INTRODUCTIONMechanisms underlying the wiring of neuronal connectivity remainpuzzling with regard to the limited repertoire of guidance cues usedto achieve the tremendous diversity of axon pathways. Tightspatiotemporal regulation of ligands in axon environment andreceptors in specific neuronal populations has been proven to becrucial to generate different axon trajectories (Yu and Bargmann,2001). In addition, modulation of axon responsiveness andreiterated use of guidance factors at successive steps of axonnavigation allow the diversification of pathway choices with arelatively small number of factors.
Beyond their function in generating permissive corridors fornavigating axon tracts, gradients of guidance cues also playinstrumental roles in the formation of topographic maps, organizingaccurate trajectories of multiple sets of axons in their target field.For example, in the retinotectal and olfactory systems, distinctlevels of sensitivity control axon positioning within gradients (Imaiet al., 2009). The mechanisms setting the sensitivity to guidancecues remain poorly explored. Class 3 secreted semaphorins (Sema3proteins) are a group of seven widely expressed chemotropicfactors, from Sema3A to Sema3G, with repulsive and attractiveactivities (Raper, 2000). Loss-of-function approaches in mice haveprovided evidence for important roles of Sema3 proteins and their
receptors, the neuropilins (Nrp proteins), in patterning neuronalconnections in various regions of developing organisms (Raper,2000). Likewise, in the motor system, the fasciculation andtrajectory of spinal motor axons that express Nrp proteins arelargely shaped by the repulsive effects of Sema3 proteins providedby 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; Varela-Echavarría et al., 1997). Intriguingly, mRNAs encoding bothreceptors and Sema3 proteins have been also detected inmotoneuron cell bodies by the time their axons elongate in theperiphery and innervate their targets (Luo et al., 1995; Püschel etal., 1995; Moret et al., 2007; Chilton and Guthrie, 2003; Meléndez-Herrera and Varela-Echavarría, 2006; Cohen et al., 2005).
Ligand/receptor co-expression emerges from recent work as akey mechanism by which growing axons lower their sensitivity toexogenous sources of the corresponding ligand (Haklai-Topper etal., 2010; Carvalho et al., 2006; Kao and Kania, 2011). Whetherregulations across ligand family members occur and, if so, whetherthey drive a diversity of emerging growth cone properties is not yetfully known. In a previous study, we brought to light an originalmechanism by which Sema3A expression in motoneurons controlsthe availability of its receptor Nrp1 at the growth cone surface, thussetting the sensitivity of their growth cone to exogenous Sema3A(Moret et al., 2007). These findings opened several issues,particularly concerning the potential contribution of othermotoneuronal Sema3 proteins and their implication during axonnavigation. We addressed these issues by investigating anotherSema3 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
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
SUMMARYThe wiring of neuronal circuits requires complex mechanisms to guide axon subsets to their specific target with high precision. Toovercome 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 guidancecues 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 aredynamically expressed, forming a code in motoneuron subpopulations, whereas their receptors, the neuropilins, are expressed inmost of them. Targeted gain- and loss-of-function approaches in the chick neural tube were performed to enable selectivemanipulation of Sema3C expression in motoneurons. We show that motoneuronal Sema3C regulates the shared Sema3 neuropilinreceptors Nrp1 and Nrp2 levels in opposite ways at the growth cone surface. This sets the respective responsiveness to exogenousNrp1- and Nrp2-dependent Sema3A, Sema3F and Sema3C repellents. Moreover, in vivo analysis revealed a context where thismodulation is essential. Motor axons innervating the forelimb muscles are exposed to combined expressions of semaphorins. Weshow first that the positioning of spinal nerves is highly stereotyped and second that it is compromised by alteration ofmotoneuronal Sema3C. Thus, the role of the motoneuronal Sema3 code could be to set population-specific axon sensitivity to limb-derived chemotropic Sema3 proteins, therefore specifying stereotyped motor nerve trajectories in their target field.
Motoneuronal Sema3C is essential for setting stereotypedmotor tract positioning in limb-derived chemotropicsemaphorinsIsabelle Sanyas, Muriel Bozon, Frédéric Moret* and Valérie Castellani*,‡
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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 influencingthe orientation of cardiac neural crest cell migration (Bagnard etal., 1998; Niquille et al., 2009; Hernández-Montiel et al., 2008).We report here novel properties of motoneuronal Sema3C on motoraxon responsiveness to environmental Sema3 proteins. Wedemonstrated by targeted manipulations in chick spinalmotoneurons that intrinsic Sema3C modulates in opposite ways theavailability of the two Sema3 receptors, Nrp1 and Nrp2, at thegrowth cone surface. Interestingly, we show that this confers toSema3C the ability to modulate axon sensitivity to different Nrp1-and Nrp2-dependent Sema3 repellents. In addition, our in vivoanalyses reveal a highly stereotyped pattern of spinal nervepositioning in the forelimb, where combinations of these Sema3repellents are detected. Finally, we uncover a key contribution ofmotoneuronal Sema3C in setting the position of LMCm-derivedventral motor tracts in the forelimb.
