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Ectodermal Wnt6 is an early negative regulator of limb chondrogenesis in the chicken embryo Geetha-Loganathan, Poongodi; Nimmagadda, Suresh; Christ, Bodo; Huang, Ruijin; Scaal, Martin Mar 25, 2010

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RESEARCH ARTICLE Open AccessEctodermal Wnt6 is an early negative regulatorof limb chondrogenesis in the chicken embryoPoongodi Geetha-Loganathan1,2*, Suresh Nimmagadda1,2, Bodo Christ1, Ruijin Huang1,3, Martin Scaal1*AbstractBackground: Pattern formation of the limb skeleton is regulated by a complex interplay of signaling centerslocated in the ectodermal sheath and mesenchymal core of the limb anlagen, which results, in the forelimb, in thecoordinate array of humerus, radius, ulna, carpals, metacarpals and digits. Much less understood is why skeletalelements form only in the central mesenchyme of the limb, whereas muscle anlagen develop in the peripheralmesenchyme ensheathing the chondrogenic center. Classical studies have suggested a role of the limb ectodermas a negative regulator of limb chondrogenesis.Results: In this paper, we investigated the molecular nature of the inhibitory influence of the ectoderm on limbchondrogenesis in the avian embryo in vivo. We show that ectoderm ablation in the early limb bud leads toincreased and ectopic expression of early chondrogenic marker genes like Sox9 and Collagen II, indicating that thelimb ectoderm inhibits limb chondrogenesis at an early stage of the chondrogenic cascade. To investigate themolecular nature of the inhibitory influence of the ectoderm, we ectopically expressed Wnt6, which is presently theonly known Wnt expressed throughout the avian limb ectoderm, and found that Wnt6 overexpression leads toreduced expression of the early chondrogenic marker genes Sox9 and Collagen II.Conclusion: Our results suggest that the inhibitory influence of the ectoderm on limb chondrogenesis acts on anearly stage of chondrogenesis upsteam of Sox9 and Collagen II. We identify Wnt6 as a candidate mediator ofectodermal chondrogenic inhibition in vivo. We propose a model of Wnt-mediated centripetal patterning of thelimb by the surface ectoderm.BackgroundThe limbs of tetrapods form as mesenchymal protru-sions of the somatic lateral plate mesoderm covered byan ectodermal sheath. The cartilaginous anlagen of thelimb skeletal elements form from the centralmost regionof the limb mesenchyme in a process called chondro-genesis (reviewed in [1]). Chondrogenesis starts byenhanced proliferation of the central limb mesenchyme,which leads to local mesenchymal condensations. Oneof the earliest markers of these presumptive chondro-cytes is the SRY-related transcription factor Sox9 [2-4].Subsequently, Sox9 - positive chondroprogenitor cellsproduce cartilage-specific extracellular matrix (ECM)components like Aggrecan and Collagen II and undergoterminal differentiation into chondrocytes. Later indevelopment, the cartilaginous matrices of the skeletalelements become vascularized and undergo ossification.Interaction of limb mesenchyme and ectoderm isimportant in the regulation of patterning, morphogen-esis and differentiation of the limb bud [5,6]. The leastunderstood patterning event is the centripetal patterningof the limb, which lays down, from the outside to theinside, epidermis, dermis, musculature, and skeleton.Into the resident limb mesenchyme, skeletal muscle pre-sursor cells immigrate from the somites and arrangearound the central chondrogenic mesenchyme to formthe limb musculature in several layers [7,8]. The mar-ginal mesenchyme in close contact with the overlyingectoderm forms the connective tissue of the dermis.A major signaling center in this process is the ecto-derm. The subectodermal mesenchyme, which will formthe dermal layer of the skin, retains its non-condensedmesenchymal morphology, whereas in deeper cell layers* Correspondence: poongodi@interchange.ubc.ca; martin.scaal@anat.uni-freiburg.de1Institute of Anatomy and Cell Biology, Department of MolecularEmbryology, University of Freiburg, Albertstrasse 17, D-79104 Freiburg,GermanyGeetha-Loganathan et al. BMC Developmental Biology 2010, 10:32http://www.biomedcentral.com/1471-213X/10/32© 2010 Geetha-Loganathan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.premyogenic and prechondrogenic condensations occur.This regionalization of condensed and