Axon guidance in the developing ocular motor systemand Duane retraction syndrome depends onSemaphorin signaling via alpha2-chimaerinJuan E. Ferrarioa, Pranetha Baskarana, Christopher Clarka, Aenea Hendrya, Oleg Lernerb, Mark Hintzea, James Allenc,John K. Chiltonc, and Sarah Guthriea,1

aMedical Research Council Centre for Developmental Neurobiology, King’s College, London SE1 1UL, United Kingdom; bIMS Health, London N1 9JY,United Kingdom; and cInstitute of Biomedical and Clinical Science, Peninsula Medical School, University of Exeter, Plymouth PL6 8BU, United Kingdom

Edited* by Lynn T. Landmesser, Case Western Reserve University, Cleveland, OH, and approved July 16, 2012 (received for review October 27, 2011)

Eye movements depend on correct patterns of connectivity be-tween cranial motor axons and the extraocular muscles. Despitethe clinical importance of the ocular motor system, little is knownof the molecular mechanisms underlying its development. We haverecently shown that mutations in the Chimaerin-1 gene encodingthe signaling protein α2-chimaerin (α2-chn) perturb axon guidancein the ocular motor system and lead to the human eye movementdisorder, Duane retraction syndrome (DRS). The axon guidance cuesthat lie upstream of α2-chn are unknown; here we identify candi-dates to be the Semaphorins (Sema) 3A and 3C, acting via the Plex-inA receptors. Sema3A/C are expressed in and around the devel-oping extraocular muscles and cause growth cone collapse ofoculomotor neurons in vitro. Furthermore, RNAi knockdown ofα2-chn or PlexinAs in oculomotor neurons abrogates Sema3A/C-de-pendent growth cone collapse. In vivo knockdown of endogenousPlexinAs orα2-chn function results in stereotypical oculomotor axonguidance defects,which are reminiscent ofDRS,whereas expressionof α2-chn gain-of-function constructs can rescue PlexinA loss offunction. These data suggest that α2-chn mediates Sema3–PlexinArepellent signaling. We further show that α2-chn is required foroculomotor neurons to respond to CXCL12 and hepatocyte growthfactor (HGF), which are growth promoting and chemoattractantduring oculomotor axon guidance. α2-chn is therefore a potentialintegrator of different types of guidance information to orchestrateocular motor pathfinding. DRS phenotypes can result from incorrectregulation of this signaling pathway.

Eye movements in vertebrates depend on the operation of sixextraocular muscles, which are innervated by three nerves.

The oculomotor nerve (OMN) innervates four of these muscles,namely, the ventral oblique (VO), ventral rectus (VR), medialrectus (MR), and dorsal rectus (DR), whereas the abducens andtrochlear nerves innervate the lateral rectus (LR) and dorsaloblique (DO), respectively (1) (Fig. 1 A–C). The arrangement ofthis “ocular motor” system is conserved across vertebrates; inhumans, incorrect development of this pattern of innervationleads to eye movement disorders such as strabismus, whichaffects 1% of the population, and may result in amblyopia orpartial blindness (2). The etiology of these axonal miswiringdisorders is poorly understood.Mutations in the α2-chn protein isoform encoded by the chi-

maerin-1 (CHN1) gene have recently been shown to be responsiblefor the DURS2 variant of a congenital form of strabismus, Duaneretraction syndrome (DRS) (3). In DRS, defects in horizontal eyemovements result from incoordination of the medial and lateralrecti muscles, which are innervated by the oculomotor and theabducens nerves, respectively (2). Neuroimaging studies suggestthat DURS2 may involve absence of the abducens concomitantwith aberrant innervation of the LR muscle by the OMN, and/orhypoplasia of both the abducens and oculomotor nerves (4, 5).Wehave previously shown that expression of α2-chn forms harboringidentified human mutations, in the oculomotor nerves of chickenembryos, leads to characteristic axon guidance defects, suggesting

a role for α2-chn in axon pathfinding (3). Such a function for α2-chn is supported by its role in corticospinal tract formation, whereit is thought to transduce ephrin-B-EphA4 axon guidance signals(6). However, the signals that lie upstream of α2-chn in the ocularmotor system are currently unknown.To address ocular motor guidance mechanisms, we previously

