RESEARCH ARTICLE 3633
Development 139, 3633-3643 (2012) doi:10.1242/dev.080051
© 2012. Published by The Company of Biologists Ltd
Motoneuronal Sema3C is essential for setting stereotyped
motor tract positioning in limb-derived chemotropic
semaphorins
Isabelle Sanyas, Muriel Bozon, Frédéric Moret* and Valérie Castellani*,‡
SUMMARY
The wiring of neuronal circuits requires complex mechanisms to guide axon subsets to their specific target with high precision. To
overcome the limited number of guidance cues, modulation of axon responsiveness is crucial for specifying accurate trajectories.
We report here a novel mechanism by which ligand/receptor co-expression in neurons modulates the integration of other guidance
cues by the growth cone. Class 3 semaphorins (Sema3 semaphorins) are chemotropic guidance cues for various neuronal projections,
among which are spinal motor axons navigating towards their peripheral target muscles. Intriguingly, Sema3 proteins are
dynamically expressed, forming a code in motoneuron subpopulations, whereas their receptors, the neuropilins, are expressed in
most of them. Targeted gain- and loss-of-function approaches in the chick neural tube were performed to enable selective
manipulation of Sema3C expression in motoneurons. We show that motoneuronal Sema3C regulates the shared Sema3 neuropilin
receptors Nrp1 and Nrp2 levels in opposite ways at the growth cone surface. This sets the respective responsiveness to exogenous
Nrp1- and Nrp2-dependent Sema3A, Sema3F and Sema3C repellents. Moreover, in vivo analysis revealed a context where this
modulation is essential. Motor axons innervating the forelimb muscles are exposed to combined expressions of semaphorins. We
show first that the positioning of spinal nerves is highly stereotyped and second that it is compromised by alteration of
motoneuronal Sema3C. Thus, the role of the motoneuronal Sema3 code could be to set population-specific axon sensitivity to limbderived chemotropic Sema3 proteins, therefore specifying stereotyped motor nerve trajectories in their target field.
INTRODUCTION
Mechanisms underlying the wiring of neuronal connectivity remain
puzzling with regard to the limited repertoire of guidance cues used
to achieve the tremendous diversity of axon pathways. Tight
spatiotemporal regulation of ligands in axon environment and
receptors in specific neuronal populations has been proven to be
crucial to generate different axon trajectories (Yu and Bargmann,
2001). In addition, modulation of axon responsiveness and
reiterated use of guidance factors at successive steps of axon
navigation allow the diversification of pathway choices with a
relatively small number of factors.
Beyond their function in generating permissive corridors for
navigating axon tracts, gradients of guidance cues also play
instrumental roles in the formation of topographic maps, organizing
accurate trajectories of multiple sets of axons in their target field.
For example, in the retinotectal and olfactory systems, distinct
levels of sensitivity control axon positioning within gradients (Imai
et al., 2009). The mechanisms setting the sensitivity to guidance
cues remain poorly explored. Class 3 secreted semaphorins (Sema3
proteins) are a group of seven widely expressed chemotropic
factors, from Sema3A to Sema3G, with repulsive and attractive
activities (Raper, 2000). Loss-of-function approaches in mice have
provided evidence for important roles of Sema3 proteins and their
University of Lyon, UCBL1, CGphiMC, UMR CNRS 5534, 16 rue Raphael Dubois,
69622 Villeurbanne, France.
*These authors contributed equally to this work
‡
Author for correspondence ([email protected])
Accepted 11 July 2012
receptors, the neuropilins (Nrp proteins), in patterning neuronal
connections in various regions of developing organisms (Raper,
2000). Likewise, in the motor system, the fasciculation and
trajectory of spinal motor axons that express Nrp proteins are
largely shaped by the repulsive effects of Sema3 proteins provided
by peripheral tissues such as the dermamyotome, the notochord,
the ectoderm and the limb (Behar et al., 1996; Huber et al., 2005;
Kitsukawa et al., 1997; Raper, 2000; Taniguchi et al., 1997; VarelaEchavarría et al., 1997). Intriguingly, mRNAs encoding both
receptors and Sema3 proteins have been also detected in
motoneuron cell bodies by the time their axons elongate in the
periphery and innervate their targets (Luo et al., 1995; Püschel et
al., 1995; Moret et al., 2007; Chilton and Guthrie, 2003; MeléndezHerrera and Varela-Echavarría, 2006; Cohen et al., 2005).
Ligand/receptor co-expression emerges from recent work as a
key mechanism by which growing axons lower their sensitivity to
exogenous sources of the corresponding ligand (Haklai-Topper et
al., 2010; Carvalho et al., 2006; Kao and Kania, 2011). Whether
regulations across ligand family members occur and, if so, whether
they drive a diversity of emerging growth cone properties is not yet
fully known. In a previous study, we brought to light an original
mechanism by which Sema3A expression in motoneurons controls
the availability of its receptor Nrp1 at the growth cone surface, thus
setting the sensitivity of their growth cone to exogenous Sema3A
(Moret et al., 2007). These findings opened several issues,
particularly concerning the potential contribution of other
motoneuronal Sema3 proteins and their implication during axon
navigation. We addressed these issues by investigating another
Sema3 whose expression profile caught our attention. Indeed,
Sema3C is expressed in restricted subsets of spinal motoneurons,
as well as in the limb. The contribution of these different sources
DEVELOPMENT
KEY WORDS: Motoneuron, Axon guidance, Semaphorin, Chick
Development 139 (19)
3634 RESEARCH ARTICLE
MATERIALS AND METHODS
Plasmid construction
The Sema3CiresEGFP bicistronic construct was derived from the pAG-NTcoll1 plasmid [kindly provided by J. Raper (Feiner et al., 1997)]. IresEGFP
fragment was amplified from ires2EGFP plasmid (Clontech, Mountain
View, USA) by PCR between suitable primers, then inserted at the XhoI
site located downstream from the chick Sema3C cDNA.