MATERIALS AND METHODSPlasmid constructionThe Sema3CiresEGFP bicistronic construct was derived from the pAG-NT-coll1 plasmid [kindly provided by J. Raper (Feiner et al., 1997)]. IresEGFPfragment was amplified from ires2EGFP plasmid (Clontech, MountainView, USA) by PCR between suitable primers, then inserted at the XhoIsite 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 foursiRNAs with the following sequences: 5�-GGAACGGACTACAAGTATA-3�, 5�-GGTTAAGCTGAGTGAGAGA-3�, 5�-GCACGCGGTGTTTG -GGATA-3� and 5�-GCCATTAATCATCCGGATA-3�. As a control, theON-TARGETplus Non-Targeting Pool from ABgene was used, containingfour siRNA with the following sequences: 5�-UGGUUUACAUGU -CGACUAA-3�, 5�-UGGUUUACAUGUUGUGUGA-3�, 5�-AGGUUU -ACAUGUUUUCUGA-3� and 5�-UGGUUUACAUGUUUUCCUA-3�,with no robust similarities to chick mRNA.
In ovo electroporationIn 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 atbrachial and thoracic levels to stage HH14-15 embryos (Hamburger andHamilton, 1992). Plasmids prepared with EndoFree maxiprep Xtra kit(Macherey-Nagel, Düren, Germany) were routinely diluted at 1.5 g/l inPBS for Sema overexpression and at 0.5 g/l in PBS for the EGFP-N3vector. siRNA cocktails were diluted at 2 g/l in PBS.
Explant cultureThe ventral one-third of spinal cords was dissected out from HH24 chickembryos at brachial level in DMEM (GIBCO). Explants were cultured onglass coverslips precoated with laminin (50 g/ml, Sigma) and poly-ornithin (10g/ml, Sigma), and grown in a previously described medium(Marthiens et al., 2005). Immunolabeling using a motoneuron axonalmarker NgCAM confirmed that fibers extending from these explants weremotoneuron axons.
Histological analysesCryosections (20 m) were obtained from embryos fixed in 4%paraformaldehyde and embedded in 7.5% gelatine/15% sucrose. In situhybridizations and whole-mount immunostaining were performed asdescribed previously (Henrique et al., 1995; Moret et al., 2007). In orderto identify the Sema3C-expressing motoneuronal pool, Sema3C in situhybridization and Isl1, FoxP1 and Lim3 immunostainings were performedon adjacent sections, as Isl1, FoxP1 and Lim3 epitopes were found to bealtered by in situ hybridization protocols. Images of Sema3C in situhybridization were changed into negative images and superimposed on
immunostaining images for the aforementioned motoneuronal markers. Forimmunostaining, cryosections, whole-mount embryos or explants wereincubated with the following antibodies diluted in 2% bovine serumalbumin 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, SanFrancisco, USA), rabbit anti-cleaved caspase 3 (1/100; Asp175; CellSignaling, Danvers, USA), rabbit anti-FoxP1 (1/50; AbCam, Cambridge,UK) and rabbit anti-GFP (1/200; Molecular Probes, Eugene, USA). Mouseanti-NgCAM (1/400, 8D9 developed by Dr Vance Lemmon), mouse anti-islet1/2 (1/50; 39.4D5 developed by Dr Thomas Jessell, ColumbiaUniversity, NY, USA), mouse anti-islet1 (1/50; 39.3F7 developed by DrThomas Jessell) and mouse anti-Lim3 (1/50; 67.4E12 developed by DrThomas Jessell) were obtained from the Developmental StudiesHybridoma Bank developed under the auspices of the NIHCD andmaintained by the University of Iowa (Department of Biological Sciences,Iowa City, USA).
For chromogenic immunostaining, suitable biotinylated antibodies, thenthe ABC complex (Vectastain), were used prior to DAB staining (Vector,Paris, France). For immunofluorescence staining, sections were incubatedwith appropriate Alexa 555 or Alexa 488 antibodies (1/500; MolecularProbes) or Fluoprobes 547 antibodies (1/200).
Nrp1, Nrp2 and NgCAM immunofluorescent labeling on explants non-permeabilized or permeabilized with 0.02% Triton X100, were detectedwith combined suitable Alexa 555 (1/500) and biotinylated antibodies(1/50). Explants were subsequently incubated with Alexa350-streptavidin(1/50; Molecular Probes).
Collapse assayConditioned media were obtained by transfection of MYC-taggedSema3A, Sema3C or Sema3F, as well as control constructs in humanembryonic 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 Semasupernatants for 45 minutes at 37°C, and fixed in 4%paraformaldehyde/0.5% sucrose. Individual axons were randomly selectedthrough NgCam or phalloidin TRITC (Sigma) fluorescent labeling andtheir morphology examined at 40� magnification as described by Falk etal. (Falk et al., 2005). Analyses were performed from a minimum of threeindependent experiments. For antibody blocking experiments, blockinggoat anti-neuropilin 1 (25 g/ml; R&D Systems, Minneapolis, USA),rabbit anti-neuropilin 2 (10 g/ml; Zymed, San Francisco, USA) antibodieswere applied 1 hour prior to the application of conditioned medium.
Analysis of cell surface protein fraction and western blotsFor cell surface protein fraction analysis, B103 cell lines were co-transfected with chick neuropilin 1 (kindly provided by J. Raper, Universityof Pennsylvania, School of Medicine, Philadelphia, USA) or mouseneuropilin 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 surfaceprotein isolation kit (Thermo Scientific, Waltham, USA). Surface and totalprotein 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 aperoxidase rabbit anti-goat (1/5000; Sigma, St Louis, USA) secondaryantibody. Results were observed in four independent experiments.