mapped the development of axon projections in the chicken em-bryo (1, 7). We found that the abducens nerve first projects to theLR on embryonic day 4 (E4) (8, 9) (Fig. 1A), whereas the trochlearand theOMN reach theDO andVO, respectively, on E5 (Fig. 1B),with theOMN sending branches to its other targets on E6–E7 (Fig.1C). Candidate guidance cues in the system are the Semaphorins,which are expressed in the developing head, with cognate Neuro-pilin receptors present in cranial motor neurons (7, 10). In addi-tion, we have previously identified two diffusible chemoattractants,the chemokine CXCL12 and hepatocyte growth factor (HGF) toplay a role in ocular motor pathfinding (11).In the present study, we demonstrate that Semaphorins play

a key role in wiring the ocularmotor system and that they signal viaα2-chn. We present in vitro and in vivo evidence that α2-chn iscritical in the pathfinding of oculomotor neurons as a downstreamcomponent of the Sema3/PlexinA repellent signaling pathway.Wealso show that α2-chn is required for axonal responses to CXCL12and HGF in vitro. Taken together, these findings suggest thatcorrect regulation of these signaling systems acts via α2-chn toensure the fidelity of axon projections. Deregulation of thispathway results in axon guidance defects similar to those observedin Duane retraction syndrome.

ResultsSema3AReceptors PlexinA1 and PlexinA2Are Expressed byOculomotorNeurons.We have previously demonstrated the expression of theclass 3 Semaphorin receptors Neuropilin-1 and -2 in ocularmotor neurons (10). Sema3s signal via complexes between theNeuropilins and PlexinAs, including PlexinA1 and -A2 (12, 13).We therefore characterized the expression patterns of PlexinAsby in situ hybridization on sections of embryonic midbrain. Atembryonic day 5, PlexinA1 is expressed in the entire oculomotornucleus, with PlexinA2 expressed in a large subset of the neurons(Fig. 1 D and E). At E6, when the OMN is branching to itstargets, the entire oculomotor nucleus maintains PlexinA1 ex-pression, including oculomotor neurons that cross the midline

Author contributions: J.E.F., J.K.C., and S.G. designed research; J.E.F., P.B., C.C., A.H., O.L.,M.H., J.A., J.K.C., and S.G. performed research; J.E.F., C.C., and S.G. analyzed data; andJ.E.F. and S.G. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: sarah.guthrie@kcl.ac.uk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1116481109/-/DCSupplemental.

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(7) (Fig. 1F), whereas only a subpopulation of neurons expressPlexinA2 (Fig. 1G). These neurons may represent the ventrolat-eral and ventromedial pools that project to the VO and the DRmuscles, respectively (14). These observations suggest that func-tional Sema3 signaling complexes are present in OMN neurons.

Sema3A and Sema3C Are Expressed in and/or Around the ExtraocularMuscles. Class 3 Semaphorins are expressed in the developingchick head at relevant stages for ocular motor axon guidance(10). We performed a more detailed in situ hybridization analysisfor Sema3A and Sema3C on sections of the periocular region atE4 and E6, corresponding with relevant stages of axon outgrowthand OMN branching, respectively (Fig. 1 H–S). At E4, abducensaxons have reached the LR, whereas oculomotor and trochlearaxons have just exited the neuroepithelium (7). At this stage,Sema3A is expressed in large mesenchymal regions includingthose adjacent to midbrain at the site of OMN exit, and in thenotochord and perinotochordal mesenchyme underlying thehindbrain, adjacent to the path of the abducens nerve (15) (Fig. 1H and M). At E6, Sema3A in situ hybridization with doublefluorescent immunostaining of nerves and muscles revealed highlevels of Sema3A expression in the mesenchyme bordering (butnot within) the extraocular muscles (Fig. 1 I–L). These Sema3A+

cells might represent muscle sheath cells and/or domains ofneural crest-derived mesenchyme cells. Overall, Sema3A wasexpressed around all six extraocular muscles including the distalVO target of the OMN (Fig. 1K).By contrast, at E4 Sema3C was not expressed throughout the

head mesenchyme, but showed high expression in the LR, theabducens target, which lies ventral to hindbrain rhombomere 2/3(8) (Fig. 1 N and T). By E6, Sema3C expression was detected inall of the extraocular muscles (Fig. 1 O–S), but by contrast withSema3A, was largely within subpopulations of muscle fibers,rather than surrounding them. At E6, as at E4, the most exten-sive domain of Sema3C expression was observed in the LRmuscle (Fig. 1S). These expression data suggest that Sema3Aand -3C are possible axon guidance cues in the innervation of theextraocular muscles.

