frizzled paper.pdf

7/25/2019 Frizzled paper.pdf

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Development/Plasticity/Repair

Frizzled3 Controls Axonal Polarity and Intermediate Target

Entry during Striatal Pathway Development

FrancescaMorello,1XAsheetaA. Prasad,1Kati Rehberg,1 Renata Vieirade Sa,1Noelia Anton-Bolanos,2

Eduardo Leyva-Diaz,2 YouriAdolfs,1 XFadel Tissir,3 Guillermina Lopez-Bendito,2 andR. JeroenPasterkamp11Department of Translational Neuroscience, Brain Center Rudolf Magnus, University Medical Center Utrecht, 3584 CG, Utrecht, The Netherlands, 2Instituto

de Neurociencias de Alicante, Consejo Superior de Investigaciones Cientficas and Universidad Miguel Hernandez, 03550 Sant Joan dAlacant, Spain, and3Institute of Neuroscience, Universite Catholique de Louvain, Institute of Neuroscience, Brussels 1200, Belgium

Thestriatum isa largebrainnucleus with animportant role in thecontrol of movement andemotions. Mediumspiny neurons (MSNs)are

striatal output neurons forming prominent descending axon tracts that target different brain nuclei. However, how MSN axon tracts inthe forebrain develop remains poorly understood. Here, we implicate the Wnt binding receptor Frizzled3 in several uncharacterized

aspects of MSN pathway formation[i.e.,anteriorposterior guidance of MSN axons in the striatum andtheir subsequent growthinto theglobus pallidus (GP), an important (intermediate) target]. In Frizzled3knock-out mice, MSN axons fail to extend along the anteriorposterioraxisofthestriatum,andmanydonotreachtheGP.Wnt5aactsasanattractantforMSNaxons invitro,isexpressedinaposteriorhigh, anterior low gradient in the striatum, and Wnt5a knock-out mice phenocopy striatal anteriorposterior defects observed inFrizzled3 mutants. This suggests that Wnt5acontrols anteriorposterior guidance of MSN axons through Frizzled3. Axonsthat reachtheGPin Frizzled3 knock-outmice fail to enter this structure. Surprisingly, entry of MSNaxonsinto theGP non cell-autonomously requiresFrizzled3, and our data suggest that GP entry may be contingent on the correct positioning of corridor guidepost cells for thalamocor-tical axons by Frizzled3. Together, these data dissect MSN pathway development and reveal (non)cell-autonomous roles for Frizzled3 in

MSN axon guidance. Further, they are the first to identify a gene that provides anteriorposterioraxon guidance in a large brain nucleusand link Frizzled3 to corridor cell development.

Key words: axon guidance; corridor cell; development; Frizzled3; striatum

IntroductionThe formation of longitudinal axon tracts in the CNS is complexand requires a myriad of molecular signals and specialized cell

types, such as intermediate target and guidepost cells. Progresshas been made in defining the mechanisms that regulate the wir-ing of long ascending and descending pathways in the brainstemand spinal cord (for review, see Mastick et al., 2010; Stoeckli,2006;Van den Heuvel and Pasterkamp, 2008;Zou, 2012). How-ever, how longitudinal axon tracts that originate in the forebrainReceived May 12, 2015; revised Aug. 18, 2015; accepted Sept. 8, 2015.

Author contributions: F.M., A.A.P., G.L.-B., and R.J.P. designed research; F.M., A.A.P., K.R., R.V.d.S., N.A.-B.,

E.L.-D., and Y.A. performed research; F.T. contributed unpublished reagents/analytic tools; F.M., A.A.P., K.R.,

R.V.d.S., N.A.-B., E.L.-D., Y.A., G.L.-B., and R.J.P. analyzed data; R.J.P. wrote the paper.

Thiswork wassupported byThe Netherlands Organization for Health Research andDevelopment(ZonMW-VIDI

and ZonMW-TOP), Stichting ParkinsonFonds, and the European Union (mdDANeurodev, FP7/20072011, Grant

222999) to R.J.P., Act ions de Recherches Concerte es (ARC-1 0/15-02 6) and Fondation medica le Reine Elisabet h toF.T., and the Spanish MINECO BFU2012-34298 to G.L.-B. (who is an EMBO Young Investigator). This study was

performedin partwithinthe frameworkof DutchTopInstitutePharmaProject T5-207toR.J.P.We thankYiminZou

forcriticallyreadingthis manuscript;Ger ArkesteinfortechnicalassistanceduringFACS;theMutantMouseRegional

Resource Center facility at University of CaliforniaDavis; Jeremy Nathans, Marten Smidt, Oscar Marin, Stewart

Anderson, Sonia Garel, and Thomas Jessell for providing mice; Jeremy Nathans for providing rabbit anti-Fzd3 anti-body; and Eljo van Battum for help with the figures.