For total protein level analysis, spinal cord extracts were obtained bydissection of ventral spinal cords from embryos electroporated withSema3CiresGFP or EGFP constructs. Tissues lysates were then analyzedby 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 widthLabeling was acquired under an Axiovert microscope (Zeiss, Germany)equipped with a Coolsnap CCD camera (Photometrics, Evry, France) or a
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LSM510 Meta Confocal Microscope (Zeiss). Fluorescence quantificationswere analyzed with the ImageJ software (National Institutes of Health,USA). For quantification of the spinal nerve thickness, NgCAMimmunofluorescence was performed on whole-mount chick embryos.Images were captured at 10� magnification in the brachial and thoracicregion. Spinal nerve outline was traced on NgCAM labeling and its widthmeasured. Normalized width reported on the histogram was defined as thequotient of motor tract widths between electroporated and non-electroporated sides.
Three-dimensional reconstructions and quantification of the limbinnervation patternThe analysis of the limb innervation phenotype was obtained on NgCam-labeled whole-mount embryos. Each control and electroporated limb wasfully scanned with a confocal microscope. Projection patterns from thecontrol and electroporated sides were then reconstructed in threedimensions. The mirror stack of the control side was obtained. Control andelectroporated sides were then superimposed with the ImageJ 3D viewerplug-in. Pseudocolors were affected to these projection patterns (controlside, magenta; electroporated side, cyan).
For quantification, confocal stacks were analyzed by a three-stepprocess. First, the scanned innervation pattern for each limb was projectedto obtain, for each limb, separate reconstitutions of dorsal and ventraltracts. Then pseudocolors were applied to these projection patterns (controlside, magenta; electroporated side, cyan) and the mirror image of thecontrol side was obtained. Finally, the control and electroporated patternswere superimposed for each embryo in order to form a composite image.For the quantifications presented in the diagrams, embryo classificationwas established after visual assessment of the dorsal or ventral tractsuperimposition: 1 corresponds to the best superimposition and 20 to themost severe mismatches of the motor tracts in the composite image. Threeindependent blind classifications were performed in order to obtain thefinal average rank for each embryo. The ranks of embryos were comparedbetween Si control and Si Sema3C conditions by a Mann-Whitneystatistical test.
Quantifications of Nrp levelsFor quantification of growth cone fluorescence, images were captured at40� magnification with constant exposure parameters below pixelsaturation. Growth cone outline was traced with NgCAM staining, whichclearly delineated filopodia and lamellipodia in both control andexperimental conditions. After background subtraction, the intensity ofneuropilin and NgCAM staining was measured within the outline and themean intensity per pixel was calculated. Intensity values were normalizedto the respective control experiments conditions: 1 corresponds to the meanlevel of neuropilins in the control conditions. Growth cones were thenclassified according to the fluorescence intensity.
Statistical analysisStatistical analyses were carried out using the 2 test for collapse assaysand Nrp cell surface quantification; and the Mann-Whitney test was usedfor immunofluorescence quantifications and nerve tract phenotype analysis.
RESULTSSpecific and dynamic Sema3C expression in thechick spinal motor columnsTo define the motoneuron subpopulations expressing Sema3C, in situhybridization was performed on chick transverse sections and theSema3C expression pattern was compared with the immunolabelingof specific Lim3, Isl1 and FoxP1 motoneuron markers from adjacentsections. Sema3C was found already expressed in motoneurons atHH24, when motor axons navigate towards their targets (Fig. 1A).At brachial level, Sema3C+ neurons are found among theLim3+/Isl1low/FoxP1– motoneuron sub-column, the medial MedialMotor Column (MMCm), projecting between the dorsal rootganglion (DRG) and the dermamyotome (Fig. 1A). Sema3C is alsoexpressed in the Lim3–/Isl1high/FoxP1+ population corresponding to
the medial part of the Lateral Motor Column (LMCm) whichprojects 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 limbmesenchyme, Sema3C expression is detected, in the brachial region,in the Lim3+/Isl1low/FoxP1– MMCm subcolumn and in theLim3–/FoxP1+ LMC population, particularly in a subset of theLim3–/Isl1high/FoxP1+ LMCm subcolumn where strong Sema3Cexpression is observed (Fig. 1B). In the thoracic region, highSema3C expression is present in the MMCm (Fig. 1B). Weakexpression is also detectable in the MMCl subcolumn (Fig. 1B).Therefore, Sema3C is dynamically expressed by motoneurons withrestricted and subcolumn-specific expression at the brachial level,suggesting a potential role during LMCm axon navigation.
Motoneuronal Sema3C modulates Nrp1 and Nrp2availability at the growth cone surfaceOur previous work (Moret et al., 2007) showed that intrinsicSema3A sets motoneuron sensitivity to exogenous Sema3A byregulating, at post-transcriptional level, the availability of its Nrp1
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Fig. 1. Sema3C expression patterns in motoneuronalsubpopulations. (A,B)In situ hybridization and fluorescentimmunostaining against Sema3C, Lim3, Isl1 and FoxP1 on adjacentcryosections of HH24 (A) and HH26/27 (B) chick spinal cord at brachialand thoracic levels. Scale bars: 50m. (C)In situ hybridization of chickcryosections for Sema3C at stage HH24. Electroporation of Sema3CsiRNA cocktail significantly downregulates Sema3C expression inmotoneurons (red arrow). Sema3CiresGFP electroporation leads toSema3C overexpression in the whole spinal cord, including inmotoneurons (red arrow). Green lines define the electroporated side ofthe spinal cord. Scale bars: 100m.