For RNA interference experiments, a SiRNA cocktail (ABgene, Epsom,
Surrey, UK) was co-electroporated with the EGFP-N3 vector (Clontech,
Mountain View, USA) in the chick spinal cord. The cocktail contained four
siRNAs with the following sequences: 5⬘-GGAACGGACTACAAGTATA3⬘, 5⬘-GGTTAAGCTGAGTGAGAGA-3⬘, 5⬘-GCACGCGGTGTTTGGGATA-3⬘ and 5⬘-GCCATTAATCATCCGGATA-3⬘. As a control, the
ON-TARGETplus Non-Targeting Pool from ABgene was used, containing
four siRNA with the following sequences: 5⬘-UGGUUUACAUGUCGACUAA-3⬘, 5⬘-UGGUUUACAUGUUGUGUGA-3⬘, 5⬘-AGGUUUACAUGUUUUCUGA-3⬘ and 5⬘-UGGUUUACAUGUUUUCCUA-3⬘,
with no robust similarities to chick mRNA.
In ovo electroporation
In ovo electroporation of chick embryos (Gallus gallus; EARL Morizeau,
Dangers, France) was performed as described previously (Creuzet et al.,
2002). The plasmids and siRNAs were introduced in the neural tube at
brachial and thoracic levels to stage HH14-15 embryos (Hamburger and
Hamilton, 1992). Plasmids prepared with EndoFree maxiprep Xtra kit
(Macherey-Nagel, Düren, Germany) were routinely diluted at 1.5 g/l in
PBS for Sema overexpression and at 0.5 g/l in PBS for the EGFP-N3
vector. siRNA cocktails were diluted at 2 g/l in PBS.
Explant culture
The ventral one-third of spinal cords was dissected out from HH24 chick
embryos at brachial level in DMEM (GIBCO). Explants were cultured on
glass coverslips precoated with laminin (50 g/ml, Sigma) and polyornithin (10g/ml, Sigma), and grown in a previously described medium
(Marthiens et al., 2005). Immunolabeling using a motoneuron axonal
marker NgCAM confirmed that fibers extending from these explants were
motoneuron axons.
Histological analyses
Cryosections (20 m) were obtained from embryos fixed in 4%
paraformaldehyde and embedded in 7.5% gelatine/15% sucrose. In situ
hybridizations and whole-mount immunostaining were performed as
described previously (Henrique et al., 1995; Moret et al., 2007). In order
to identify the Sema3C-expressing motoneuronal pool, Sema3C in situ
hybridization and Isl1, FoxP1 and Lim3 immunostainings were performed
on adjacent sections, as Isl1, FoxP1 and Lim3 epitopes were found to be
altered by in situ hybridization protocols. Images of Sema3C in situ
hybridization were changed into negative images and superimposed on
immunostaining images for the aforementioned motoneuronal markers. For
immunostaining, cryosections, whole-mount embryos or explants were
incubated with the following antibodies diluted in 2% bovine serum
albumin blocking solution: goat anti-neuropilin 1 (1/50; R&D Systems,
Minneapolis, USA), rabbit anti-neuropilin 2 (1/50; Zymed, San Francisco,
USA), mouse anti-neurofilament 160 kDa (1/50; RMO-270; Zymed, San
Francisco, USA), rabbit anti-cleaved caspase 3 (1/100; Asp175; Cell
Signaling, Danvers, USA), rabbit anti-FoxP1 (1/50; AbCam, Cambridge,
UK) and rabbit anti-GFP (1/200; Molecular Probes, Eugene, USA). Mouse
anti-NgCAM (1/400, 8D9 developed by Dr Vance Lemmon), mouse antiislet1/2 (1/50; 39.4D5 developed by Dr Thomas Jessell, Columbia
University, NY, USA), mouse anti-islet1 (1/50; 39.3F7 developed by Dr
Thomas Jessell) and mouse anti-Lim3 (1/50; 67.4E12 developed by Dr
Thomas Jessell) were obtained from the Developmental Studies
Hybridoma Bank developed under the auspices of the NIHCD and
maintained by the University of Iowa (Department of Biological Sciences,
Iowa City, USA).
For chromogenic immunostaining, suitable biotinylated antibodies, then
the ABC complex (Vectastain), were used prior to DAB staining (Vector,
Paris, France). For immunofluorescence staining, sections were incubated
with appropriate Alexa 555 or Alexa 488 antibodies (1/500; Molecular
Probes) or Fluoprobes 547 antibodies (1/200).
Nrp1, Nrp2 and NgCAM immunofluorescent labeling on explants nonpermeabilized or permeabilized with 0.02% Triton X100, were detected
with combined suitable Alexa 555 (1/500) and biotinylated antibodies
(1/50). Explants were subsequently incubated with Alexa350-streptavidin
(1/50; Molecular Probes).
Collapse assay
Conditioned media were obtained by transfection of MYC-tagged
Sema3A, Sema3C or Sema3F, as well as control constructs in human
embryonic kidney cells (HEK 293T) using Exgen (Euromedex, Les Ulys,
France), cultured in DMEM medium with 10% heat-inactivated FBS,
0.05% penicillin and streptomycin. After 24 hours of culture at 37°C,
ventral spinal cord explants were incubated with control and Sema
supernatants for 45 minutes at 37°C, and fixed in 4%
paraformaldehyde/0.5% sucrose. Individual axons were randomly selected
through NgCam or phalloidin TRITC (Sigma) fluorescent labeling and
their morphology examined at 40⫻ magnification as described by Falk et
al. (Falk et al., 2005). Analyses were performed from a minimum of three
independent experiments. For antibody blocking experiments, blocking
goat anti-neuropilin 1 (25 g/ml; R&D Systems, Minneapolis, USA),
rabbit anti-neuropilin 2 (10 g/ml; Zymed, San Francisco, USA) antibodies
were applied 1 hour prior to the application of conditioned medium.
Analysis of cell surface protein fraction and western blots
For cell surface protein fraction analysis, B103 cell lines were cotransfected with chick neuropilin 1 (kindly provided by J. Raper, University
of Pennsylvania, School of Medicine, Philadelphia, USA) or mouse
neuropilin 2 (kindly provided by A. Püschel, University of Münster,
Germany) and Sema3CiresEGFP or EGFP constructs, using EXGEN 500
(Euromedex, Strasbourg, France) according to manufacturer’s instructions.