Fig. 1. Schematic diagram of ocular motor development, and expressionpatterns of Sema3A, Sema3C, PlexinA1, and PlexinA2. (A–C) Diagrams ofdevelopment of the ocular motor system in the chicken embryo at E4–E6 ina lateral view of a chick head (from ref. 7). Abducens nerve (VI) is shown ingreen, oculomotor nerve (III) in blue, trochlear nerve (IV) in purple, andmuscles are shown in red. Muscle abbreviations are as presented in the text.MB, midbrain; HB, hindbrain. (D–G) Transverse sections through E5–E6 chickenmidbrain (stages as labeled), showing in situ hybridization for PlexinA1 orPlexinA2 as labeled or PlexinA2/Islet1 double in situ hybridization (Islet1mRNA detection, in red). (H–S) In situ hybridization for Sema3A or Sema3C, aslabeled, in the chick head at E4 or E6. (H and N) Sagittal sections through thehead at E4. (M and T) Transverse sections at rhombomere 2/3 level. (I–L andO–

S) Transverse sections through the periocular region at E6, showing in situhybridization either singly or in combination with immunostaining for nerves(neurofilament H; green) and muscles (sarcomeric myosin; red). (Scale bar, 150μm in D–G; 400 μm in H, M, N, and T; and 80 μm in all remaining panels.)

Fig. 2. In vivo phenotypes in the ocular motor system following electro-poration of shRNAs for PlexinA1 and α2-chn. Confocal montages at E6 of theperiocular regions of chicken embryos electroporated at E2 with controlplasmid (A) or with shRNAs to knock down expression of PlexinA1 (B), α2-chn(C), or both (D). Whole mounts were immunostained to show axons in greenand muscles in red. Midbrain is Left and ventral at Bottom in all panels. Whitearrows show defasciculating or overshooting axons; arrowheads show axonsectopically directed toward the LR muscle. Asterisk shows ciliary ganglion;muscle abbreviations are as presented in text. (Scale bar, 200 μm in A–D.)

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In Vivo Knockdown of PlexinA or α2-chn Signaling Produces SimilarAxon Guidance Defects. To test a possible role of Sema3–Plexininteractions in OMN guidance in vivo, we electroporated chickenembryos with fluorescently tagged shRNA constructs that knockdown expression of PlexinA1 or PlexinA2, or with a scrambledshRNA control construct. OMNs expressing control constructsformed normal projections to the four target muscles (Fig. 2A andTable 1) as previously shown (3). We found that knockdown ofPlexinA1 or PlexinA2 resulted in striking and reproducible axonguidance defects, including frequent defasciculation of OMNaxons away from the nerve, aberrant “branches” toward the LRmuscle, or axonal overshooting of specific muscles. For PlexinA1shRNA, branching to LR was seen in 22/26 cases, other defasci-culations in 21/26 cases, and overshooting in 6/26 cases, with 20/26embryos (77%) showing two or more defects simultaneously (Fig.2B and Table 1). For silencing of PlexinA2, defasciculation waspresent in 9/17 cases (Table 1) and no embryo displayed morethan one guidance error. These overexuberant growth and de-fasciculation defects suggest that PlexinA signaling controls OMNtopographic targeting via a repulsive mechanism.We have previously shown that expression of gain-of-function

α2-chn constructs causes stalling and defasciculation of theOMN (3). We therefore used RNAi-mediated knockdown of α2-chn in vivo, using target sequences specific to α2-chn, and not itsrelated isoform α1-chn (Fig. S1). α2-chn knockdown resulted inphenotypes that closely resemble those following PlexinAknockdown, including multiple nerve defasciculations and mus-cle overshooting (Fig. 2C and Table 1). All cases (10/10) showedectopic branches toward the LR and 50% exhibited two or morephenotypes (Table 1). In extreme cases, α2-chn knockdownresulted in widespread defasciculation and projection towardother branchial arch muscles (Fig. S2). The similar phenotypesproduced by silencing either PlexinAs or α2-chn in the OMNimplicates these molecules in the same signaling pathway.We next knocked down simultaneously (Fig. S1) PlexinA1 and