The authors declare no competing financial interests.

Significance Statement

Striatal axon pathways mediate complex physiological functions and are an important therapeutic target, underscoring the needto define how these connections are established. Remarkably, the molecular programs regulating striatal pathway development

remain poorly characterized. Here, we determine the embryonic ontogeny of the two main striatal pathways (striatonigral andstriatopallidal) and identify novel (non)cell-autonomous roles for the axon guidance receptor Frizzled3 in uncharacterized as-

pects of striatal pathway formation (i.e., anteriorposterior axon guidance in the striatum and axon entry into the globus palli-dus). Further, ourresults link Frizzled3to corridor guidepostcell development andsuggest that an abnormal distributionof these

cells has unexpected, widespread effects on the development of different axon tracts (i.e., striatal and thalamocortical axons).

The Journal of Neuroscience, October 21, 2015 35(42):1420514219 14205


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are established is less well understood. Furthermore, our under-standing of how intermediate targets and guidepost cells developinto choicepoints and permissive corridors for growing axons israther rudimentary(Dickson and Zou, 2010;Garel and Lopez-Bendito, 2014).

Among the most prominent descending longitudinal fibersystems in the forebrain areaxon projections from mediumspiny

neurons (MSNs) in the striatum. The striatum is part of the basalganglia and controls the output of this structure through twodistinct axon tracts: the directand indirect pathways. These path-ways arise from morphologically similar but biochemically andfunctionally distinct subpopulations of MSNs that are intermin-gled within the striatum. The direct pathway originates fromstriatonigral MSNs that, in mice, project axons to theentopedun-cular nucleus and substantia nigra (SN). In contrast, axons fromstriatopallidal MSNsform the indirect pathway and innervate theglobus pallidus (GP) and indirectly influence the SN via the sub-thalamic nucleus (Gerfen and Surmeier, 2011). The striatonigral(direct) and striatopallidal (indirect) pathways provide antago-nistic but balanced outputs that control psychomotor function

(Graybiel, 2005). Changes in striatal connectivity cause behav-ioraldeficitsand underlie disorders,such as Huntingtons disease(Crittenden and Graybiel, 2011; Gerfen and Surmeier, 2011;Russo and Nestler, 2013), underscoring the need to understandhow these connections are established and maintained.

Surprisingly little is known about the extracellular cuesthat regulate the development of MSN axon projections.The transmembrane protein OL-protocadherin (OL-pc orPcdh10) cell-autonomously mediates MSN axon out-growth, whereas interactions between the chemorepellentsemaphorin3E (Sema3E) and its receptor plexinD1 regulatestriatonigral axon pathfinding(Chauvet et al., 2007;Uemuraet al., 2007). The transcriptional regulators Islet1 (Isl1) andCOUP-TF-interacting protein 2 (Ctip2) control plexinD1ex-

pression in embryonic MSNs, whereas Isl1 also regulatessema3Eexpression along the trajectory of striatonigral axons(Arlotta et al., 2008; Ehrman et al., 2013; Lu et al., 2014).Isl1/ mice show altered striatonigral pathfinding in partphenocopying defects observed in sema3E/ mice (Ehrmanet al., 2013). Knock-out mice for the transcription factor earlyB-cell factor 1(Ebf1) also show reduced striatonigral pathwaydevelopment, but the extrinsic effectors involved are un-known (Lobo et al., 2006,2008). Despite this recent progressin our understanding of the intrinsic regulation of MSNaxon pathway development, the extrinsic cues involved re-main largely unknown. For example, molecules controllinganteriorposterior (AP) guidance and GP navigation have yet

to be determined.Here, we determine the ontogeny of the mouse striatal effer-

ent pathways using BAC transgenic reporter mice and identify arequirement forthe Wntbinding receptor Frizzled3(Fzd3) (Zou,2012;Tissir and Goffinet, 2013)in striatal pathway formation.We find that, within the striatum, Fzd3, and its ligand Wnt5a arerequired in vivo for extension of MSN axons along the AP axis. In

addition, Fzd3 noncell-autonomously regulates the entry ofMSN axons into an important (intermediate) target, the GP, andthepositioningof corridor cells.Corridorcellsare a populationof guidepost cells for thalamocortical axons located in betweenthe GP and the medial ganglionic eminence (MGE). The mecha-nisms that regulate corridor cell development remain incom-pletely understood (Bielle and Garel, 2013). Our data indicate

that corridor cells repel MSNaxons andthat their mislocalizationin the GP ofFzd3/mice may render the GP nonpermissive forMSN axon growth. Conclusively, our data reveal previously un-characterized aspects of striatal pathway development and iden-tify cell autonomous and non cell-autonomous roles for Fzd3 inMSN axon guidance. Further, they implicate Fzd3 in the poorlydefined mechanisms underlying corridor cell development.