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receptor at the growth cone surface. We therefore asked whethermotoneuronal Sema3C modulates its receptor expression. Sema3Chas been shown to bind to both Nrp1 and Nrp2. Its guidanceactivities are thought to be mediated by Nrp1/Nrp2 heterodimers orby Nrp2/Nrp2 homodimers (Chen et al., 1998; Takahashi et al.,1998). Nrp1 was found expressed in all spinal motoneurons andNrp2 was predominantly expressed in LMCl but also in LMCmmotoneurons (Mauti et al., 2006; Moret et al., 2007). We performedtargeted Sema3C gain- and loss-of-function experiments in the chickspinal cord and investigated in explant cultures modulations of Nrp1and Nrp2 levels in motor growth cones using immunocytochemistry.Unilateral overexpression of intrinsic Sema3C was obtained byelectroporation of Sema3CiresGFP in the chick spinal cord (Fig. 1C).An EGFP plasmid was used as a control for these experiments. Theefficiency of Sema3C overexpression was checked by in situhybridization as no efficient antibody against chick Sema3C isavailable (Fig. 1C). High levels of Nrp1 and Nrp2 were observed inmost motor axons after permeabilization (supplementary materialFig. S1A,B,C,D). Nrp1 cell surface labeling was detected in allmotor axons. By contrast, 50% of motor axons lacked Nrp2 cellsurface staining. Moreover, different levels of Nrp1 and Nrp2 levelswere observed between axons from the same explant, suggesting thatregulatory mechanisms generate a diversity of neuropilin levels atthe surface of motor growth cones (Fig. 2A). Motor growth conescould then be classified into different categories from null/low cellsurface 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 theincrease of the low Nrp1 one (Fig. 2B,D). Surprisingly oppositeeffects were found for Nrp2, as the percentage of high-Nrp2+ motoraxons was increased following Sema3C overexpression (Fig. 2C,E).
Next, we performed loss-of-function experiments through theelectroporation of a custom cocktail of four siRNAs directedagainst chick Sema3C. EGFP expression vector wassimultaneously co-electroporated to monitor electroporated cells.The knock down efficiency was again assessed through in situhybridization (Fig. 1C). These loss-of-function experiments led usto mirror results to those obtained after Sema3C gain-of-function,i.e. an upregulation of Nrp1 levels and a downregulation of Nrp2levels at the surface of motor growth cone (Fig. 2F-I). Indeed, lossof intrinsic Sema3C in motoneurons resulted in a significantincrease of the motoneuron population expressing intermediateNrp1 levels (fluorescence intensity <0.5-1.0), at the expense of thenon-expressing category (fluorescence intensity <0.5) (Fig. 2F,H).On the contrary, loss of Sema3C in motoneurons led to a decreaseof the motoneuron population expressing high Nrp2 surface levelsin favor of the intermediate categories (fluorescence intensity 0.5-1.5 and 1.5-3.0) (Fig. 2G,I). Taken together, these results revealedthat motoneuronal Sema3C has a differential impact on Nrp levelsat the growth cone surface and could thus take part in the diversityof Nrp expression levels by motor axons.
Interestingly, gain of Sema3C in motoneurons did not affect thetotal Nrp1 and Nrp2 protein pools, assessed by permeabilization ofthe axons prior to immunostaining (supplementary material Fig.S1A-D). Moreover, no modification of the Nrp1 or Nrp2 isoformpatterns could be detected after a gain of motoneuronal Sema3C inwestern blot and RT-PCR analysis of electroporated spinal cordextracts (supplementary material Fig. S1E; data not shown).Together, these data show that neuronal Sema3C does not modulatetranscription, translation or axonal transport of its receptors Nrp1and Nrp2, but specifically acts on their availability at the growth
cone surface. To confirm these results in a heterologous system,neuroblastoma B103 cells were co-transfected with Nrp1, Nrp2 andSema3CiresGFP or EGFP constructs, surface proteins werebiotinylated and Nrp proteins were assessed in the biotinylatedfraction by western blot. As expected, Sema3C overexpression didnot affect the total protein pool but resulted in decreased Nrp1 andincreased Nrp2 levels in the cell surface fraction (Fig. 2J,K).
Sema3C induces motoneuron growth conecollapseAs Nrp receptors are shared between Sema3 family members(Giger et al., 2000), we investigated whether and, if so, howSema3C, through regulation of Nrp1 and Nrp2 levels, could impactthe sensitivity of motoneurons to Sema3C, Sema3A and Sema3F.First, we investigated the currently unknown responsiveness ofspinal motoneurons to environmental Sema3C, which can exertboth attractive and repulsive effects depending on the cell type(Hernández-Montiel et al., 2008; Tamariz et al., 2010). Weobserved in brachial explant cultures that treatment by Sema3C-conditioned medium (Sema3CCM) induced the collapse of abouthalf 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 Nrp1and Nrp2, as motoneurons displayed significantly less collapseresponse to Sema3CCM after selective blocking of Nrp1 or Nrp2with antibodies (Fig. 3C,D; P<0.001, 2 test). In addition, in theseculture explants, Sema3FCM induced a 60% collapse rate within themotoneuron population (data not shown). Therefore, all threestudied Sema3 proteins, Sema3C, Sema3F and Sema3A (Moret etal., 2007) display collapse properties on motor growth cones. Thisculture model thus allowed us to assess the modulatory property ofmotoneuronal Sema3C on the responsiveness to all three Sema3repellents.