α2-chn and found that the OMN manifested an even strongerphenotype, with multiple defasciculating and overshooting axons(Fig. 2D and Table 1); 80% of embryos showed two or moreguidance defects. PlexinA2 and α2-chn simultaneous knockdownproduced defects comparable to those with α2-chn shRNA alone(Table 1), whereas combinatorial knockdown of both PlexinAsproduced effects comparable to knockdown of PlexinA1 alone,(50% embryos with two or more errors). Analysis of PlexinA1/α2-chn double knockdown embryos at an earlier time point showedOMN defasciculation toward the LR, suggesting that pathfindingdefects occur relatively early (Fig. S2 and Table S1).

Therefore, all three types of knockdown produced similarphenotypes, with striking defasciculation in the vicinity of theciliary ganglion and frequent ectopic branching to the LR mus-cle. In particular, the α2-chn loss-of-function phenotype clearlycontrasts with the α2-chn gain-of-function phenotype, entailingectopic branching/overshooting and axon stalling, respectively.Both phenotypes are consistent with a correct level of α2-chnsignaling being required for topographic nerve-muscle targeting.

α2-chn Signaling Mediates Growth Cone Collapse Induced by Sema3Aand Sema3C in Oculomotor Neurons. As knockdown of PlexinAs orα2-chn caused ocular motor guidance defects in vivo, we nexttested whether Sema3/C might signal via PlexinAs or α2-chn inoculomotor neurons. We generated E5 OMN primary cultures(11) and found that treating them with Sema3A or -3C resultedin a growth cone collapse response (Fig. 3 A–D), which typifiesthe action of repellent/inhibitory molecules (16).We tested the effects of expressing gain-of-function α2-chn

mutant forms or PlexinA/α2-chn shRNAs on OMN growth conecollapse responses. Mutations characterized in DURS2 patientssuch as the G228S mutation confer gain of function on α2-chn,leading to a hyperactivation of downstream signaling pathways(3). OMN neurons transfected with the G228S-α2-chn isoformmanifested a higher percentage of collapsed growth cones, perse, than GFP-transfected control neurons (Fig. 3D). Applicationof Sema3A led to a significant increase in the percentage ofcollapsed growth cones in GFP-transfected but not in G228S-α2-chn–transfected neurons (Fig. 3D). However, the enhancedgrowth cone collapse induced by hyperactivation of α2-chn aloneprovides circumstantial evidence that α2-chn participates ina signaling cascade downstream of repellent guidance cues suchas Sema3A/3C.By contrast, we found that knocking down PlexinA or α2-chn

abrogated OMN growth cone collapse responses to Sema3A andto Sema3C compared with neurons expressing a control shRNAplasmid (Fig. 3E). The percentage of collapsed growth cones ineither case was not significantly different from that exhibited byPlexinA1/A2 shRNA-transfected neurons treated with control, Fcprotein. Therefore, silencing either PlexinA1 or PlexinA2 abro-gates Sema3A and Sema3C-dependent growth cone collapseequivalently, suggesting that both receptors are involved in Sema3responses. OMN neurons transfected with α2-chn shRNAs (orcontrol: scrambled shRNA) and challenged with Sema3A orSema3C showed a similar pattern of response. α2-chn shRNAexpression abrogated OMN growth cone collapse responses toSema3A/C (Fig. 3E) and reduced the percentage of collapse to anequivalent level as for PlexinAs shRNAs. These data suggest that

Table 1. Summary of phenotypes resulting from in vivo electroporation experiments

Overgrowth phenotype Stalling

Plasmid nDefasciculation

to LRDefasciculation

elsewhere OvershootingTwo or

more defects Full Partial

Control shRNA 11α2-chn shRNA 10 10 5 3 5PlexinA1shRNA 26 22 21 6 20PlexinA2shRNA 17 2 9G228S α2-chn 1 μg/μL 6 1 6G228S α2-chn 0.5 μg/μL 10 5 5PlexinA1shRNA + G228S α2-chn 7 1 3PlexinA2 shRNA + G228S α2-chn 10 2α2-chn shRNA + PlexinA1 shRNA 5 5 4 1 4α2-chn shRNA + PlexinA2 shRNA 4 4PlexinA2 shRNA + PlexinA1 shRNA 8 8 4 1 4

Total numbers of embryos analyzed (n) for each individual construct or combination and the main phenotypes associated are shown.LR, lateral rectus muscle. Blank cells represent 0s (defects not observed).