Surface proteins were biotinylated and isolated with the Pierce cell surface
protein isolation kit (Thermo Scientific, Waltham, USA). Surface and total
protein pools were analyzed by western blot using goat anti-neuropilin 1
(1/1000; R&D Systems, Minneapolis, USA) and goat anti-neuropilin 2
(1/1000; R&D Systems, Minneapolis, USA) primary antibodies, and a
peroxidase rabbit anti-goat (1/5000; Sigma, St Louis, USA) secondary
antibody. Results were observed in four independent experiments.
For total protein level analysis, spinal cord extracts were obtained by
dissection of ventral spinal cords from embryos electroporated with
Sema3CiresGFP or EGFP constructs. Tissues lysates were then analyzed
by western blot using goat anti-neuropilin 1 (1/1000; R&D Systems,
Minneapolis, USA) primary antibody and a peroxidase rabbit anti-goat
(1/5000; Sigma, St Louis, USA) secondary antibody.
Microscopy and quantification of spinal nerve width
Labeling was acquired under an Axiovert microscope (Zeiss, Germany)
equipped with a Coolsnap CCD camera (Photometrics, Evry, France) or a
DEVELOPMENT
of Sema3C during motor axon navigation remains undetermined.
Sema3C has been identified as a guidance cue for cortical,
mesencephalic and sympathetic axons, in addition to influencing
the orientation of cardiac neural crest cell migration (Bagnard et
al., 1998; Niquille et al., 2009; Hernández-Montiel et al., 2008).
We report here novel properties of motoneuronal Sema3C on motor
axon responsiveness to environmental Sema3 proteins. We
demonstrated by targeted manipulations in chick spinal
motoneurons that intrinsic Sema3C modulates in opposite ways the
availability of the two Sema3 receptors, Nrp1 and Nrp2, at the
growth cone surface. Interestingly, we show that this confers to
Sema3C the ability to modulate axon sensitivity to different Nrp1and Nrp2-dependent Sema3 repellents. In addition, our in vivo
analyses reveal a highly stereotyped pattern of spinal nerve
positioning in the forelimb, where combinations of these Sema3
repellents are detected. Finally, we uncover a key contribution of
motoneuronal Sema3C in setting the position of LMCm-derived
ventral motor tracts in the forelimb.
Motoneuronal Sema3C and axon guidance
RESEARCH ARTICLE 3635
LSM510 Meta Confocal Microscope (Zeiss). Fluorescence quantifications
were analyzed with the ImageJ software (National Institutes of Health,
USA). For quantification of the spinal nerve thickness, NgCAM
immunofluorescence was performed on whole-mount chick embryos.
Images were captured at 10⫻ magnification in the brachial and thoracic
region. Spinal nerve outline was traced on NgCAM labeling and its width
measured. Normalized width reported on the histogram was defined as the
quotient of motor tract widths between electroporated and nonelectroporated sides.
Three-dimensional reconstructions and quantification of the limb
innervation pattern
Quantifications of Nrp levels
For quantification of growth cone fluorescence, images were captured at
40⫻ magnification with constant exposure parameters below pixel
saturation. Growth cone outline was traced with NgCAM staining, which
clearly delineated filopodia and lamellipodia in both control and
experimental conditions. After background subtraction, the intensity of
neuropilin and NgCAM staining was measured within the outline and the
mean intensity per pixel was calculated. Intensity values were normalized
to the respective control experiments conditions: 1 corresponds to the mean
level of neuropilins in the control conditions. Growth cones were then
classified according to the fluorescence intensity.
Statistical analysis
Statistical analyses were carried out using the 2 test for collapse assays
and Nrp cell surface quantification; and the Mann-Whitney test was used
for immunofluorescence quantifications and nerve tract phenotype analysis.
RESULTS
Specific and dynamic Sema3C expression in the
chick spinal motor columns
To define the motoneuron subpopulations expressing Sema3C, in situ
hybridization was performed on chick transverse sections and the
Sema3C expression pattern was compared with the immunolabeling
of specific Lim3, Isl1 and FoxP1 motoneuron markers from adjacent
sections. Sema3C was found already expressed in motoneurons at
HH24, when motor axons navigate towards their targets (Fig. 1A).
At brachial level, Sema3C+ neurons are found among the
Lim3+/Isl1low/FoxP1– motoneuron sub-column, the medial Medial
Motor Column (MMCm), projecting between the dorsal root
ganglion (DRG) and the dermamyotome (Fig. 1A). Sema3C is also
expressed in the Lim3–/Isl1high/FoxP1+ population corresponding to
Fig. 1. Sema3C expression patterns in motoneuronal
subpopulations. (A,B)In situ hybridization and fluorescent
immunostaining against Sema3C, Lim3, Isl1 and FoxP1 on adjacent
cryosections of HH24 (A) and HH26/27 (B) chick spinal cord at brachial
and thoracic levels. Scale bars: 50m. (C)In situ hybridization of chick
cryosections for Sema3C at stage HH24. Electroporation of Sema3C
siRNA cocktail significantly downregulates Sema3C expression in
motoneurons (red arrow). Sema3CiresGFP electroporation leads to
Sema3C overexpression in the whole spinal cord, including in
motoneurons (red arrow). Green lines define the electroporated side of
the spinal cord. Scale bars: 100m.
the medial part of the Lateral Motor Column (LMCm) which
projects to the ventral forelimb (Fig. 1A). At thoracic level, Sema3C+
cells lay within both MMCm and MMCl (HMC) neurons (Fig. 1A).
At HH26/27, while LMC motor axons progress in the limb
mesenchyme, Sema3C expression is detected, in the brachial region,
in the Lim3+/Isl1low/FoxP1– MMCm subcolumn and in the
Lim3–/FoxP1+ LMC population, particularly in a subset of the
Lim3–/Isl1high/FoxP1+ LMCm subcolumn where strong Sema3C
expression is observed (Fig. 1B). In the thoracic region, high
Sema3C expression is present in the MMCm (Fig. 1B). Weak
expression is also detectable in the MMCl subcolumn (Fig. 1B).
Therefore, Sema3C is dynamically expressed by motoneurons with
restricted and subcolumn-specific expression at the brachial level,
suggesting a potential role during LMCm axon navigation.