Intrinsic Sema3C sets motoneuronal collapseresponse to environmental Sema3A, Sema3C andSema3FIn vitro, the collapse response consists in an on/off retraction of thegrowth cone, which occurs when the signaling triggered byuniform exposure to sufficient concentration of repellent exceeds athreshold. In agreement with the association/dissociationequilibrium principle, an increase in neuropilin receptor levels atthe growth cone surface should favor the association with theSema3 ligand. Accordingly, if the growth cone acquires anincreased sensitivity, then the concentration of ligand that issufficient to induce the collapse should be lower. Reciprocally,decreasing neuropilin receptor levels may lead to a reducedsensitivity to Sema3 repellents. To test this hypothesis, we analyzedthe percentage of collapsed growth cones in motoneuronal Sema3Cgain- or loss-of-function contexts, following Sema3 exposure. Weobserved that intrinsic Sema3C overexpression strongly decreasedthe percentage of collapse to Sema3A (Fig. 3A,B; P<0.01, 2 test).Consistently, Sema3C knock down leads to a 14% increase incollapse response after motor explants treatment with a half doseof Sema3A supernatant (Fig. 3E; P<0.001, 2 test). Besides, ourobservations that Sema3C overexpression increased Nrp2 levelsraised the possibility that the functional outcome of this regulationcould be an increase of sensitivity to Sema3F. Indeed, when thecultures were treated with a half dose of Sema3FCM, Sema3Coverexpression resulted in significant increase in motor axoncollapse rate (Fig. 3A,B; P<0.01, 2 test). In addition, Sema3Cknock down significantly decreased the percentage of motoneuron
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Fig. 2. Sema3C gain and loss of function in motoneurons modulate Nrp1 and Nrp2 levels at the cell surface. (A)Histogram showing thediversity of cell-surface Nrp1 and Nrp2 immunofluorescence levels in control EGFP growth cones. (B,C,F,G) Histograms showing cell surface Nrp1and Nrp2 immunofluorescence of non-permeabilized growth cones from control EGFP and Sema3CiresGFP explants (B,C) or Si Control and SiSema3C explants (F,G). Growth cones were classified in four categories, depending on the fluorescence level. Fluorescence intensities werenormalized to EGFP+ conditions. **P<0.01, ***P<0.001, with 2 test. (D,E,H,I) Representative growth cones immunostained for Nrp1 and Nrp2under each condition. NgCAM is used as a specific marker of motoneuron axons. Scale bars: 10m. (J,K)Detection by western blot of Nrp1 andNrp2 proteins in biotinylated (cell surface) and total pool fractions from B103 cells co-transfected with Nrp1, Nrp2 and Sema3CiresGFP or controlEGFP constructs. D
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collapse to a full dose of Sema3F (Fig. 3E; P<0.001, 2 test).Altogether, consistent with the modulations of Nrp levels, intrinsicSema3C expression oversensitizes motor growth cones toexogenous Sema3F while desensitizing them to Sema3A.
Next, we examined the outcome of the opposite intrinsicSema3C-mediated modulations of Nrp1 and Nrp2 on the growthcone sensitivity to environmental Sema3C. Indeed, motoneuronsoverexpressing Sema3CiresGFP exhibited a significant 20%decrease in their collapse response to Sema3C when comparedwith control ones (Fig. 3A,B; P<0.01, 2 test). By contrast, wedetected no significant effect of Sema3C-targeting siRNAelectroporation on Sema3C-induced collapse (Fig. 3E). Theseresults thus reveal a complex interplay between motoneuronalSema3C and environmentally repulsive Sema3 proteins.
Proper intrinsic Sema3C expression is required forthe fasciculation of motoneuronal tractsNext, we investigated how these cross-regulations between intrinsicand environmental Sema3 proteins could impact motor axonpathfinding in vivo. We reported previously that manipulations ofintrinsic Sema3A, impacting motoneuron sensitivity to exogenousSema3A, led to trajectory defects of the proximal part of
motoneuron tracts (Moret et al., 2007). Such early gross defectswere not observed after gain and loss of function of motoneuronalSema3C (supplementary material Fig. S2A). Nevertheless, moredetailed analyses of spinal motor tracts in whole-mount embryosrevealed that Sema3C takes part in the fasciculation of the proximalpart of spinal nerves. Indeed, gain and loss of motoneuronalSema3C at the brachial and thoracic levels resulted in modest butsignificant increased and decreased spinal nerves caliber,respectively (supplementary material Fig. S2B,C,E,F).Immunofluorescence analysis against activated caspase 3 showedthat these effects were not correlated with changes in motoneurondeath (supplementary material Fig. S2D).
Motor axon tracts exhibit a remarkablestereotyped position in the limb targetIntegration of multiple guidance cues is emerging as an obligatorymechanism for axon navigation. We hypothesized that themodulatory properties of intrinsic Sema3C uncovered here couldallow motor axons to properly interpret complex gradients ofSema3 repellents. The chick forelimb appeared as an attractivebiological model with which to investigate this idea. At HH24,motor axons leave the brachial plexus to invade the limb and are
growth cones labeled with phalloïdin-TRITC in collapse assays with control,Sema3C-, Sema3A- or Sema3F-conditioned medium (CM). Scale bars:10m. (B)Histogram showingpercentages of growth cone collapse ofmotoneurons electroporated (+) or nonelectroporated (–) either with Ctrl EGFPor Sema3CiresGFP constructs, andtreated with control, Sema3CCM,Sema3ACM or Sema3FCM. The dose ofconditioned medium is indicated inbrackets. (C)Histogram showing collapsepercentages of wild-type growth conestreated with control, Sema3CCM,Sema3ACM or Sema3FCM followingcontrol (–), anti Nrp1 or anti-Nrp2antibody blocking. Nrp1 and Nrp2 arerequired for Sema3C collapse response.(D)Morphology of wild-type growthcones labeled with phalloidin-TRITC incollapse assays with control, Sema3CCM,Sema3ACM or Sema3FCM in control (–),Nrp1 (anti-Nrp1) or Nrp2 (anti-Nrp2)blocking conditions. Scale bar: 10m.(E)Histogram showing percentages ofgrowth cone collapse of motoneuronselectroporated with a control or aSema3C-targeting siRNA cocktail andtreated with control, Sema3ACM,Sema3CCM or Sema3FCM. The dose ofconditioned medium is indicated inbrackets. The number of growth conesexamined in each condition is indicatedon the respective bars. **P<0.01,***P<0.001, ns, non significant, with 2
test.