non-condensedmesoderm appears to be under the control of the ecto-derm, since factors produced by the ectoderm preventdifferentiation of nearby mesoderm to cartilage [9-11].Classical ablation experiments have shown that in theabsence of the limb ectoderm, connective tissue and car-tilage develop whereas skeletal muscle differentiation isblocked [12,13], indicating a muscle-promoting activityof the limb ectoderm. In an earlier study, we providedevidence that the muscle-promoting property of thelimb ectoderm is based on ectodermal Wnt signaling,namely Wnt6, which promotes Myf-5-dependent myo-genesis [14]. In the absence of ectodermal Wnt signal-ing, the somitic precursor cells are incapable to formmuscle, whereas they are still able to form endothelia[15].In the paraxial mesoderm, Wnt6 is known to act viathe canonical Wnt pathway, in which, upon Wnt ligandbinding, a transmembranous Frizzled receptor triggersan intracellular signaling cascade leading to the accumu-lation of cytoplasmic beta-catenin and the activation ofthe LEF/TCF-transcription complex to promote targetgene expression [16,17]. A number of studies indicatethat canonical Wnt signaling suppresses chondrogenicdifferentiation [18-20], probably by direct interaction ofbeta-catenin with the early chondrogenic marker Sox9[21,22].To date, the molecular nature of the cartilage-inhibit-ing signals from the ectoderm, and the role of these sig-nals during the molecular regulation of cartilageformation in the avian limbs, have not been established.Recently, it has been shown that Wnt3a along with FGFsignaling is able to inhibit limb chondrogenesis [23]. Inthis study, we identified Wnt6 to be an ectodermal fac-tor inhibiting limb chondrogenesis in the chick embryo.We found that Wnt6, which is secreted by the limbectoderm, inhibits cartilage formation at an early stageof chondrogenesis upstream of Sox9. We propose amodel that ectodermal Wnt signaling promotes myogen-esis, but inhibits cartilage formation outside the central-most limb mesenchyme, thus positioning the skeletalanlagen central to the muscular sheath of the limb. Thisimplies a centripetal gradient of ectodermal Wnt signal-ing involved in limb pattern formation.ResultsLimb ectoderm inhibits chondrogenesis at an early stageupstream of Sox9It is established from in vitro studies that the limb ecto-derm is an inhibitor of chondrogenesis [9,11]. However,the molecular mechanism of the inhibitory activity ofthe ectoderm is still unclear. In order to investigate atwhat stage of the chondrogenic cascade ectodermalsignals interfere with limb skeletal development in thechick, we removed the dorsal ectoderm of HH-stage20 - 21 embryo wing buds, and analyzed the expressionof a number of chondrogenic marker genes after 24 hand 48 h of reincubation. We found that ectodermremoval leads to a robust upregulation of the earlychondrogenic marker genes Sox9 (n = 9 for 24 h, n = 7for 48 h) and ColIIA (coding for Collagen II, n = 8 for24 h, n = 9 for 48 h) (Fig. 1). Moreover, the expressiondomain of both markers was expanded towards the dor-sal margin of the limb, where the ectoderm had beenremoved. This argues for an inhibitory role of the dorsalsurface ectoderm during the earliest stages of chondrocytespecification.Ectodermal Wnt6 inhibits limb chondrogenesisupstream of Sox9We next searched for signals mediating the inhibitoryeffect of limb surface ectoderm on avian chondrogen-esis. We showed earlier that Wnt6 is an ectodermal sig-naling molecule promoting limb myogenesis [14]. AsWnt6 is expressed throughout the limb ectoderm inchick at stages of early chondrogenesis [24,25], it is agood candidate gene to study Wnt-mediated inhibitionof chondrogenesis in vivo. We injected CHO-Wnt6-transgenic cells underneath the ectoderm of wing budsat the same stages as described above for ectoderm abla-tion. After injection of Wnt-6 cells in HH stage 19 - 21wing buds, Alcian blue staining after 4 days of reincuba-tion revealed a drastic shortening of the proximal skele-tal elements. The severity of the reduction in limb sizedepended on the amount of Wnt-producing cellsapplied, arguing for a dose-dependent action of Wnt sig-naling (Fig. 2, n = 18). Likewise, we monitored theexpression of the early marker genes, Sox9 (n = 7 for24 h, n = 6 for 48 h) and ColIIA (n = 7 for 24 h, n = 9for 48 h), after Wnt6-cell-injection at 24 h and 48 hpost surgery. Conversely