Motoneuronal Sema3C modulates Nrp1 and Nrp2
availability at the growth cone surface
Our previous work (Moret et al., 2007) showed that intrinsic
Sema3A sets motoneuron sensitivity to exogenous Sema3A by
regulating, at post-transcriptional level, the availability of its Nrp1
DEVELOPMENT
The analysis of the limb innervation phenotype was obtained on NgCamlabeled whole-mount embryos. Each control and electroporated limb was
fully scanned with a confocal microscope. Projection patterns from the
control and electroporated sides were then reconstructed in three
dimensions. The mirror stack of the control side was obtained. Control and
electroporated sides were then superimposed with the ImageJ 3D viewer
plug-in. Pseudocolors were affected to these projection patterns (control
side, magenta; electroporated side, cyan).
For quantification, confocal stacks were analyzed by a three-step
process. First, the scanned innervation pattern for each limb was projected
to obtain, for each limb, separate reconstitutions of dorsal and ventral
tracts. Then pseudocolors were applied to these projection patterns (control
side, magenta; electroporated side, cyan) and the mirror image of the
control side was obtained. Finally, the control and electroporated patterns
were superimposed for each embryo in order to form a composite image.
For the quantifications presented in the diagrams, embryo classification
was established after visual assessment of the dorsal or ventral tract
superimposition: 1 corresponds to the best superimposition and 20 to the
most severe mismatches of the motor tracts in the composite image. Three
independent blind classifications were performed in order to obtain the
final average rank for each embryo. The ranks of embryos were compared
between Si control and Si Sema3C conditions by a Mann-Whitney
statistical test.
receptor at the growth cone surface. We therefore asked whether
motoneuronal Sema3C modulates its receptor expression. Sema3C
has been shown to bind to both Nrp1 and Nrp2. Its guidance
activities are thought to be mediated by Nrp1/Nrp2 heterodimers or
by Nrp2/Nrp2 homodimers (Chen et al., 1998; Takahashi et al.,
1998). Nrp1 was found expressed in all spinal motoneurons and
Nrp2 was predominantly expressed in LMCl but also in LMCm
motoneurons (Mauti et al., 2006; Moret et al., 2007). We performed
targeted Sema3C gain- and loss-of-function experiments in the chick
spinal cord and investigated in explant cultures modulations of Nrp1
and Nrp2 levels in motor growth cones using immunocytochemistry.
Unilateral overexpression of intrinsic Sema3C was obtained by
electroporation of Sema3CiresGFP in the chick spinal cord (Fig. 1C).
An EGFP plasmid was used as a control for these experiments. The
efficiency of Sema3C overexpression was checked by in situ
hybridization as no efficient antibody against chick Sema3C is
available (Fig. 1C). High levels of Nrp1 and Nrp2 were observed in
most motor axons after permeabilization (supplementary material
Fig. S1A,B,C,D). Nrp1 cell surface labeling was detected in all
motor axons. By contrast, 50% of motor axons lacked Nrp2 cell
surface staining. Moreover, different levels of Nrp1 and Nrp2 levels
were observed between axons from the same explant, suggesting that
regulatory mechanisms generate a diversity of neuropilin levels at
the surface of motor growth cones (Fig. 2A). Motor growth cones
could then be classified into different categories from null/low cell
surface expression (fluorescence intensity <0.5) to high expression
(fluorescence intensity >1.5 or 3) of either Nrp1 or Nrp2.
Interestingly, Sema3C overexpression led to a drop in Nrp1 level,
more specifically by the decrease of the high Nrp1 category and the
increase of the low Nrp1 one (Fig. 2B,D). Surprisingly opposite
effects were found for Nrp2, as the percentage of high-Nrp2+ motor
axons was increased following Sema3C overexpression (Fig. 2C,E).
Next, we performed loss-of-function experiments through the
electroporation of a custom cocktail of four siRNAs directed
against chick Sema3C. EGFP expression vector was
simultaneously co-electroporated to monitor electroporated cells.
The knock down efficiency was again assessed through in situ
hybridization (Fig. 1C). These loss-of-function experiments led us
to mirror results to those obtained after Sema3C gain-of-function,
i.e. an upregulation of Nrp1 levels and a downregulation of Nrp2
levels at the surface of motor growth cone (Fig. 2F-I). Indeed, loss
of intrinsic Sema3C in motoneurons resulted in a significant
increase of the motoneuron population expressing intermediate
Nrp1 levels (fluorescence intensity <0.5-1.0), at the expense of the
non-expressing category (fluorescence intensity <0.5) (Fig. 2F,H).
On the contrary, loss of Sema3C in motoneurons led to a decrease
of the motoneuron population expressing high Nrp2 surface levels
in favor of the intermediate categories (fluorescence intensity 0.51.5 and 1.5-3.0) (Fig. 2G,I). Taken together, these results revealed
that motoneuronal Sema3C has a differential impact on Nrp levels
at the growth cone surface and could thus take part in the diversity
of Nrp expression levels by motor axons.
Interestingly, gain of Sema3C in motoneurons did not affect the
total Nrp1 and Nrp2 protein pools, assessed by permeabilization of
the axons prior to immunostaining (supplementary material Fig.
S1A-D). Moreover, no modification of the Nrp1 or Nrp2 isoform
patterns could be detected after a gain of motoneuronal Sema3C in
western blot and RT-PCR analysis of electroporated spinal cord
extracts (supplementary material Fig. S1E; data not shown).
Together, these data show that neuronal Sema3C does not modulate
transcription, translation or axonal transport of its receptors Nrp1
and Nrp2, but specifically acts on their availability at the growth
Development 139 (19)
cone surface. To confirm these results in a heterologous system,
neuroblastoma B103 cells were co-transfected with Nrp1, Nrp2 and
Sema3CiresGFP or EGFP constructs, surface proteins were
biotinylated and Nrp proteins were assessed in the biotinylated
fraction by western blot. As expected, Sema3C overexpression did
not affect the total protein pool but resulted in decreased Nrp1 and
increased Nrp2 levels in the cell surface fraction (Fig. 2J,K).
Sema3C induces motoneuron growth cone
collapse
As Nrp receptors are shared between Sema3 family members
(Giger et al., 2000), we investigated whether and, if so, how
Sema3C, through regulation of Nrp1 and Nrp2 levels, could impact
the sensitivity of motoneurons to Sema3C, Sema3A and Sema3F.