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α2-chn is required downstream of Plexins for OMN responses toSema3A and -3C axon guidance signals.Control experiments were performed by exposing OMN neu-

rons expressing α2-chn shRNAs to Ephrin-A5-Fc, which can alsocause growth cone collapse of cranial motor neurons (17). Ephrin-A5–dependent collapse in OMN neurons was not abrogated by

α2-chn knockdown, suggesting that downregulating α2-chn doesnot affect growth cone collapse in general, but is specific toSema3-dependent collapse (Fig. S3A). We also wanted to elimi-nate the possibility that α2-chn knockdown might alter levels ofPlexinA expression and found that in OMN neurons cotrans-fected with α2-chn shRNAs and tagged PlexinAs, there was noreduction in the levels of PlexinA1 or -A2 protein or a change intheir distribution (Fig. S3 B and C). Therefore, the likeliest in-terpretation of our data is that α2-chn is required downstream ofSema3A/C–Plexin signaling in oculomotor neurons.In view of the strong expression of Sema3C by the LR muscle,

which is the abducens target (Fig. 1 N and T), we also tested theeffects of Sema3C and Sema3A on abducens neurons in explantcocultures and found that Sema3C and Sema3A were respectivelychemoattractive and chemorepulsive for abducens axons (Fig.S4). These data suggest that a balance of these influences, witha predominance of Sema3C-mediated attraction, might guideabducens axons to their LR target. By contrast, OMNaxons wouldbypass the LR, due to repulsion by both Sema3C and Sema3A.

α2-chn Signaling Mediates the Growth-Promoting Effects of CXCL12and HGF in Oculomotor Neurons. Given the documented roles ofCXCL12 and HGF in promoting axon outgrowth, branching, andchemoattraction in the ocular motor system (11), we thereforetested the effects of α2-chn loss of function and gain of functionon these responses. We found that OMN neurons expressingcontrol shRNAs showed a significant increase in axon outgrowthin response to CXCL12 and HGF; this effect was abolished byα2-chn knockdown (Fig. 3F). Transfection with the G228S-α2-chn gain-of-function construct produced an increase in out-growth per se compared with GFP-transfected control neurons(Fig. 3G). G228S-α2-chn–transfected neurons that were treatedwith CXCL12 or HGF failed to show any further increase inoutgrowth, consistent with the idea that these ligands act via α2-chn and that activation of either component produces the samelevel of outgrowth.To eliminate the possibility that these effects represent a non-

specific alteration of axon outgrowth, we tested the effects of ma-nipulating α2-chn on growth modulation by a growth-promotingmolecule, glial cell-line–derived neurotrophic factor (GDNF) (18).GDNF significantly increased axon outgrowth in OMN neuronstransfected with control shRNAs, which was not impaired by α2-chn shRNAs, suggesting that α2-chn does not mediate GNDFsignaling (Fig. S3D). Treatment with GNDF led to an additionalenhancement of outgrowth relative to G228S-α2-chn transfectionalone, again suggesting that α2-chn and GDNF act within separatesignaling pathways (Fig. S3D). Taken together, these data suggestthat α2-chn mediates the effects of relevant oculomotor axonguidance ligands: Sema3s, CXCL12, and HGF. The requirementfor α2-chn in both repellent and attractant signaling raises thepossibility that α2-chn integrates such signaling pathways.