to the ectoderm ablationresults shown above, we observed a severe downregula-tion of both, Sox9 and ColIIA expression, in the chon-drogenic regions of the manipulated wing buds (Fig. 3).Injection of control cells did neither change skeletalmorphology, nor alter the expression of any markergene examined (data not shown). This suggests thatWnt6 can mimick the inhibitory effect of the ectodermon early stages of chondrogenesis. Given its expressionthroughout the limb ectoderm at the stages examined(Fig. 4), Wnt6, possibly together with other Wnts withyet unknown ectodermal expression, is a likely candidateto inhibit limb chondrogenesis in vivo.In summary, our results provide evidence that chon-drogenesis in the avian limb bud is negatively regulatedby Wnt6-signaling from the surface ectoderm, which isacting on the earliest stages of limb chondrogenesisGeetha-Loganathan et al. BMC Developmental Biology 2010, 10:32http://www.biomedcentral.com/1471-213X/10/32Page 2 of 8Figure 1 In situ hybridizations of chicken wing buds with probes against the chondrogenic markers Sox9 (A, B) and Coll2a (C, D), afterremoval of the dorsal ectoderm at HH-stage 20-21 and 24 or 48 h reincubation. On the left side is the control limb, the right limb hasbeen manipulated. Arrowheads indicate the site of ectoderm removal. A Expression of Sox9 after 24 h. Expression is stronger on the operatedside. a Transverse section of the control limb showing low Sox9 expression. a’ Transverse section of the operated limb showing strong Sox9expression, which is extending ectopically towards the dorsal surface of the limb. B Expression of Sox9 after 48 h. Expression is stronger on theoperated side. b Transverse section of the control limb showing normal Sox9 expression. b’ Transverse section of the operated limb showingenhanced Sox9 expression, which is extending ectopically towards the dorsal surface of the limb. C Expression of ColIIA after 24 h. Expression isstronger on the operated side. Dislocated fragments of gold foil are still visible. c Transverse section of the control limb showing low ColIIAexpression. c’ Transverse section of the operated limb showing strong ColIIA expression, which is extending ectopically towards the dorsalsurface of the limb. D Expression of ColIIA after 48 h. Expression is stronger on the operated side. d Transverse section of the control limbshowing normal ColIIA expression. d’ Transverse section of the operated limb showing enhanced ColIIA expression, which is extending ectopicallytowards the dorsal surface of the limb. Scale bar 500 μm.Geetha-Loganathan et al. BMC Developmental Biology 2010, 10:32http://www.biomedcentral.com/1471-213X/10/32Page 3 of 8upstream of Sox9, thus limiting cartilage formation tothe prospective skeletal anlagen in the centralmost limbmesenchyme.DiscussionOur results show that the limb ectoderm is a negativeregulator of the earliest stages of chondrogenesis indeveloping chick limbs upstream of Sox9. We further-more demonstrate that Wnt6, which is, in the chick,expressed in both dorsal and ventral limb ectoderm[24,25], inhibits chondrogenesis upstream of Sox9expression, arguing for an early inhibiting activity ofectodermal Wnt6 prior to chondrocyte differentiation.At present Wnt6 is the only Wnt known to beexpressed throughout the avian limb ectoderm [25] (Fig.4). Our data do not exclude that other Wnts, likeWnt7a expressed in the dorsal ectoderm [26] or otheryet unidentifed ectodermal Wnts, also participate in thisfunction. This redundancy is very probable in themouse, where six Wnt genes (Wnt3, Wnt4, Wnt6,Wnt7b, Wnt9b, Wnt10a) have been described to becoexpressed in the limb ectoderm [27], and severalWnts, including Wnt3a, have been shown to be inhibi-tors of chondrogenesis [23,28].A regulatory role of the ectoderm during differentia-tion of the limb mesenchyme has already been proposedby Blechschmidt in 1963 [29]. Classical studies haveexperimentally confimed a role of the limb ectodermduring cartilage differentiation [30-32], whereas a regu-latory role of the muscle anlagen on skeletogenesis canbe excluded as muscle-less limbs form normal skeleton[33]. In vitro investigations have suggested that the limbectoderm produces a diffusible factor which inhibitschondrogenic differentiation in the underlying mesench-yme. Limb ectoderm from stage 23/24 wing buds hasbeen shown to inhibit cartilage differentiation of