First, we investigated the currently unknown responsiveness of
spinal motoneurons to environmental Sema3C, which can exert
both attractive and repulsive effects depending on the cell type
(Hernández-Montiel et al., 2008; Tamariz et al., 2010). We
observed in brachial explant cultures that treatment by Sema3Cconditioned medium (Sema3CCM) induced the collapse of about
half of the motor growth cones (Fig. 3A,B; P<0.01, 2 test). Thus,
secreted Sema3C is a repulsive signal for a brachial subpopulation.
Moreover, we found that this collapse response requires both Nrp1
and Nrp2, as motoneurons displayed significantly less collapse
response to Sema3CCM after selective blocking of Nrp1 or Nrp2
with antibodies (Fig. 3C,D; P<0.001, 2 test). In addition, in these
culture explants, Sema3FCM induced a 60% collapse rate within the
motoneuron population (data not shown). Therefore, all three
studied Sema3 proteins, Sema3C, Sema3F and Sema3A (Moret et
al., 2007) display collapse properties on motor growth cones. This
culture model thus allowed us to assess the modulatory property of
motoneuronal Sema3C on the responsiveness to all three Sema3
repellents.
Intrinsic Sema3C sets motoneuronal collapse
response to environmental Sema3A, Sema3C and
Sema3F
In vitro, the collapse response consists in an on/off retraction of the
growth cone, which occurs when the signaling triggered by
uniform exposure to sufficient concentration of repellent exceeds a
threshold. In agreement with the association/dissociation
equilibrium principle, an increase in neuropilin receptor levels at
the growth cone surface should favor the association with the
Sema3 ligand. Accordingly, if the growth cone acquires an
increased sensitivity, then the concentration of ligand that is
sufficient to induce the collapse should be lower. Reciprocally,
decreasing neuropilin receptor levels may lead to a reduced
sensitivity to Sema3 repellents. To test this hypothesis, we analyzed
the percentage of collapsed growth cones in motoneuronal Sema3C
gain- or loss-of-function contexts, following Sema3 exposure. We
observed that intrinsic Sema3C overexpression strongly decreased
the percentage of collapse to Sema3A (Fig. 3A,B; P<0.01, 2 test).
Consistently, Sema3C knock down leads to a 14% increase in
collapse response after motor explants treatment with a half dose
of Sema3A supernatant (Fig. 3E; P<0.001, 2 test). Besides, our
observations that Sema3C overexpression increased Nrp2 levels
raised the possibility that the functional outcome of this regulation
could be an increase of sensitivity to Sema3F. Indeed, when the
cultures were treated with a half dose of Sema3FCM, Sema3C
overexpression resulted in significant increase in motor axon
collapse rate (Fig. 3A,B; P<0.01, 2 test). In addition, Sema3C
knock down significantly decreased the percentage of motoneuron
DEVELOPMENT
3636 RESEARCH ARTICLE
RESEARCH ARTICLE 3637
Fig. 2. Sema3C gain and loss of function in motoneurons modulate Nrp1 and Nrp2 levels at the cell surface. (A)Histogram showing the
diversity of cell-surface Nrp1 and Nrp2 immunofluorescence levels in control EGFP growth cones. (B,C,F,G) Histograms showing cell surface Nrp1
and Nrp2 immunofluorescence of non-permeabilized growth cones from control EGFP and Sema3CiresGFP explants (B,C) or Si Control and Si
Sema3C explants (F,G). Growth cones were classified in four categories, depending on the fluorescence level. Fluorescence intensities were
normalized to EGFP+ conditions. **P<0.01, ***P<0.001, with 2 test. (D,E,H,I) Representative growth cones immunostained for Nrp1 and Nrp2
under each condition. NgCAM is used as a specific marker of motoneuron axons. Scale bars: 10 m. (J,K)Detection by western blot of Nrp1 and
Nrp2 proteins in biotinylated (cell surface) and total pool fractions from B103 cells co-transfected with Nrp1, Nrp2 and Sema3CiresGFP or control
EGFP constructs.
DEVELOPMENT
Motoneuronal Sema3C and axon guidance
3638 RESEARCH ARTICLE
Development 139 (19)
Fig. 3. Intrinsic Sema3C impacts
growth cone responsiveness to
various Sema3 repellents.
(A)Morphology of Sema3CiresGFP+
growth cones labeled with phalloïdinTRITC in collapse assays with control,
Sema3C-, Sema3A- or Sema3Fconditioned medium (CM). Scale bars:
10m. (B)Histogram showing
percentages of growth cone collapse of
motoneurons electroporated (+) or non
electroporated (–) either with Ctrl EGFP
or Sema3CiresGFP constructs, and
treated with control, Sema3CCM,
Sema3ACM or Sema3FCM. The dose of
conditioned medium is indicated in
brackets. (C)Histogram showing collapse
percentages of wild-type growth cones
treated with control, Sema3CCM,
Sema3ACM or Sema3FCM following
control (–), anti Nrp1 or anti-Nrp2
antibody blocking. Nrp1 and Nrp2 are
required for Sema3C collapse response.
(D)Morphology of wild-type growth
cones labeled with phalloidin-TRITC in
collapse assays with control, Sema3CCM,
Sema3ACM or Sema3FCM in control (–),
Nrp1 (anti-Nrp1) or Nrp2 (anti-Nrp2)
blocking conditions. Scale bar: 10m.
(E)Histogram showing percentages of
growth cone collapse of motoneurons
electroporated with a control or a
Sema3C-targeting siRNA cocktail and
treated with control, Sema3ACM,
Sema3CCM or Sema3FCM. The dose of
conditioned medium is indicated in
brackets. The number of growth cones
examined in each condition is indicated
on the respective bars. **P<0.01,
***P<0.001, ns, non significant, with 2
test.
Proper intrinsic Sema3C expression is required for
the fasciculation of motoneuronal tracts
Next, we investigated how these cross-regulations between intrinsic
and environmental Sema3 proteins could impact motor axon
pathfinding in vivo. We reported previously that manipulations of
intrinsic Sema3A, impacting motoneuron sensitivity to exogenous
Sema3A, led to trajectory defects of the proximal part of
motoneuron tracts (Moret et al., 2007). Such early gross defects
were not observed after gain and loss of function of motoneuronal
Sema3C (supplementary material Fig. S2A). Nevertheless, more
detailed analyses of spinal motor tracts in whole-mount embryos
revealed that Sema3C takes part in the fasciculation of the proximal
part of spinal nerves. Indeed, gain and loss of motoneuronal
Sema3C at the brachial and thoracic levels resulted in modest but
significant increased and decreased spinal nerves caliber,
respectively (supplementary material Fig. S2B,C,E,F).