Materials andMethodsAnimals and tissue treatment.All animal use and care were in accordancewith local regulations and institutional guidelines. Mice of either sex

were used and maintained in a 12 h light-dark cycle and housed 14 percage with food and waterad libitum. C57BL/6 mice were obtained from

Charles River.Frizzled3knock-out mice were kindly provided by JeremyNathans (Johns Hopkins University School of Medicine, Baltimore)(Wang et al., 2002), Pitx3-Cre mice by Marten Smidt (University of

Amsterdam) (Smidt et al., 2012),Islet1-Cremice by Sonia Garel (EcoleNormale Superieure, Paris, France) (Srinivas et al., 2001); with permis-sion from Thomas Jessell, Ryk knock-outmice by StevenStacker(LudwigInstitute for Cancer Research, Melbourne, Australia) (Halford et al.,2000), Gbx2-CreERT2 miceby James Li (University of Connecticut Health

Center, Farmington, CT)(Chen et al., 2009), andNkx2-1-Cremice byOscar Marin (MRC Center for Developmental Neurobiology, London)(Xu et al.,2008); withpermission from Stewart Anderson.Wnt5a knock-out mice (Yamaguchi et al., 1999)and ROSA26-DTAmice(Ivanova etal., 2005) were obtained from The Jackson Laboratory, and Drd2-EGFP,

Chrm4-EGFP, andDrd2-Cre(ER44) BAC transgenic mice from Mutant

Mouse Regional Resource Center (www.gensat.org)(Gong et al., 2003,2007).Frizzled3fl/flmice were as described previously (Chai et al., 2014).The morning of detection of the vaginal plug was defined as embryonic(E) day 0.5, and the day of birth as postnatal (P) day 0. For in situhybridization experiments, embryonic brains were directly frozen, and20 m sections were cut on a cryostat. For immunohistochemistry, em-bryonic brains were collected in PBS and fixed by immersion for 0.5 8 hin 4% PFA in PBS at 4C. Postnatal mice were transcardially perfused

with ice-cold saline followed by 4% PFA and postfixed for 2 h. Samplesintended for immunohistochemistry on cryosections were washed inPBS and cryoprotected in 30% sucrose. Brains were frozen and stored at80C. Cryostat sections were cut at 1625 m. To generate vibratomesections, fixed brains were embedded in 3% low melting point agarose inPBS and sectioned coronally at 150 m using a vibratome (Leica).

Enzymatic dissociation and FACS of striatal neurons. E15.5 or E17.5

embryos from hemizygous D2-EGFPorM4-EFGPmicewerecollected inice-cold L-15 (Invitrogen) followed by dissection of the striatum in L-15with 10% FCS. Striatal tissue was dissociated using the Papain Dissocia-tion System (Worthington Biochem), as previously described (Lobo etal., 2006). Cells were resuspended in L-15 containing 25 g/ml DnaseI(Sigma) and filtered through a 70 m mesh. Cells were treated with

propidium iodide(20 g/ml, Invitrogen) to label dead cells andsortedina Cytopeia Infux cell sorter (528/38 filter for EGFP fluorescence). Wild-type cells were used to calibratethe FITCH andpropidium iodidesignals.Approximately 70,000100,000 striatal cells were obtained per embryofrom each FACS run. EGFP cells were directly collected in RLT lysisbuffer (QIAGEN).

RNA extraction and quantitative PCR.Total RNA extraction was per-

formed from FACS purified striatal neurons using the RNeasy Micro Kit

(QIAGEN) according to the manufacturers protocol. RNA quality andrelative concentration were determined using a spectrophotometer andBioanalyzer Nanochip (Agilent). Each experimental sample consisted of

Correspondence should be addressed to Dr. R. Jeroen Pasterkamp, Department of Translational Neuroscience,

BrainCenterRudolfMagnus,University Medical Center Utrecht,Universiteitsweg 100,3584 CG,Utrecht,The Neth-

erlands. E-mail:[email protected].

F.Morellospresentaddress:Departmentof Biosciences,VikkiBiocenter,Universityof Helsinki,Helsinki,Finland.

