpf. (E) Model of
wild-type Plexin A3 function. During intraspinal motor axon guidance, a
diffusible repulsive cue (semaphorin; orange), secreted from the
posterior somite, spreads anteriorly and posteriorly. This repulsion
directs Plexin A3-sensitive growth cones (RoP, MiP) towards the midsegmental exit zone. After they exit from the spinal cord, CaP, MiP and
RoP motoneurons navigate towards their respective muscle targets.
(F) In sidetracked mutants, MiP and RoP motoneurons are insensitive to
semaphorin repulsion during intraspinal guidance and extend
aberrantly. After they exit from the spinal cord and navigate towards
their targets, mutant CaP, MiP and RoP motoneurons exhibit exuberant
branching, suggesting that Plexin A3 signaling is also important to
prevent precocious spreading of motor axonal branches. CaP, caudal
primary; MiP, middle primary; RoP, rostral primary.
periphery (Feldner et al., 2005; Sato-Maeda et al., 2006). We
therefore examined two other semaphorin ligands, Sema3Fa and
Sema3Ab. In sensory and sympathetic neurons, the Plexin A3Neuropilin 2 complex preferentially transduces the activities of
Sema3F semaphorins (Yaron et al., 2005). Whereas sema3Fa is
expressed diffusely in ventral somites (Fig. 4C), Sema3Ab is
localized in a discrete band in the posterior part of each somite (Fig.
4D) (Roos et al., 1999), more consistent with our model. However,
systematic studies to examine the expression patterns of all
semaphorin genes, as well as additional functional studies, will be
required to identify the precise mechanisms by which Plexin A3
mediates intraspinal axonal guidance.
Independent of the model, our studies, as well as those by Tanaka
et al. in this issue (Tanaka et al., 2007), provide the first genetic
demonstration that Plexin A3 signaling plays a crucial role during
intraspinal motor axon guidance. Although previous studies have
demonstrated a role for class 3 semaphorins and neuropilins in later
stages of motor axon guidance [i.e. in the periphery (Huber et al.,
2003)], our results clearly implicate a plexin signaling pathway in
the very early steps, when growth cones orient towards the
segmental exit point. The finding that sidetracked encodes Plexin A3
provides two novel insights. First, our results show that motor axons
can exit the spinal cord at seemingly random positions. Previous
studies had suggested that sites of axonal entry or exit from the
spinal cord are restricted and prefigured by the presence of
specialized neural crest derivatives – the boundary cap cells
(Niederlander and Lumsden, 1996). Our results suggest that the
neural tube is competent to form exit points along its entire length,
but that the precise locations of segmental exit points are determined
by the growth cone.
Second, intraspinal guidance towards spinal exit points is
governed, at least in part, by repulsive guidance. In the chick, elegant
rotation experiments have shown that inverting rhombomeres in
their anteroposterior orientation does not alter the growth of motor
axons towards their anterior-lying exit points, which suggested that
exit points represent a chemoattractive, intermediate target guiding
motor axons (Guthrie and Lumsden, 1992). Our results clearly
demonstrate that intraspinal guidance requires Plexin A3 signaling,
which propagates the repulsive activities of semaphorins. Our model
is consistent with recent results obtained by Feldner et al using
plexin A3 as well as semaphorin 3A morpholinos (Feldner et al.,
2007), as well as by Tanaka et al. (Tanaka et al., 2007). Although this
does not exclude the co-existence of a chemoattractive mechanism,
our results present compelling genetic evidence that a particular
guidance mechanism – repulsion – guides the intraspinal migration
of motor growth cones.
Our studies also reveal a later role for Plexin A3 in restricting
axonal branching. In sidetracked (plexin A3) mutants, branches
invaded the myotome prior to the time period when wild-type
motor axons do (Fig. 3F). This phenotype is somewhat reminiscent
of the behavior that mossy fibers display in plexin A4 mutant mice
(Suto et al., 2007). There, it has been proposed that plexin A4
prevents mossy fibers from invading the entire CA3 region,
thereby restricting them to a narrow zone (Suto et al., 2007). Thus,
similar to the situation in the CNS, sidetracked (plexin A3)
signaling in the periphery appears to be crucial in order to prevent
precocious spreading of motor axonal branches into future
synaptic muscle fields, possibly coordinating pre- and postsynaptic development.
We would like to thank V. Schneider and J. Birely for help with the in situ
hybridizations; C. Moens (HHMI, WashU) for providing sema3Fa; and D.
Gilmour (EMBL) for comments on the manuscript. We would also like to thank
Development 134 (18)
H. Tanaka and H. Okamoto for communicating unpublished results. This work
was supported by grants from the American Heart Association (K.A.P.), the
National Science Foundation (M.G.) and the National Institute of Health
(M.G.).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/18/3251/DC1
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DEVELOPMENT
Plexin A3 in motor axon guidance