Immunofluorescence analysis against activated caspase 3 showed
that these effects were not correlated with changes in motoneuron
death (supplementary material Fig. S2D).
Motor axon tracts exhibit a remarkable
stereotyped position in the limb target
Integration of multiple guidance cues is emerging as an obligatory
mechanism for axon navigation. We hypothesized that the
modulatory properties of intrinsic Sema3C uncovered here could
allow motor axons to properly interpret complex gradients of
Sema3 repellents. The chick forelimb appeared as an attractive
biological model with which to investigate this idea. At HH24,
motor axons leave the brachial plexus to invade the limb and are
DEVELOPMENT
collapse to a full dose of Sema3F (Fig. 3E; P<0.001, 2 test).
Altogether, consistent with the modulations of Nrp levels, intrinsic
Sema3C expression oversensitizes motor growth cones to
exogenous Sema3F while desensitizing them to Sema3A.
Next, we examined the outcome of the opposite intrinsic
Sema3C-mediated modulations of Nrp1 and Nrp2 on the growth
cone sensitivity to environmental Sema3C. Indeed, motoneurons
overexpressing Sema3CiresGFP exhibited a significant 20%
decrease in their collapse response to Sema3C when compared
with control ones (Fig. 3A,B; P<0.01, 2 test). By contrast, we
detected no significant effect of Sema3C-targeting siRNA
electroporation on Sema3C-induced collapse (Fig. 3E). These
results thus reveal a complex interplay between motoneuronal
Sema3C and environmentally repulsive Sema3 proteins.
Motoneuronal Sema3C and axon guidance
RESEARCH ARTICLE 3639
sorted out according to their pool-specific identity (Swanson and
Lewis, 1982; Tsuchida et al., 1994). At HH26, LMCl motoneuron
axons have invaded the limb and project into the dorsal part of the
limb, forming the radialis profundus nerve, whereas LMCm
motoneurons, expressing intrinsic Sema3C, project their axons
ventrally and form the interosseus nerve and the median nerve
(Tsuchida et al., 1994; Swanson and Lewis, 1982). To gain insights
into the degree of stereotypy of nerve positioning in the limb, a
series of confocal images of three whole-mount HH26 NgCAMimmunolabeled chick embryos was taken to reconstruct the whole
nerve patterns of the limb. Remarkably, as illustrated by the perfect
matching of all three median and interosseus nerves projecting
ventrally, the positioning of motor branches showed a high degree
of fidelity, with very low inter-individual variations (Fig. 4A,B).
Next, using in situ hybridization on whole-mounted and crosssectioned embryos, we observed that Sema3C is expressed by the
central limb mesenchyme, whereas Sema3A and Sema3F are
expressed by the peripheral one (Fig. 4C,D). This then highlighted
the existence of complex expression of Sema3 proteins in the limb
mass (Fig. 4C,D).
Sema3C expression by LMCm motoneurons
contributes to specify the fine positioning of
spinal nerves in the limb
Having characterized an appropriate in vivo context for our
purpose, we then asked whether Sema3C expression by LMCm
motoneurons modulates their reading of the Sema3 combinations
and properly positions their tracts in the limb. We herein took
advantage of the chick model that enables the manipulation of only
one side of the neural tube and superimposed left and right limb
innervations (Fig. 5A). In Si Control electroporated embryos, we
could observe that ventral and dorsal innervation patterns of the left
and right forelimbs were perfectly symmetrical, thus confirming
the highly stereotyped forelimb innervation pattern (Fig. 5B;
supplementary material Movie 1). By sharp contrast, in the Si
Sema3C electroporated embryos, although the radialis profundus
nerves (dorsal) arising from Sema3C– LMCl could be normally
superimposed, important mismatches were detected for the median
and interosseus nerves (ventral), which arise from the LMCm pool
in which Sema3C was knocked down (Fig. 5B; supplementary
material Movie 2). The electroporation level of each embryo was
assessed by co-electroporation of EGFP with siRNA cocktails
(supplementary material Fig. S3A). We then classified Si Control
and Si Sema3C embryos for the amplitude of innervation mismatch
between control and electroporated sides. Significantly higher
levels of mismatch were found in the Si Sema3C condition for the
trajectories of the median and interosseus (ventral) nerves (Fig.
5C,D; supplementary material Fig. S2B; P<0.01, Mann Whitney
test). By contrast, the trajectory of the nerve radialis profundus
(dorsal) was found to be unaffected by loss of motoneuronal
Sema3C (Fig. 5C,E; supplementary material Fig. S2B, ‘ns’ Mann
Whitney test). Finally, overexpressing Sema3C did not impact the
stereotypy of the limb nerve patterns, suggesting that LMCl
motoneurons, which do not endogenously express Sema3C, are not
responsive to Sema3C gain of function (Fig. 5C).
Altogether, our results then demonstrate that Sema3C takes part
in the developmental program by which motoneurons set a proper
sensitivity to a combination of target-derived Sema3 repellents,
which controls the stereotyped positioning of ventral motor nerves
in the limb (Fig. 6).
DISCUSSION
Our study reveals that motoneuronal Sema3C balances the
respective strength of Sema3 repellents through the control of Nrp
growth cone surface level. Altering motoneuronal Sema3C levels
results in defective positioning of LMCm motor tracts in the limb,
DEVELOPMENT
Fig. 4. The chick forelimb displays a stereotyped
innervation pattern. (A)Procedure to obtain a
composite image of the limb innervation from three
different embryos. (B)Confocal stack projections of
NgCam-labeled forelimb innervation from three chick
embryos. The composite image shows the highly
stereotyped projection pattern of motor tracts in the
limb. Scale bar: 100m. (C)In situ hybridization of
Sema3C, Sema3A and Sema3F in chick whole-mounted
forelimb (in blue) and limb cryosections (in gray).
Dorsoventral, mediolateral and anteroposterior
orientations are indicated on the upper panels. Scale
bars: 100m (cryosections); 500m (whole mounts).