A. A. Prasads present address: School of Psychology, University of New South Wales, Sydney, NSW, Australia.

E.Leyva-Diazspresentaddress:Departmentof Biochemistryand MolecularBiophysics,HowardHughesMedical

Institute, Columbia University Medical Center, New York, NY 10032.DOI:10.1523/JNEUROSCI.1840-15.2015

Copyright 2015 the authors 0270-6474/15/3514206-15$15.00/0

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pooled RNA derived from at least two hemizygous D2-EGFPor M4-EGFPembryos. cDNA synthesis, cRNA double amplification, and qual-ity control and fragmentation were performed using an automatedsystem (Caliper Life Sciences) and starting with 70 ng total RNA fromeach sample, as described previously (Chakrabarty et al., 2012). TheqPCR was performed in a 10 l reaction solution using Roche SYBRGreen I (LC-FastStart DNA MasterPlus) and 0.5 M of each primer.

PCRs were performedin a 7900HTFast Real-TimePCR System(AppliedBiosystems) (95C for 3 min, followed by 45 cycles of denaturation at95C, annealing at 55C, and extension at 72C for 30 s each). Primer

sequences are as follows: Fzd1-10and Gapdh(Shah et al., 2009), Drd2(Bice et al., 2008), andHprt1(QuantiTec primer Assay; QIAGEN). Todetect Chmr4, the following primers were used: 5-AGCCGAGCATTAAGAAACCTCCAC-3 (forward); and 5-TCATTGGAAGTGTCCTTGTCAGCC-3 (reverse). Samples were run in triplicate, and a com-mon threshold signal was chosen manually in the linear amplificationrange of all samples by inspecting the log-transformed fluorescence sig-nals plotted against cycle number using SDS software (Applied Biosys-

tems). For each gene, relative expression was calculated using the ddCtmethod (Livak and Schmittgen, 2001)correcting for the amplification effi-ciency of each primer pair and using Gapdh and Hprt1 as reference genes.

Explant, dissociated neuron, and hemislice cultures. Three-dimensionalcollagen matrix assays were performed as described previously(Schmidtet al., 2012). In brief, E13.5-E14.5 wild-type or Fzd3/ and littermatecontrol embryos were collected in ice-cold L-15 and the striatum, GP, orcorridor cells were carefully dissected in ice-cold L-15 containing 2%FCS. Small explants were then cut from the dissected tissue at 200350m and plated in custom-made rat tail collagen. Striatal explants wereplaced 300500m from either (1) aggregates of HEK293 cells tran-siently transfected with EGFP, Wnt5a, or Wnt5b constructs using Lipo-fectamine2000; or (2)GP or corridor cell explants in 4-welltissueculturedishes (Nunc). Explants were cultured in Neurobasal with B27 supple-

ment (Invitrogen), HEPES, -mercaptoethanol, and antibiotics, and in-cubated in a humidified incubator at 37C and 5% CO2for 23 d. Then,

cultures were fixed in 4% PFA for 1 h at room temperature and immu-nostained with anti--tubulin (Covance) and anti-Nkx2-1 antibodies(Biopat), as previously described (Schmidt et al., 2012). Staining wasvisualized using epifluorescent illumination on a Zeiss Aksioskop A1 orby confocal laser scanning microscopy (Olympus FV1000). Nkx2-1staining was used to monitor the success of GP dissection and only co-cultures containing the entire GP were analyzed. Quantifications wereperformed on explants deriving from at least three independent experi-ments. To calculate proximal/distal (P/D) ratios in cocultures, the totalarea covered by striatal axons in the proximal and distal quadrants of thecultures was determined and used to calculate P/D ratios per explant(Schmidt et al., 2012). For quantification of corridor-striatal explantcocultures, the length of the 40 longest axons extending into the quad-

rants proximal and distal to the corridor explant was measured withImageJ software. Once the mean P/D ratio of each explant was deter-mined, a guidance index was calculated as described previously(Bielle etal., 2011b). Data were statistically analyzed by unpaired two-tailedMannWhitney test, one-way ANOVA ( 5%) or KruskalWallis testand expressed as mean SEM.

To generate dissociated striatal neuron cultures, the striatum was mi-crodissected from E15.5D2-EGFPandM4-EGFPembryos. Tissue fromseveral different embryos was pooled and incubated in trypsin inDMEM/F-12 for 20 min at 37C. Trypsin activity was inhibited throughaddition of 20% FCS in DMEM/F12 and tissue was triturated with afire-polished glass pipette. Then, cells were pelleted by mild centrifuga-tion and resuspended in Neurobasal medium supplemented with B27,antibiotics, and glutamine. A single-cell suspension was generated byforcing the cells through a 70 m filter, after which cells were plated onpoly-D-lysine- and laminin-coated glass coverslips in a 12-well plate for3 d in Neurobasal medium. Cultures were fixed in 4% PFA for 10 min atroom temperatureand coimmunostained with rabbitanti-Fzd3 (1:500, agift from Jeremy Nathans) and chicken anti-EGFP (1:500, Abcam) anti-bodies. Images were captured on a Zeiss Aksioskop A1 or by confocallaser scanning microscopy (Olympus FV1000). The length of axonsemerging from EGFP-labeled MSNs was determined in three randomlyselected areas in each culture using OpenLab (Improvision) software.Data from three independent experiments were statistically analyzed byunpaired two-tailed Studentsttest and expressed as mean SEM.