Combinatorial Manipulation of Sema–Plexin and α2-chn Signaling inVivo Suggests an Interaction of These Pathways. We next testedwhether some of these signaling components interacted in vivo bycombinatorial manipulation of PlexinA and α2-chn. We con-firmed that electroporation of 1 μg/μL of G228S-α2-chn plasmidproduced a strong stalling phenotype (3) (Fig. 4A). When theconcentration of G228S-α2-chn was reduced to 0.5 μg/μL, someembryos showed a partial phenotype, with some axons stalling atthe DR, but the remainder continued to project toward the othermuscle targets (Fig. 4B).To test a possible α2-chn/PlexinA interaction, we coelec-

troporated PlexinA shRNA (loss of function) together with theG228S-α2-chn (gain of function) plasmids. As silencing ofSema3–PlexinA signaling (upstream of α2-chn) led to defasci-culation and overbranching phenotypes, we hypothesized thatactivation of α2-chn might suppress such aberrant growth. In

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Fig. 3. α2-chn and PlexinAs mediate growth cone collapse (GCC) and axonoutgrowth in oculomotor neurons in vitro. (A–D) Illustrative examples ofentire oculomotor neurons (A), and normal (B), or collapsed growth cones (C),visualized using an antibody to Islet1/2 or phalloidin to label F-actin. (Scalebar, 20 μm.) (D) Histograms showing percentage of collapsed growth cones incultures transfected with either control GFP or G228S-α2-chn plasmids, inresponse to treatment with control (Fc) or Sema3A. Data were analyzed bytwo-way ANOVA. Post hoc analysis shows that Sema3A induces GCC incontrol (GFP transfected) neurons (*P < 0.01) andG228S-α2-chn also increasesthe percentage of GCC in absence of any treatment: #P < 0.01. (E) Histogramsshowing collapsed growth cones in oculomotor cultures transfected withscrambled control (SCR), PlexinA1, -A2, or α2-chn shRNA. Cultures werechallenged with either control (Fc reagent), Sema3A, or Sema3C. Data wereanalyzed by two-way ANOVA. Post hoc analysis shows that Sema3A orSema3C induce GCC in control (SCR shRNA transfected) neurons (*P < 0.001)and PlexinA1/A2 and α2-chn shRNA reduce Sema3A/3C-induced GCC to con-trol levels; in all cases P > 0.05. # denotes that all these conditions are sig-nificantly different from Sema3A/C-induced GCC (P < 0.05). (F and G) His-tograms showing outgrowth of oculomotor neurons transfected with eithera control plasmid (SCR shRNA or GFP), α2-chn shRNA (F), or G228S-α2-chn (G)and treated with CXCL12 or HGF. Data were analyzed by a pairwise multiplecomparison procedure (Holm–Sidak method). (F) In SCR shRNA-expressingneurons, both CXCL12 and HGF increase outgrowth relative to controls (*P <0.001) and α2-chn shRNA-transfection abrogates this increase (#P < 0.001). (G)In GFP-transfected neurons both CXCL12 (ψ P = 0.044) and HGF (ψ P = 0.012)increase outgrowth relative to controls. Neurons transfected with G228S-α2-chn display an increase in outgrowth in absence of treatment (@P < 0.001).This increase is similar in CXCL12 and HGF-treated neurons. All histogramsshow mean ± SEM.

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agreement with this idea, coelectroporation of both plasmidsproduced a rescue of the PlexinA knockdown phenotype andrestored normal morphology (Fig. 4 C and D and Table 1).These phenotypes were indistinguishable from those obtainedusing control shRNA or GFP plasmids (Fig. 2A) (3). Coex-pression of G228S-α2-chn provided a more complete rescue ofPlexinA2 shRNA than of PlexinA1 shRNA (Table 1). Togetherwith our Plexin/α2-chn loss-of-function experiments, these “res-cue” experiments suggest that α2-chn is a downstream executorof Sema3–PlexinA-dependent axon guidance within the OMN.

DiscussionIn this study, we demonstrate that ocular motor axon guidancedepends on the function of Sema3A/C ligands, which signal viaPlexinAs and α2-chimaerin. Such signaling in oculomotor neu-rons in vitro produces a repulsive response, whereas abducensneurons display a differential growth promotion or repulsionresponse to Sema3C and Sema3A, respectively. Silencing of ei-ther PlexinAs or α2-chn signaling in vivo leads to very similaroculomotor axon guidance phenotypes, which are reminiscent ofDRS (Fig. 5A). Expression of constitutively active forms of α2-chn can rescue PlexinA loss-of-function phenotypes, supportingthe conclusion of a shared signaling pathway (summarized in Fig.5A). α2-chn also mediates the outgrowth-promoting effects of

CXCL12 and HGF in oculomotor neurons; α2-chn may there-fore act as an intermediate to balance and interpret positive andnegative guidance cues in the ocular motor system in vivo.