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sorted out according to their pool-specific identity (Swanson andLewis, 1982; Tsuchida et al., 1994). At HH26, LMCl motoneuronaxons have invaded the limb and project into the dorsal part of thelimb, forming the radialis profundus nerve, whereas LMCmmotoneurons, expressing intrinsic Sema3C, project their axonsventrally and form the interosseus nerve and the median nerve(Tsuchida et al., 1994; Swanson and Lewis, 1982). To gain insightsinto the degree of stereotypy of nerve positioning in the limb, aseries of confocal images of three whole-mount HH26 NgCAM-immunolabeled chick embryos was taken to reconstruct the wholenerve patterns of the limb. Remarkably, as illustrated by the perfectmatching of all three median and interosseus nerves projectingventrally, the positioning of motor branches showed a high degreeof fidelity, with very low inter-individual variations (Fig. 4A,B).Next, using in situ hybridization on whole-mounted and cross-sectioned embryos, we observed that Sema3C is expressed by thecentral limb mesenchyme, whereas Sema3A and Sema3F areexpressed by the peripheral one (Fig. 4C,D). This then highlightedthe existence of complex expression of Sema3 proteins in the limbmass (Fig. 4C,D).
Sema3C expression by LMCm motoneuronscontributes to specify the fine positioning ofspinal nerves in the limbHaving characterized an appropriate in vivo context for ourpurpose, we then asked whether Sema3C expression by LMCmmotoneurons modulates their reading of the Sema3 combinationsand properly positions their tracts in the limb. We herein tookadvantage of the chick model that enables the manipulation of onlyone side of the neural tube and superimposed left and right limbinnervations (Fig. 5A). In Si Control electroporated embryos, wecould observe that ventral and dorsal innervation patterns of the leftand right forelimbs were perfectly symmetrical, thus confirming
the highly stereotyped forelimb innervation pattern (Fig. 5B;supplementary material Movie 1). By sharp contrast, in the SiSema3C electroporated embryos, although the radialis profundusnerves (dorsal) arising from Sema3C– LMCl could be normallysuperimposed, important mismatches were detected for the medianand interosseus nerves (ventral), which arise from the LMCm poolin which Sema3C was knocked down (Fig. 5B; supplementarymaterial Movie 2). The electroporation level of each embryo wasassessed by co-electroporation of EGFP with siRNA cocktails(supplementary material Fig. S3A). We then classified Si Controland Si Sema3C embryos for the amplitude of innervation mismatchbetween control and electroporated sides. Significantly higherlevels of mismatch were found in the Si Sema3C condition for thetrajectories of the median and interosseus (ventral) nerves (Fig.5C,D; supplementary material Fig. S2B; P<0.01, Mann Whitneytest). By contrast, the trajectory of the nerve radialis profundus(dorsal) was found to be unaffected by loss of motoneuronalSema3C (Fig. 5C,E; supplementary material Fig. S2B, ‘ns’ MannWhitney test). Finally, overexpressing Sema3C did not impact thestereotypy of the limb nerve patterns, suggesting that LMClmotoneurons, which do not endogenously express Sema3C, are notresponsive to Sema3C gain of function (Fig. 5C).
Altogether, our results then demonstrate that Sema3C takes partin the developmental program by which motoneurons set a propersensitivity to a combination of target-derived Sema3 repellents,which controls the stereotyped positioning of ventral motor nervesin the limb (Fig. 6).
DISCUSSIONOur study reveals that motoneuronal Sema3C balances therespective strength of Sema3 repellents through the control of Nrpgrowth cone surface level. Altering motoneuronal Sema3C levelsresults in defective positioning of LMCm motor tracts in the limb,
3639RESEARCH ARTICLEMotoneuronal Sema3C and axon guidance
Fig. 4. The chick forelimb displays a stereotypedinnervation pattern. (A)Procedure to obtain acomposite image of the limb innervation from threedifferent embryos. (B)Confocal stack projections ofNgCam-labeled forelimb innervation from three chickembryos. The composite image shows the highlystereotyped projection pattern of motor tracts in thelimb. Scale bar: 100m. (C)In situ hybridization ofSema3C, Sema3A and Sema3F in chick whole-mountedforelimb (in blue) and limb cryosections (in gray).Dorsoventral, mediolateral and anteroposteriororientations are indicated on the upper panels. Scalebars: 100m (cryosections); 500m (whole mounts).(D)Schematic representation of environmentalsemaphorin expression pattern in the chick forelimb onNgCam-labeled HH26 chick cryosections.
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where Sema3 repellents form complex combinations, thusdemonstrating that crosstalk between intrinsic and extrinsic Sema3proteins regulates the positioning of motor axon tracts in theirtarget field (Fig. 6).