For transplantation experiments, striatal explants (200 m) were mi-crodissected from E14.5Fzd3/ embryos or littermate controls, and asmall DiI crystal (Invitrogen) was placed in each explant, as describedpreviously (Uemura et al., 2007). Striatal explants containing DiI crystals

were then placedinto therostralpart of thestriatum in E14.5wild-typeorFzd3/ hemislices. Hemislices consist of half brains cultured on mem-brane inserts (Corning) with the cortical surface facing up ( Pasterkamp

Figure 1. Development of striatopallidal and striatonigral axon pathways. Immunohisto-chemistryforEGFP(green)incoronalsectionsfrom DopaminereceptorD2 (D2)-EGFPmice(A,D,G, I) andin sagittal sections from Cholinergic receptor, muscarinic4 (M4)-EGFPmice(B, C, E, F,

H,J) at the indicated developmental stages. B,C,E,F,H,J, Rostral is to the left. Sections arecounterstained with DAPI (blue). InD2-EGFPandM4-EGFPmice, the striatopallidal (indirect)and striatonigral (direct) pathways are labeled, respectively. Arrows indicate the striatonigralpathway. At leastn 3 embryos pertimepointper genotype were analyzed.Hip,Hippocam-pus; SNr,substantia nigra, parsreticulata;STR, striatum. Scalebars:A, B, D, E,G, I,100m; C,F, 40m;H,J, 200m.

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et al., 2003;Schmidt et al., 2014). Transplantcultures were grown in Neurobasal mediumsupplemented with B27, antibiotics, and glu-tamineand maintained for 3 d in culture. Thencultures were fixed in PBS containing 4% PFAand 4% sucrose for 1 h at room temperature,washed in PBS, incubated in blocking solution(1% PBS, 1% FCS, 1 mg/ml digitonin) for 1 h

at room temperature, and incubated in pri-mary antibodies overnight 4C. Cultures werewashed extensively in PBS, incubated with Al-exaFluor secondary antibodies for 3 h at roomtemperature, rinsed in PBS, and embedded inFluosave (Calbiochem) or 95% glycerol. Toquantify the transplantation experiments, thepresence of DiI-labeled axons from striatalexplants was assessed using Photoshop soft-ware. TheGP was visualizedby Nkx2-1immu-nostaining, and fluorescence intensity ofstriatal axons was measured in three bins cor-responding to regions rostral to the GP, in theGP, and caudal to the GP followed by back-ground correction. Data from at least threeindependent experiments were statistically an-alyzed by KruskalWallis test, and data werepresented as mean SEM.

In situhybridization and immunohistochem-istry. Nonradioactive in situ hybridization wasperformed as described previously(Pasterkampet al., 2007). Digoxigenin-labeled Fzd3, Wnt5a,Sema3A, Sema3F, ephrinA5, andNetrin-1 in situprobes were as described previously (Kolk et al.,2009;Fenstermaker et al., 2010;Schmidt et al.,2014).For Sema3Eprobes, the followingprimerswere used to generate probe templates: 5-CCGTACTGTGCCTGGGATGGC-3 (forward), 5-GGGGTTGGCATACTTCCACTT-3 (reverse);

and for Ebf1 5-GCTCACTTTGAGAAGCAGCCG-3 (forward), 5-CGTACCTTCCGAGGGGTCAAG-3 (reverse). Probe hybridization wasperformed overnight, and sense probes were in-cluded as specificity controls. Sections subjected tothe entire in situ hybridization procedure, but withno or sense probe added, did not exhibit specifichybridization signals.

Immunohistochemistry on cryostat or vi-bratome sections was as described previously(Kolk et al., 2009). In brief, sections were incu-bated in blocking solution (1% PBS, 1% BSA,0.1% Triton X-100) for 30 min at room temper-aturefollowed by incubationin primary antibod-ies in blocking buffer overnight at 4C. The next

day, sections were rinsedseveraltimes in PBSandincubated with appropriate AlexaFluor second-ary antibodies for 1 h at room temperature. Sec-tions were counterstained with DAPI (Sigma),washed extensively in PBS, and embedded inProlong Gold Antifade reagent (Invitrogen).Staining was visualized using epifluorescent illu-mination on a Zeiss AksioskopA1 or by confocallaser scanning microscopy (Olympus FV1000).