cul-tured limb mesenchyme cells even without direct con-tact but acting over some distance, thus arguing for asecreted mechanism of inhibition [9,10]. This is in linewith the long range inhibitory activity of ectodermalWnt6 in the limb suggested by our experiments.The sequential steps of chondrogenesis are character-ized by the expression of typical marker genes. Initially,the uniform limb mesenchyme aggregates in the centralcore of the limb mesenchyme to form precartilaginousmesenchymal condensations, which roughly presage thefuture skeletal elements. These prechondrocyticmesenchymal cells produce high levels of hyaluronicFigure 2 Alcian-blue staining of the cartilaginous skeleton of embryonic chicken wings. A-C Control wings. A1-C1 Wings after injection ofWnt6-CHO cells at HH-stage 20-21 and 4 days reincubation. Skeletal elements are shorter in length compared to the control wings. A1 to C1illustrate increasingly severe defects depending on the quantity and spreading of injected cells, A1 being the mildest, C1 being the severestphenotype. A1 only the humerus is affected. B1 stylopod and zeugopod are affected, C1 the entire limb is affected. Scale bar 500 μm.Geetha-Loganathan et al. BMC Developmental Biology 2010, 10:32http://www.biomedcentral.com/1471-213X/10/32Page 4 of 8Figure 3 In situ hybridization of chicken wing buds with probes against chondrogenic markers Sox9 (A, B) and Coll2a (C, D), afterinjection of Wnt6-CHO cells at HH-stage 20-21 and 24 or 48 h reincubation. On the left side is the control limb, the right limb has beenmanipulated. Dotted line indicates the site of injection. A Expression of Sox9 after 24 h. Expression is severely reduced on the operated side.a Transverse section of the control limb showing normal Sox9 expression. a’ Transverse section of the operated limb showing weak Sox9expression. B Expression of Sox9 after 48 h. Expression is reduced on the operated side. b Transverse section of the control limb showing normalSox9 expression. b’ Transverse section of the operated limb showing reduced Sox9 expression. C Expression of ColIIA after 24 h. Expression islocally reduced on the operated side. c Transverse section of the control limb showing normal ColIIA expression. c’ Transverse section of theoperated limb showing weak ColIIA expression. D Expression of ColIIA after 48 h. Expression is reduced on the operated side. d Transversesection of the control limb showing normal ColIIA expression. d’ Transverse section of the operated limb showing reduced ColIIA expression.Scale bar 500 μm.Geetha-Loganathan et al. BMC Developmental Biology 2010, 10:32http://www.biomedcentral.com/1471-213X/10/32Page 5 of 8acid and cell adhesion proteins like N-CAM and N-Cad-herin. During chondrogenic differentiation, the nascentchondrocytes express the nuclear transcription factorSox9, which is required for the expression of the carti-lage-specific marker gene ColIIA1, and start to depositthe cartilage matrix including Collagens II, IX, XI, andaggrecan. Subsequently, the balance between chondro-cyte differentiation and proliferation is regulated byinteraction of members of the BMP, FGF and Ihh path-ways, which eventually leads to chondrocyte hypertro-phy and osteogenesis (reviewed in [1]).Akiyama et al. [34] have shown that Sox9 is requiredduring at least two steps of cartilage formation, firstduring mesenchymal condensation and second, as aninducer of the related factors Sox5 and Sox6, duringovert chondrocyte differentiation. In line with this, earlyinactivation of Sox9 leads to a loss of cartilage and bone[34]. Canonical Wnt signaling, which is known to inhibitchondrogenesis [18,19], is antagonizing Sox9 activity atposttranslational level [21]. Accordingly, forced expres-sion of beta-catenin, a member of the canoncial Wntpathway, in prechondrogenic cells leads to shortened ormissing skeletal elements [19], a skeletal phenotypesimilar to the skeletal defects described here after Wnt6overexpression. Moreover, stabilization of beta-cateninin mouse limbs leads to repression of Sox9, whereasdeletion of beta-catenin results in an expansion of theSox9 expression domain in the limb bud mesenchyme[35]. This argues for the hypothesis that the inhibitoryeffect of Wnt6 on chondrogenesis observed in thisreport could be transduced by the canoncial Wnt path-way, as has been described for the epithelializing activityof Wnt6 in the somites [17]. However, as Wnt6 has alsobeen shown to act via non-canonical signaling [36],further studies on the downstream events leading tochondrogenic inhibition will be required to substantiatethis hypothesis.As we found that Wnt signaling not only inhibits Sox9expression, but also expression of the early Sox9 targetgene ColIIA, we argue that Wnt6 signaling negativelyregulates chondrogenesis at a very early step, likely dur-ing mesenchymal condensation. This is in agreementwith a recent study identifying Sox9 as a target ofWnt3a -mediated inhibition of cartilage formation [23].Interestingly, also overexpression of Sox9 has beenobserved to result in shortened skeletal elements, likelydue to inhibition of cell proliferation and differentiation[21]. This illustrates that the chondrogenesis-promotingactivity of Sox9 is finely balanced. As activated beta-catenin and Sox9 are interacting in a negative feedbackloop [21], correct Sox9 activity depends on an appropri-ate level of canonical Wnt signaling.In accordance with this, we report here that the sever-ity of limb skeletal defects is correlated with the amountof Wnt6 expressing cells implanted. This finding, anddata from earlier work from our laboratory [14], arguefor a fine-tuned, dose-dependent regulatory activity ofectodermal Wnt signaling on the development of both,muscle and cartilage. Our results support a model ofcentripetal patterning of the limb by ectodermal Wntsignaling: Within the peripheral limb mesenchyme,which is close to the ectodermal source of Wnt6, immi-grated muscle precursor cells originating from thesomites receive high doses of Wnt ligand, and differenti-ate into muscle [14], whereas the chondrogenic pathwayis inhibited. The deeper, centrally located autochthonousmesenchyme, which is farther away from the ectodermalsource of Wnt6, receives low doses of Wnt ligand, thusallowing for Sox9 expression and cartilage formation inthe central limb. Our data are in line with a previouslyproposed model combining Wnt and FGF signaling inproximodistal and centripetal limb patterning [23]. Inextension to this model, our results indicate that ecto-dermal Wnt signaling inhibits chondrogenesis upstreamof Sox9, and provide in vivo data identifying Wnt6 as acandidate inhibitor. Recent results indicate that in addi-tion to the Wnt gradient, local expression of Wnt-inhi-bitors like Wif-1 at the cartilage-mesenchyme-interfaceFigure 4 Comparison of the expression domains of Sox9 and Wnt6 in the limb. Slightly oblique transverse sections of wing buds atHH-stage 24. A Sox9 expression is limited to the chondrogenic region, which is located in the center of the limb bud mesenchyme. B Wnt6 isexpressed in the entire circumference of the limb bud ectoderm. Scale bar 500 μmGeetha-Loganathan et al. BMC Developmental Biology 2010, 10:32http://www.biomedcentral.com/1471-213X/10/32Page 6 of 8impedes Wnt-mediated inhibition of cartilage formationin the skeletogenic regions [37], possibly thus sharpen-ing the circumference of the forming skeletal elements.Using Axin2lacZ/+ mouse mesenchyme as reporter,Nusse and coworkers measured the reach of ectodermalWnt signaling leading to a mesenchymal intracellularresponse to be 100 μm, which is in line with a Wnt-dependent centripetal patterning activity originating inthe ectoderm [23]. Thus our data support the hypothesisthat ectodermal Wnt signaling acts on the limbmesenchyme in a centripetal dosage gradient which isinvolved in specifying mesenchymal differentiation intoperipheral muscle and central cartilage.ConclusionIn this paper, we investigated the molecular nature ofthe inhibitory influence of the ectoderm on limb chon-drogenesis, which has been suggested by a number ofclassical experiments in the chick. Our data providenovel insight into two aspects of limb skeletaldevelopment:1. So far it has been unknown at what level of thelimb chondrogenic cascade the negative effect of thelimb ectoderm interferes. Our results demonstrate thatthe ectoderm inhibits chondrogenesis at a very earlystage of chondrogenesis upstream of Sox9 and CollagenII-expression.2. Even though an inhibitory role of Wnt signaling onchondrogenesis has been known through a number ofstudies, no in vivo data using feasible candidate Wntshave been available. To our knowledge Wnt6 is presentlythe only Wnt expressed throughout the limb ectoderm atthe critical stages of early chondrogenesis. We