Remarkable fidelity of motor axon tract positionin the limbThe establishment of precise connections between motor pools andindividual muscles relies on a series of guidance decisions leadingmotor axons to segregate from each other at choice points alongtheir navigation. Likewise, more than 30 different limb muscles arecontacted by motor axon subsets. The tract arising from the LMCmotor column and reaching the limb first separates into dorsalLMCl and ventral LMCm tracts. Next, several branchesindividualize from the main tract, navigating in the limb width tocontact single muscles. Studies in the field essentially concentratedon the primary LMCl/LMCm dorsoventral choice and established
that it is specified by a combination of guidance cues, includingephrins, 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 guidancechoices that prepare motoneuron pools for the specific innervationof their target muscles. Our 3D reconstruction of limb innervationpatterns revealed a remarkable fidelity of this terminal guidanceprocess, which demonstrates that robust mechanisms exist to setthe accurate positioning of spinal tracts in limb quadrants, thusachieving the fine-tuning of motor axon connectivity.
Contribution of semaphorin/neuropilin co-expression in the terminal steps of motor axonguidanceWe provide evidence that the control of growth cone sensitivity toenvironmental semaphorins by motoneuronal semaphorin/neuropilin co-expression is part of the developmental program that
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Fig. 5. Intrinsic Sema3C takes part in thestereotyped projection pattern of the chickforelimb. (A)Procedure to obtain composite images ofthe limb innervation from electroporated and controlsides of an embryo. (B)Superimposed 3D reconstructionsof left (magenta) and right (cyan) forelimb innervationsfrom embryos electroporated with Si control or SiSema3C. The complete limb innervation (limb tract), theventral tracts or the dorsal tracts are displayed.Downregulation of Sema3C expression in LMCmmotoneurons shifts the position of the LMCm-derivedventral tracts (yellow arrow). (C)Higher magnification ofconfocal stack projections displaying the ventral or dorsalforelimb tracts in Si control, Si Sema3C or Sema3CiresGFPconditions. Downregulation of intrinsic Sema3C leads toaltered projection patterns of the ventral median andinterosseus nerves in the forelimb (yellow arrows). Scalebars: 100m. (D,E)Graphs comparing the amplitude ofventral tract (D) or dorsal tract (E) mismatches between Sicontrol and Si Sema3C embryos. Eleven Si control andnine Si Sema3C embryos were classified according to thelevel of mismatch observed in the innervationsuperimposition of the control and electroporated sides.The scale goes from 1 for the best to 20 for the mostsevere mismatch. Ranks are reported on the graphs. Thered lines indicate the average rank in each condition.**P<0.01, ns, non significant with Mann-Whitney test.
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controls terminal motor axon guidance. The regulatory mechanismdescribed here has the potential to create subtle differences ofguidance responses between motor axons subsets, thusindividualizing them within a common environment. First,motoneuronal Sema3 proteins show different modulatory effects onmotor growth cone responsiveness. We have previously observedthat motoneuronal Sema3A sets the sensitivity to Nrp1-dependentSema3A by regulating cell surface levels of Nrp1 (Moret et al.,2007). Here, we demonstrate that, by contrast, motoneuronalSema3C modulates both Nrp1 and Nrp2 levels, and therebyresponsiveness to both Nrp1- and Nrp2-dependant Sema3 proteins.As a result, regulation of receptor availability by motoneuronalSema3 proteins can have a wide range of functional outcomes.
Second, Sema3 members have dynamic and specific expressionin motoneurons subpopulations. For example, Sema3C is expressedin LMCm but not in LMCl motoneurons.
Third, the effects of motoneuronal Sema3 proteins are confinedto restricted subpopulations. Our in vitro analysis show that Nrp1is exposed at the surface of all motor growth cones, but Nrp2 ononly half of them. Modulation of Nrp2 by Sema3C was limited tothe population that did expose Nrp2 at the surface. The proportionof axons that do not expose Nrp2 at their surface was not changedby Sema3C overexpression or knock down. Correlatively,alteration of Sema3C expression in motoneurons affected onlymotor axon positioning of LMCm population that endogenouslyexpresses it. Ectopic Sema3C expression in LMCl motoneuronswhich normally do not express it had no impact on their projection.LMCl motoneurons that are insensitive to Sema3C regulation maytherefore represent the population with undetectable Nrp2 surfaceexpression in the explant cultures. Overall, our results areconsistent with a population-specific effect of motoneuronal Sema3proteins in motor axon guidance. This diversity of motoneuronalSema effects has to be considered in the light of previous report ofSema3 expression in motoneurons. Interestingly, a complex andintriguing Sema3 code with specific semaphorin combinations inrestricted motoneuron pools has been reported (Moret et al., 2007;Cohen et al., 2005). We could hypothesize that motoneuronalSema3 proteins other than Sema3A and Sema3C also differentiallyset responsiveness to environmental Sema3 proteins gradients.Therefore, by defining the specific cell-surface Nrp levels and
sensitivities to environment-derived Sema3 proteins, such a Semacode has the potential to confer pool-specific guidance responses.Consistently, our analysis reveals a striking diversity of Nrp levelsat the motor growth cone surface. Such a diversity, which is likelyto mirror a wide range of axon sensitivity, could be set by themotoneuronal Sema code in order to allow terminal navigation.