(D)Schematic representation of environmental
semaphorin expression pattern in the chick forelimb on
NgCam-labeled HH26 chick cryosections.
3640 RESEARCH ARTICLE
Development 139 (19)
where Sema3 repellents form complex combinations, thus
demonstrating that crosstalk between intrinsic and extrinsic Sema3
proteins regulates the positioning of motor axon tracts in their
target field (Fig. 6).
Remarkable fidelity of motor axon tract position
in the limb
The establishment of precise connections between motor pools and
individual muscles relies on a series of guidance decisions leading
motor axons to segregate from each other at choice points along
their navigation. Likewise, more than 30 different limb muscles are
contacted by motor axon subsets. The tract arising from the LMC
motor column and reaching the limb first separates into dorsal
LMCl and ventral LMCm tracts. Next, several branches
individualize from the main tract, navigating in the limb width to
contact single muscles. Studies in the field essentially concentrated
on the primary LMCl/LMCm dorsoventral choice and established
that it is specified by a combination of guidance cues, including
ephrins, semaphorins and the trophic factor GDNF (Eberhart et al.,
2000; Huber et al., 2005; Luria et al., 2008; Dudanova et al., 2010).
By contrast, very little is known about the subsequent guidance
choices that prepare motoneuron pools for the specific innervation
of their target muscles. Our 3D reconstruction of limb innervation
patterns revealed a remarkable fidelity of this terminal guidance
process, which demonstrates that robust mechanisms exist to set
the accurate positioning of spinal tracts in limb quadrants, thus
achieving the fine-tuning of motor axon connectivity.
Contribution of semaphorin/neuropilin coexpression in the terminal steps of motor axon
guidance
We provide evidence that the control of growth cone sensitivity to
environmental semaphorins by motoneuronal semaphorin/
neuropilin co-expression is part of the developmental program that
DEVELOPMENT
Fig. 5. Intrinsic Sema3C takes part in the
stereotyped projection pattern of the chick
forelimb. (A)Procedure to obtain composite images of
the limb innervation from electroporated and control
sides of an embryo. (B)Superimposed 3D reconstructions
of left (magenta) and right (cyan) forelimb innervations
from embryos electroporated with Si control or Si
Sema3C. The complete limb innervation (limb tract), the
ventral tracts or the dorsal tracts are displayed.
Downregulation of Sema3C expression in LMCm
motoneurons shifts the position of the LMCm-derived
ventral tracts (yellow arrow). (C)Higher magnification of
confocal stack projections displaying the ventral or dorsal
forelimb tracts in Si control, Si Sema3C or Sema3CiresGFP
conditions. Downregulation of intrinsic Sema3C leads to
altered projection patterns of the ventral median and
interosseus nerves in the forelimb (yellow arrows). Scale
bars: 100m. (D,E)Graphs comparing the amplitude of
ventral tract (D) or dorsal tract (E) mismatches between Si
control and Si Sema3C embryos. Eleven Si control and
nine Si Sema3C embryos were classified according to the
level of mismatch observed in the innervation
superimposition of the control and electroporated sides.
The scale goes from 1 for the best to 20 for the most
severe mismatch. Ranks are reported on the graphs. The
red lines indicate the average rank in each condition.
**P<0.01, ns, non significant with Mann-Whitney test.
Motoneuronal Sema3C and axon guidance
RESEARCH ARTICLE 3641
Fig. 6. Model of the interplay between intrinsic and environmental Sema3 proteins expressions for the establishment of the highly
stereotyped motoneuron projection pattern in the forelimb. Expression of Sema3C in LMCm motoneurons (green) decreases Nrp1 availability
and increases Nrp2 on LMCm-derived growth cones, modulating their sensitivity to Sema3A, Sema3C and Sema3F as a result. This can be seen by
looking at combined Sema3 gradients in the forelimb. This modulation contributes to the fine positioning of LMCm nerve tracts in the ventral
forelimb, as reflected by their shift in the Sema3C knock down condition (double-headed arrows).
sensitivities to environment-derived Sema3 proteins, such a Sema
code has the potential to confer pool-specific guidance responses.
Consistently, our analysis reveals a striking diversity of Nrp levels
at the motor growth cone surface. Such a diversity, which is likely
to mirror a wide range of axon sensitivity, could be set by the
motoneuronal Sema code in order to allow terminal navigation.
Emerging growth cone properties for Sema3C/
Nrp1/Nrp2 co-expression
Together with other recent work, our data point out the versatility
of ligand/receptor co-expression in axon guidance signaling.
Ephrin/Eph, Sema3A/Nrp1, Sema6A/PlexinA4 co-expression all
inhibited axon responsiveness to the exogenous ligand (Carvalho
et al., 2006; Hornberger et al., 1999; Haklai-Topper et al., 2010).
Such modulations are essential during motor axon navigation.
Likewise, EphA cis-attenuation in LMCm neurons and EphB cisattenuation in LMCl neurons ensure proper dorsoventral
segregation of motor axons in the limb (Kao and Kania, 2011).
Sema3A/Nrp1 regulates motor axon pathfinding in peripheral
tissues (Moret et al., 2007). We show here that the effect of
motoneuronal Sema3C is not limited to its environmental
counterpart, as it also influenced the sensitivity to Sema3A and
Sema3F. By showing that motoneuronal Sema3C upregulates
growth cone responsiveness to Sema3F, our work provides the first
example of ligand/receptor co-expression with an oversensitization
outcome. Intrinsic Sema3C has even dual effects, as it also
decreased the sensitivity to other Sema3 proteins (Sema3A). This
study also highlights precise regulatory potentialities of intrinsic
Sema3C. We showed that Sema3C overexpression reduced
motoneuron collapse response to Sema3C exposure, consistent
with a decreased sensitivity of their growth cones. However,
Sema3C knock down did not significantly alter the global collapse
response to Sema3C. This may reflect a heterogeneous situation at
the cell scale. Consistent with previous studies (Chen et al., 1998;
Takahashi et al., 1998), we showed that collapsing effects of
Sema3C on motor axons require both Nrp1 and Nrp2 receptor
activity. Opposing regulation of Nrp1 and Nrp2 level by intrinsic
Sema3C could possibly either positively or negatively impact the
amount of Nrp1/Nrp2 heterodimers, depending on the respective
initial levels of neuropilin receptors in the cell. Therefore, in the
DEVELOPMENT
controls terminal motor axon guidance. The regulatory mechanism
described here has the potential to create subtle differences of
guidance responses between motor axons subsets, thus
individualizing them within a common environment. First,
motoneuronal Sema3 proteins show different modulatory effects on
motor growth cone responsiveness. We have previously observed
that motoneuronal Sema3A sets the sensitivity to Nrp1-dependent
Sema3A by regulating cell surface levels of Nrp1 (Moret et al.,
2007). Here, we demonstrate that, by contrast, motoneuronal
Sema3C modulates both Nrp1 and Nrp2 levels, and thereby
responsiveness to both Nrp1- and Nrp2-dependant Sema3 proteins.