Sema3A and Sema3C Act via PlexinAs to Orchestrate Ocular MotorGuidance. For the OMN, the Sema3A/C expression patterns andin vivo effects of PlexinA knockdown suggest that OMN ectopicgrowth toward the LR depends on a failure of the OMN to re-spond to Sema3-mediated repulsion. Our model is therefore thatSema3C-dependent growth promotion/attraction leads to abdu-cens innervation of the LR muscle, whereas the OMN is repelledfrom this target by Sema3C (Fig. 5B). Sema3A/C-dependentrepulsion or inhibition of growth then acts to ensure correcttopography of OMN axon projections to the muscles.We found that PlexinAs are required for Sema3A/C signaling

in the OMN and that PlexinA1 is widely expressed there, whereasPlexinA2 is localized in subnuclei. Although Neuropilin-2 andNeuropilin-1 are expressed within the oculomotor nucleus (10),a further characterization of the precise coreceptor complexeson ocular motor neurons will be required. An additional factor tobe taken into account is that OMN neurons themselves expressSema3s (7), which in other contexts are capable of acting in cis ortrans with axonal receptors to modulate interactions betweenaxons, or with their environment.

α2-chn Mediates Axon Guidance Signaling in the Ocular Motor System.Several lines of evidence suggest that α2-chn mediates Sem-aphorin signaling in the OMN. In vivo phenotypes followingknockdown of PlexinAs or α2-chn are closely similar, suggestingthat α2-chn might be recruited to PlexinA/Neuropilin receptorcomplexes in OMN neurons, as is the case in dorsal root gan-glion neurons where α2-chn is required for Sema3A-inducedgrowth cone collapse (19). However, α2-chn’s known associationwith tyrosine kinase receptors (20) may suggest the involvementof an additional signaling component(s). As α2-chn is requiredfor OMN responses to CXCL12 and HGF, this raises the pos-sibility that PlexinAs might associate with the HGF receptor

Fig. 4. Coelectroporation of PlexinA2 shRNA and G228S-α2-chn producesa normal oculomotor axon projection in vivo. Confocal image montages atE6 of the periocular regions of chicken embryos electroporated at E2 eitherwith 1 μg/μL (A) or 0.5 μg/μL (B) of G228S-α2-chn, and combinatorial elec-troporation of G228S-α2-chn together with PlexinA2 shRNA (C) or PlexinA1shRNA (D). Immunostaining was with antibodies to GFP for electroporatedaxons (green) or to sarcomeric myosin for muscles (red). Midbrain is Left,ventral at Bottom in all panels. White solid arrow shows region where themajority of oculomotor axons stall. Asterisk shows ciliary ganglion; muscleabbreviations are as in text. (Scale bar, 200 μm).

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Plexin / α2-chn shRNA G228S α2-chnPlexin shRNA + G228S α2-chn Normal projection

Fig. 5. Schematic diagram illustrating in vivo phenotypes resulting frommanipulations in the chicken embryo. (A) Phenotypes resulting from abro-gation of PlexinA or α2-chn signaling, expression of α2-chn gain-of-functionmutant construct, or rescue experiment with Plexin loss of function, α2-chngain of function. (B) Model from E4–E6, based on timing of axon projectionsshown in Fig. 1 A–C. Model of the role of chemoattraction and repulsion intargeting axon projections with color-coded cues; hatching represents ex-pression of multiple cues.

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Met, as has been shown in some contexts for PlexinBs (21).Therefore, the in vivo phenotypes resulting from α2-chn knock-down may represent a composite of loss of responsiveness toSema3s and CXCL12/HGF.In other axon guidance systems such as the corticospinal tract,

α2-chn can function downstream of ephrinB-EphA4 signaling(6). Whereas OMN neurons do not express EphA4, they collapsein response to ephrin-A5, which has been suggested in somecontexts to activate EphB receptors (22). EphB1 and B2 areexpressed in the ocular motor system, and although we did notdetect ephrinA expression in the extraocular muscles (23), a rolefor ephrin-Eph signaling remains a formal possibility.