The following primary antibodies were used:

rabbit anti-EGFP (1:500, Invitrogen, A11122),

chicken anti-EGFP (1:500, Abcam AB13970), rabbit anti-DARPP32 (1:500,

Santa Cruz Biotechnology SC-11365), rabbit anti-Nkx2-1 (1:2000, Biopat),

rabbit anti-Frizzled3 (1:500; a gift from Jeremy Nathans), rabbit anti-

tyrosine hydroxylase (TH) (1:1000; Pel-Freeze), rat anti-OL-protocadherin(1:2000, Millipore, clone 5G10), mouse anti-III-tubulin (1:3000, Sigma,

T8660), mouse anti-Islet1 (1:100; Developmental Studies Hybridoma

Bank, 394D5), mouse anti-neurofilament (1:50; Developmental Studies

Hybridoma Bank, 2H3), rat anti-Ctip2 (1:500, Abcam, ab18465), and

rabbit anti-Foxp1 (1:250; Abcam, ab16645). Secondary antibodies were

AlexaFluor-488, AlexaFluor-555,or AlexaFluor-594conjugated (1:500, Sigma).To assess and quantify MSN axon orientation defects, sagittal sections

from E17.5-E18.5 mutant embryos (n 3) and littermate controls

Figure 2. Molecular profiling ofFrizzled expression in striatopallidal and striatonigral MSNs. A, Scatter plots showingthe distribution of EGFP-positive MSNs purified from E15.5 and E17.5 D2-EGFPembryos compared with reference tissue. EGFP-

positive neurons (outlined area) were selected by FACS. PI, Propidium iodide; wt, wild-type. BD, Quantitative PCR was used to

determine relative expression ofDopamine receptor D2(Drd2; marks striatopallidal MSNs),Cholinergic receptor, muscarinic 4

(Chrm4; marks striatonigral MSNs), andFrizzled3in D2-EGFP or M4-EGFP cells obtained by FACS at E15.5 or E17.5. Drd2is

expressedinD2-EGFP butnotinM4-EGFP cells,whereasChrm4 isexpressedinM4-EGFP butnotD2-EGFP cells, confirm-

ingthespecificityoftheFACSprocedure.Of10Frizzledstested,only Fzd2 (datanotshown)and Fzd3 showeddetectableexpression

inE15.5andE17.5MSNs.n 3independentexperiments.**p0.01(two-wayANOVA).***p 0.001(two-wayANOVA).EJ,

Coimmunostaining for EGFP and Frizzled3 in dissociatedstriatal neuron cultures generated from E15.5M4-EGFPor D2-EGFPmice

analyzed at 3 DIV. Arrows indicate axons.M4-EGFP n 3 litters,D2-EGFP n 3 litters. Scale bars: EJ, 40m.



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(n 3) were immunostained with anti-DARPP32 antibodies and coun-terstained with DAPI. Images were captured from these sections at iden-tical medial to lateral locations in the middle portion of the striatumusing a Zeiss Axioskop A1 microscope. Then, using Axiovision software(Zeiss), angles were determined between baseline (a line dividing thestriatum in a dorsal and ventral half, represents 0) and the trajectory ofthe initial axon segment just proximal to the DARPP32-positive cellbody. Fifty to 100 DARPP32-positive neurons were measured per em-bryo, andangles were grouped andcalculated as percentage of total (Fen-stermaker et al., 2010). For assessing DARPP32 axon density at the GP,two to five embryos were analyzed at E17.5-E18.5 and two or three well-spaced (80 m) sections at the same AP level were imaged and assessed.A 250 m rectangle was placed 50 m from the dorsal surface of theNkx2-1-positive GP andDARPP32 axon densitywas assessed in the rect-angle using ImageJ software(Kolk et al., 2009). In each embryo, signals

were normalized to DARPP32 staining in thecortex, which wassimilar inwild-type and mutant mice. Data were averaged per embryo, and datafrom several individual animals were pooled. Finally, data from mutant

mice were normalized to control and statisti-callyanalyzedby unpaired two-tailed Studentsttest.

Quantification and statistics methods.Statis-tical analyses were performed using IBM SPSSStatistics by Studentsttest, one-way ANOVA,or KruskalWallis. All data in this manuscriptare derived from at least three independently

performed experiments. All data were ex-pressed as mean SEM, and significance wasdefined asp 0.05.