As a result, regulation of receptor availability by motoneuronal
Sema3 proteins can have a wide range of functional outcomes.
Second, Sema3 members have dynamic and specific expression
in motoneurons subpopulations. For example, Sema3C is expressed
in LMCm but not in LMCl motoneurons.
Third, the effects of motoneuronal Sema3 proteins are confined
to restricted subpopulations. Our in vitro analysis show that Nrp1
is exposed at the surface of all motor growth cones, but Nrp2 on
only half of them. Modulation of Nrp2 by Sema3C was limited to
the population that did expose Nrp2 at the surface. The proportion
of axons that do not expose Nrp2 at their surface was not changed
by Sema3C overexpression or knock down. Correlatively,
alteration of Sema3C expression in motoneurons affected only
motor axon positioning of LMCm population that endogenously
expresses it. Ectopic Sema3C expression in LMCl motoneurons
which normally do not express it had no impact on their projection.
LMCl motoneurons that are insensitive to Sema3C regulation may
therefore represent the population with undetectable Nrp2 surface
expression in the explant cultures. Overall, our results are
consistent with a population-specific effect of motoneuronal Sema3
proteins in motor axon guidance. This diversity of motoneuronal
Sema effects has to be considered in the light of previous report of
Sema3 expression in motoneurons. Interestingly, a complex and
intriguing Sema3 code with specific semaphorin combinations in
restricted motoneuron pools has been reported (Moret et al., 2007;
Cohen et al., 2005). We could hypothesize that motoneuronal
Sema3 proteins other than Sema3A and Sema3C also differentially
set responsiveness to environmental Sema3 proteins gradients.
Therefore, by defining the specific cell-surface Nrp levels and
population of motoneurons, intrinsic Sema3C expression could
contribute to generate diversity of growth cone responsiveness to
environmental Sema3C.
The outcome of such multiple functional potentialities of
intrinsic Sema3C on axon tract positioning by exogenous Sema3
sources is likely to depend on the localization of these sources and
their relative repulsive strength on growth cones. The in vivo
consequences of Sema3C overexpression and knock down on the
guidance of motor axon tracts in the forelimb may reflect such
complexity. We showed that Sema3C overexpression did not alter
LMCm axon trajectory in the ventral forelimb. In vitro, it modified
motor growth cone responses, with a more or less pronounced
effect depending on the Sema. Together, it suggests that the
outcome of strong desensitization to Sema3A and Sema3C
combined to the moderate oversensitization to Sema3F is null in
the ventral forelimb (supplementary material Fig. S4). By contrast,
the inward deviation of the LMCm motor tract following intrinsic
Sema3C knock down reflects that the moderate desensitization to
Sema3F combined with the oversensitization to Sema3A observed
in vitro potentiates the repulsive force exerted by the ventral
forelimb periphery (supplementary material Fig. S4).
Our finding that Sema3C intrinsic effects rely on the regulation
of Nrp1 and Nrp2 raises an even broader potential for Sema3C to
regulate the responsiveness to other Nrp ligands, such as VEGF
(Neufeld et al., 2002; Erskine et al., 2011). Until now, the
mechanisms underlying the integration of multiple extracellular
guidance signals are poorly known and Sema3C thus appears as an
interesting example of how a neuron-derived factor can participate
in the program integrating guidance information by the growth
cone.
The mechanisms underlying the regulation by co-expressed
ligand/receptor pairs appear to differ. Ephrin/Eph and
Sema6A/PlexinA4 ligand-receptor cis-interactions were shown to
mask receptors to prevent trans-binding with the ligand and/or to
inhibit the receptor phosphorylation, thus suppressing downstream
signaling. In both cases, the levels of cell surface receptor were not
affected (Haklai-Topper et al., 2010; Carvalho et al., 2006). By
contrast, our biochemical analysis of the biotinylated cell surface
fraction of Nrp proteins indicates that the modulation of Nrp
availability relies on the proteins level at the plasma membrane.
Moreover, in neither cell lines nor motor growth cones was the
total pool of Nrp proteins found to be altered by Sema3C
misexpression, indicating that this regulation may rely on
trafficking of plasma membrane and intracellular receptor pools.
Interesting issues will be to decipher the currently unknown
molecular pathways that regulate the trafficking of Nrp receptors,
and their regulation by Semaphorin/Nrp co-expression. Finally,
multiple functional properties could emerge from ligand/receptor
co-expression. Interesting issues will also be to assess whether
cross-regulation of semaphorin signaling influences a wider range
of axonal properties, such as inter-axonal communications, shortterm desensitization (Piper et al., 2005) and adaptation to
exogenous ligand sources (Ming et al., 2002). In addition to their
role in axon guidance, Sema3 proteins and neuropilins are involved
in diverse developmental processes, such as angiogenesis, and in
pathological contexts, such as cancer. Sema3/neuropilin coexpression has been widely noted, suggesting that the regulatory
pathway uncovered here could have broader implications.
Acknowledgements
We thank J. Raper for the kind gift of Sema3A and chick Nrp1 constructs, A
Püschel for the Nrp2 construct and J. Falk for his helpful comments on this
work.
Development 139 (19)
Funding
This work was supported by the Agence National de la Recherche (ANR) CNS
program, by Fondation pour la Recherche Médicale (FRM labeled team), by
Association Française contre les Myopaties (AFM) and by PhD fellowships from
Ministere de l’Education Nationale de la Recherche et de Technologie (MENRT)
and FRM.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.080051/-/DC1
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DEVELOPMENT
Motoneuronal Sema3C and axon guidance