PlexinA, α2-Chimaerin, and DRS. Neuroimaging studies on DRSpatients show heterogeneity of ocular motor projection pheno-types (4). As Duane patients, who are heterozygous for CHN1,carry one wild-type and one mutant allele, this situation mayresemble the experimental paradigm reported here in whichlower quantities of G228S-α2-chn plasmid (0.5 μg/μL) wereexpressed, generating a hypoplasia-like phenotype of the OMN.Interestingly, G228S-α2-chn exerted a dual effect in vitro, bothpromoting axon outgrowth (in long-term assays) as well asgrowth cone collapse (in acute assays). This dual function mightdepend on the subcellular localization of α2-chn, its proximity todifferent cytoskeletal regulators, or even degradation (24), whichmight be differentially regulated by guidance cues. We also ob-served that silencing α2-chn or PlexinAs (loss of function)resulted in ectopic branching of the OMN to the LR muscle,again resembling DRS. A complete understanding of this het-erogeneity of phenotypes will require further studies of itsintracellular regulation.

α2-chn May Integrate Axon Guidance Signals During Pathfinding tothe Extraocular Muscles.Our favored hypothesis is that ocularmotorinnervation depends on a balance of chemoattraction by HGF andCXCL12 and chemorepulsion by Sema3A/C, which ensures topo-graphic targeting and is dependent on α2-chn (Fig. 5B). The appar-ently dual role of α2-chn inmediating “positive” as well as “negative”cellular responses might depend not only on its established role in

Rac inactivation, but also on regulation of the rate of Rac cycling,and/or its association with other cytoskeletal effectors. In thecontext of the ocular motor projection, CXCL12 might attenuateSema3A/C-dependent repulsion (25), as well as collaboratingwith HGF (11) via a pathway involving α2-chn to allow the fidelityof axon projections to individual extraocular muscles. In sum, wepropose α2-chn to be a unique integrator of guidance cues inmotor neuron pathfinding.

Materials and MethodsChicken embryos were obtained (26) and then used for immunostaining, insitu hybridization, immunohistochemistry (17), or in ovo electroporation aspreviously described (27). All experiments were approved by the ethical re-view committee of King’s College London. siRNA sequences for α2-chn weredesigned using the Whitehead Institute siRNA Selection Program (28),synthesized, cloned into the pRFPRNAiC miRNA vector (29), and validated(Fig. S1). Knockdown of PlexinAs used previously validated sequences (30).The G228S-α2-chn construct was previously described (3). Whole-mount em-bryos were staining with Ab for sarcomeric myosin (mf20; 1:200; De-velopmental Studies Hybridoma Bank (DSHB), together with either Ab for GFP(1/500; Abcam) or RFP (1/500; Abcam) and followed by the correspondingsecondary antibodies (Alexa Fluor 488 or 568 conjugated; 1:500; Invitrogen).

Oculomotor neurons were cultured as previously described (11). Neuronswere transfected by nucleofection (Lonza) following manufacturer’s in-structions. For growth cone collapse assays, neurons were cultured for 24 hand treated with Sema3A-Fc, Sema3C-Fc, Ephrin-A5-Fc, or Fc protein (R&DSystems). Cultures were fixed, immunostained, and scored for growth conecollapse. For axon outgrowth assays, neurons were cultured for 48 h inCXCL12, HGF, or GDNF (all from R&D Systems). Neurons were immunostainedas above and axon lengths quantitated. For extended details of methods seeSI Materials and Methods.

ACKNOWLEDGMENTS. We thankUweDrescher, Ivo Lieberam, Robert Knight,and QueeLim Ch’ng for valuable advice and discussion; Subathra Poopalasun-daram for technical assistance; and Dominic Davenport, Rita Silva-Camilo,Reetu Parbhakar, Jessica McLean, and Yasmin Issop for generation of prelim-inary data. This work was generously funded by a Wellcome Trust grant (toS.G.), a GKT (Guy’s, King’s and St. Thomas’s) Prize studentship (to O.L.), and bya South West Regional Development Agency studentship (to J.A.). C.C. wasfunded by a Fight for Sight PhD studentship. A.H. was funded by the MedicalResearch Council.

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