ResultsDevelopment of striatopallidal andstriatonigral axon projectionsMSNs account for the vast majority of allstriatal neurons (90%95%) and are theoutput projection neurons of the stria-tum. The remaining 5%10% of striatalneurons are interneurons with local con-nections within the striatum. The twoclasses of MSNs that occupy the striatum,striatopallidal and striatonigral neurons,are morphologically indistinguishableand intermingled in the striatum. As a re-sult of theseanatomical features, relativelylittle is known about the spatiotemporaldevelopment of striatopallidal and stria-tonigral MSNs and their axon projec-tions. Here we exploited BAC transgenicmouse lines in which striatopallidal andstriatonigral MSNs are specifically and in-dependently labeled with GFP(Gong etal., 2003). In Dopamine receptor D2(Drd2)-EGFP (D2-EGFP) mice, striato-

pallidal MSNs and their axons are labeled,whereas in Cholinergic receptor, musca-rinic 4 (Chrm4)-EGFP(M4-EGFP) micestriatonigral MSNs and axons are labeled.These mouse lines have been used previ-ously to examine functional and molecu-lar properties of postnatal and adultstriatopallidal and striatonigral neurons(Lobo, 2009), but a comprehensive analy-sis at embryonic and early postnatal stagesis lacking. The bulk of MSNs is generatedfrom E13 onwards (Passante et al., 2008),and we therefore started our analysis at

E13.5. At E13.5, EGFP

cells were observed in the striatum ofbothD2-EGFPandM4-EGFPembryos(Fig. 1A, B). InD2-EGFPmice, these cells were largely confined to the striatum, whereascellular EGFP expression in M4-EGFPmice was broader withlabeling in surrounding structures, such as the ganglionic emi-nences. In both lines, EGFP striatal axons were readily discern-ible and extended posteriorly to the GP at E13.5 (Fig. 1AC). Inaddition, the first striatonigral axons already contacted their tar-get, the SN, at this stage (Fig. 1C). Comparison ofD2-EGFPand

M4-EGFPmouse embryos showed that the development of thestriatopallidal pathway appeared delayed compared with that ofthe striatonigral pathway. For example, at E15.5 and E17.5, aprominent striatonigral bundle was present in M4-EGFPmice,

and numerous EGFP

axons projected into midbrain. In con-trast, despite the presence of a large number of strongly labeledEGFP neurons in the striatum, a relatively small subset of

Figure3. Frizzled3/miceshowdefectsinstriatalpathwaydevelopment.A,Schematicsofsagittalsectionsoftheembryonicbrain showing the striatum (STR) and its axon projections in green. Red lines and box indicate the origin ofBM. BI, Immuno-histochemistry for DARPP32 (green) in coronal sections from E17.5Fzd3/ andFzd3/mice obtained from different rostro-caudal levels. Sections are counterstained with DAPI (blue). White arrows indicate the striatonigral bundle. Red arrow indicatesaccumulation of striatal axonsat thelevel of theGP. AC,Anteriorcommissure;Hip, hippocampus;STR, striatum.J, K, Immunohis-tochemistry for EGFP in coronal sections from E13.5Fzd3/;D2-EGFPandFzd3/;D2-EGFPembryos. DAPI in blue. Red arrowindicates accumulation of striatopallidal axons. White dotted lines indicate GP. Red dotted line indicates striatal axons. L,M,Immunohistochemistry for EGFP and Nkx2-1 (red) in coronal sections from E17.5 Fzd3/;M4-EGFPand Fzd3/;M4-EGFPembryos.WhitedottedlinesindicateGP.AsterisksindicateaxonbundlesattheGP.Numberofmiceanalyzed: BI,E17.5 Fzd3/,n 10; E17.5Fzd3/,n 12;J, K,E13.5 Fzd3/;D2-EGFPorFzd3/;D2-EGFP,n 3; L,M,E17.5 Fzd3/;M4-EGFPorFzd3/;M4-EGFP,n 5. Scale bars: BI, 190m;JM, 95m.



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EGFP axons extended into the GP atthese stages in D2-EGFPmice (Fig. 1DH). However, by P7, patterns of innerva-tion resembled those detected in themature brain in both lines. The GP washeavily innervated inD2-EGFPmice, andEGFP axons traversed the GP in tight

axon fascicles inM4-EGFPmice to furtherfasciculate into a compact axon bundle atmore caudal levels and innervate the SN(Fig. 1 I,J). Together, these results revealpreviously unexplored aspects of stria-tonigral and striatopallidal axon pathwaydevelopment and identifyD2-EGFPand

M4-EGFPmice as valuable tools for theanalysis of striatal axon pathfinding andtarget innervation.