present invivo data showing that injection of Wnt6-expressing cellsinto chick limb buds inhibits chondrogenesis upstream ofSox9 and Collagen II-expression.Together, our data suggest that Wnt6 is a candidatemediator of the inhibitory influence of the ectoderm onlimb chondrogenesis. We propose a model that ectoder-mal Wnt signaling limits chondrogenesis to the centrallimb mesenchyme, whereas it promotes myogenesis inthe peripheral mesenchyme, thus regulating centripetalpattern formation in the avian limb bud.MethodsPreparation of chick embryosFertilized chicken eggs were incubated at 38°C, and theembryos were staged according to Hamburger andHamilton [38].Removal of limb ectodermFor ectoderm removal, the ectoderm was stained with nileblue sulphate in ovo using a blunt glass needle coated with2.5% agarose containing 2% nile blue. The ectoderm (ofHH- stage 20-21) was peeled from the mesenchyme onthe dorsal side of the forelimb bud as far as possibletowards the edges without disturbing the apical ectoder-mal edge [14,15]. After removal of ectoderm, the limb wascovered with gold foil to prevent regeneration of the ecto-derm. Embryos were reincubated for 24 h/48 h, fixed andprocessed for whole mount in situ hybridization.Injection of Wnt-6 cellsTransfection and processing of CHO cells producingWnt-6 protein were done as described earlier [14]. CHOcontrol cells were used in parallel. Confluent cultureswere harvested, cells were washed in phosphate-bufferedsaline (PBS), pelleted and resuspended in a minimalvolume of medium. Experiments were performed onembryos at HH- stage 19-21. For cell injection, the ecto-derm of the limb was punctured with a tungsten needleand concentrated cell suspensions were locally appliedwith a micropipette. The relative amount of cellsinjected and the localization of cells was monitored inwhole mount embryos by careful inspection of injectedembryos after surgery and prior to staining. Embryoswere reincubated for various time periods (1-4 days)and processed for Alcian blue staining or for wholemount in situ hybridization.Whole-mount in situ hybridizationEmbryos were washed in PBS and fixed overnight in 4%paraformaldehyde at 4°C, washed twice in PBT, dehy-drated in methanol and stored at -20°C. Whole-mountin situ hybridization was performed as previouslydescribed [39]. Selected embryos were embedded in 4%agar and sectioned with a vibratome at 50 μm. The fol-lowing probes were used in this study:Sox9 (381 bp,kindly provided by Dr. Craig Smith, Melbourne, AUS),chicken Collagen IIA (890 bp, kindly provided byDr. William Upholt, Farmington, CT).Alcian blue cartilage stainingWhole-mount alcian blue staining was performed tovisualize the skeletal phenotype of the manipulatedembryos. Specimens were firstly stained with 0.015%alcian blue in 80% ethanol and 20% acetic acid for 1-3days, fixed and dehydrated in ethanol for 1 day, andcleared and stored in 100% methylsalicylate [40].AcknowledgementsWe thank Ute Baur, Ulrike Pein and Günter Frank for excellent technicalassistance. This work was funded by the Deutsche Forschunggemeinschaft(SFB592, A1 and GRK1104 to B.C. and M.S, and the European Network ofExcellence MYORES to B.C. and M.S).Author details1Institute of Anatomy and Cell Biology, Department of MolecularEmbryology, University of Freiburg, Albertstrasse 17, D-79104 Freiburg,Geetha-Loganathan et al. BMC Developmental Biology 2010, 10:32http://www.biomedcentral.com/1471-213X/10/32Page 7 of 8Germany. 2Present address: Department of Oral Health Sciences, LifeSciences Institute, University of British Columbia, Life Sciences Centre, 2350Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada. 3Present address:Insitute of Anatomy, Department of Neuroanatomy, Nussallee, D-53115Bonn, Germany.Authors’ contributionsPG-L performed the largest part of the experiments and participated in thedesign of the study and in the writing of the manuscript. SN participated inthe performance of the experiments and documentation of data. RH and BCparticipated in the design of the study, evaluation of results andinterpretation of data. MS participated in the design of the study, evaluationof results and interpretation of data, and wrote the manuscript. All authorsread and approved the final manuscript.Received: 18 September 2009 Accepted: 25 March 2010Published: 25 March 2010References1. 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