Emerging growth cone properties for Sema3C/Nrp1/Nrp2 co-expressionTogether with other recent work, our data point out the versatilityof ligand/receptor co-expression in axon guidance signaling.Ephrin/Eph, Sema3A/Nrp1, Sema6A/PlexinA4 co-expression allinhibited axon responsiveness to the exogenous ligand (Carvalhoet 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 cis-attenuation in LMCl neurons ensure proper dorsoventralsegregation of motor axons in the limb (Kao and Kania, 2011).Sema3A/Nrp1 regulates motor axon pathfinding in peripheraltissues (Moret et al., 2007). We show here that the effect ofmotoneuronal Sema3C is not limited to its environmentalcounterpart, as it also influenced the sensitivity to Sema3A andSema3F. By showing that motoneuronal Sema3C upregulatesgrowth cone responsiveness to Sema3F, our work provides the firstexample of ligand/receptor co-expression with an oversensitizationoutcome. Intrinsic Sema3C has even dual effects, as it alsodecreased the sensitivity to other Sema3 proteins (Sema3A). Thisstudy also highlights precise regulatory potentialities of intrinsicSema3C. We showed that Sema3C overexpression reducedmotoneuron collapse response to Sema3C exposure, consistentwith a decreased sensitivity of their growth cones. However,Sema3C knock down did not significantly alter the global collapseresponse to Sema3C. This may reflect a heterogeneous situation atthe cell scale. Consistent with previous studies (Chen et al., 1998;Takahashi et al., 1998), we showed that collapsing effects ofSema3C on motor axons require both Nrp1 and Nrp2 receptoractivity. Opposing regulation of Nrp1 and Nrp2 level by intrinsicSema3C could possibly either positively or negatively impact theamount of Nrp1/Nrp2 heterodimers, depending on the respectiveinitial levels of neuropilin receptors in the cell. Therefore, in the
3641RESEARCH ARTICLEMotoneuronal Sema3C and axon guidance
Fig. 6. Model of the interplay between intrinsic and environmental Sema3 proteins expressions for the establishment of the highlystereotyped motoneuron projection pattern in the forelimb. Expression of Sema3C in LMCm motoneurons (green) decreases Nrp1 availabilityand increases Nrp2 on LMCm-derived growth cones, modulating their sensitivity to Sema3A, Sema3C and Sema3F as a result. This can be seen bylooking at combined Sema3 gradients in the forelimb. This modulation contributes to the fine positioning of LMCm nerve tracts in the ventralforelimb, as reflected by their shift in the Sema3C knock down condition (double-headed arrows).
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population of motoneurons, intrinsic Sema3C expression couldcontribute to generate diversity of growth cone responsiveness toenvironmental Sema3C.
The outcome of such multiple functional potentialities ofintrinsic Sema3C on axon tract positioning by exogenous Sema3sources is likely to depend on the localization of these sources andtheir relative repulsive strength on growth cones. The in vivoconsequences of Sema3C overexpression and knock down on theguidance of motor axon tracts in the forelimb may reflect suchcomplexity. We showed that Sema3C overexpression did not alterLMCm axon trajectory in the ventral forelimb. In vitro, it modifiedmotor growth cone responses, with a more or less pronouncedeffect depending on the Sema. Together, it suggests that theoutcome of strong desensitization to Sema3A and Sema3Ccombined to the moderate oversensitization to Sema3F is null inthe ventral forelimb (supplementary material Fig. S4). By contrast,the inward deviation of the LMCm motor tract following intrinsicSema3C knock down reflects that the moderate desensitization toSema3F combined with the oversensitization to Sema3A observedin vitro potentiates the repulsive force exerted by the ventralforelimb periphery (supplementary material Fig. S4).
Our finding that Sema3C intrinsic effects rely on the regulationof Nrp1 and Nrp2 raises an even broader potential for Sema3C toregulate the responsiveness to other Nrp ligands, such as VEGF(Neufeld et al., 2002; Erskine et al., 2011). Until now, themechanisms underlying the integration of multiple extracellularguidance signals are poorly known and Sema3C thus appears as aninteresting example of how a neuron-derived factor can participatein the program integrating guidance information by the growthcone.
The mechanisms underlying the regulation by co-expressedligand/receptor pairs appear to differ. Ephrin/Eph andSema6A/PlexinA4 ligand-receptor cis-interactions were shown tomask receptors to prevent trans-binding with the ligand and/or toinhibit the receptor phosphorylation, thus suppressing downstreamsignaling. In both cases, the levels of cell surface receptor were notaffected (Haklai-Topper et al., 2010; Carvalho et al., 2006). Bycontrast, our biochemical analysis of the biotinylated cell surfacefraction of Nrp proteins indicates that the modulation of Nrpavailability relies on the proteins level at the plasma membrane.Moreover, in neither cell lines nor motor growth cones was thetotal pool of Nrp proteins found to be altered by Sema3Cmisexpression, indicating that this regulation may rely ontrafficking of plasma membrane and intracellular receptor pools.Interesting issues will be to decipher the currently unknownmolecular pathways that regulate the trafficking of Nrp receptors,and their regulation by Semaphorin/Nrp co-expression. Finally,multiple functional properties could emerge from ligand/receptorco-expression. Interesting issues will also be to assess whethercross-regulation of semaphorin signaling influences a wider rangeof axonal properties, such as inter-axonal communications, short-term desensitization (Piper et al., 2005) and adaptation toexogenous ligand sources (Ming et al., 2002). In addition to theirrole in axon guidance, Sema3 proteins and neuropilins are involvedin diverse developmental processes, such as angiogenesis, and inpathological contexts, such as cancer. Sema3/neuropilin co-expression has been widely noted, suggesting that the regulatorypathway uncovered here could have broader implications.
AcknowledgementsWe thank J. Raper for the kind gift of Sema3A and chick Nrp1 constructs, APüschel for the Nrp2 construct and J. Falk for his helpful comments on thiswork.
FundingThis work was supported by the Agence National de la Recherche (ANR) CNSprogram, by Fondation pour la Recherche Médicale (FRM labeled team), byAssociation Française contre les Myopaties (AFM) and by PhD fellowships fromMinistere de l’Education Nationale de la Recherche et de Technologie (MENRT)and FRM.
Competing interests statementThe authors declare no competing financial interests.
Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.080051/-/DC1
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