Molecular profiling of Frizzleds instriatopallidal and striatonigral

neuronsTheD2-EGFPandM4-EGFPmouse linesnot only provide a tool for visualizing stri-atal pathways (Fig. 1) but also enable thepurification of MSNs by FACS (Lobo etal., 2006). We exploited this property tosearch for molecular cues involved in stri-atal pathway development. Pure popula-tions of striatopallidal and striatonigralMSNs were obtained from E15.5 andE17.5 D2-EGFP and M4-EGFP mice byFACS (Fig. 2A). At these developmentalstages, striatal axons are actively navigat-ing toward and into their target structures

(Fig. 1). Analysis of the expression ofDrd2 and Chmr4, markers for striato-pallidal and striatonigral MSNs, respec-tively, confirmed the specificity of thepurification procedure (Fig. 2 B, C).Next, we performed quantitative PCR(qPCR) to detect expression of mem-bers of different axon guidance ligandand receptor families (Kolodkin andPasterkamp, 2013) (data not shown).Among the most strongly expressedcandidates was the Wnt binding andplanar cell polarity receptor Frizzled3

(Fzd3), which was detected at equal lev-els in E15.5 and E17.5 striatopallidal andstriatonigral MSNs (Fig. 2D). Fzd3 haspreviously been implicated in the devel-opment of longitudinal axon bundles that, analogous to stri-atal projections, extend along the AP axis of the CNS (Zou,2012). In addition, Fzd3 is enriched in the embryonic stria-tum, and the striatum ofFzd3/ embryos displays increasedcell death from E18 onwards, hinting at reduced target inner-vation by striatal projections (Wang et al., 2002). Together,these observations identify Fzd3 as a strong candidate for con-trolling striatal axon development. To further examine therole of Fzd3 in MSNs, we performed immunohistochemistry

for Fzd3 in dissociated neuron cultures from E15.5D2-EGFPandM4-EGFPmice. Fzd3 was expressed in the somata, axons,and growth cones of most EGFP striatopallidal and stria-

tonigral MSNs (Fig. 2EJ). Thus, Fzd3 is expressed in MSNsand their axons during the period of axonal growth andpathfinding.

Frizzled3 is required for growth of striatal axons toward andinto the GPTo examine whether Fzd3 is required for striatal pathway devel-opment in vivo, we performed immunohistochemistry fordopamine- and cAMP-regulated phosphoprotein (DARPP32) in

E17.5 wild-type andFzd3/

embryos. DARPP32 is expressed in95% of MSNs but not by other cell types in the striatum(An-derson and Reiner, 1991). Although expression of DARPP32 was

Figure 4. MSN differentiation is intact inFrizzled3/ mice. Immunocytochemistry for OL-protocadherin (OL-pc) (A, B),COUP-TF-interacting protein 2 (Ctip2) (E, F,J, L),orforkheadboxproteinp1(Foxp1)(G,H,M,P,Q, T)oncoronalsectionsofE13.5or E16.5Fzd3/ orFzd3/ embryos. Several sections are counterstained with DAPI. C, D,In situhybridization forearly B-cell

factor1 (Ebf1)oncoronalsectionsofE16.5Fzd3/

or Fzd3/

embryos. Fzd3/

mice have a slightlyenlargedbrainbutshowno obvious defect in theexpressionor distributionof various striatal markers (OL-pc, Ctip2, Foxp1,Ebf1).Cx, Cortex; STR,striatum.Scale bars:A, B, 400m; C, D, 400m; EH, 550m; IL, 650m;MO, QS, 500m; P, T, 250m.



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decreased in the striatum ofFzd3/ embryos(Fig. 3AE), anal-ysis of the expression of various other genes known to be ex-pressed in MSNs (OL-pc, Ebf1, Ctip2, forkhead box protein p1

(Foxp1), Isl1, Netrin-1) did not reveal a marked loss of MSNs(Figs. 4,Fig. 10). This is in line with previous work showing thatthe proliferation, distribution, and number of striatal neurons issimilar in wild-typeand Fzd3 mutant mice until E18(Wang et al.,2002). Analysis of DARPP32 axons revealed that in wild-typemice MSN axons projected caudally in the striatum toward theGP, entered the GP forming large bundles that eventually tar-geted the midbrain. In contrast, in Fzd3/ embryos striatal ax-ons accumulated at the GP and did not enter this nucleus norproject caudally toward the SN(Fig. 3AI). In mice, striatopalli-dal axons target the GP, whereas striatonigral axons traverse theGP to innervate the entopeduncular nucleus and the SN (Gerfenand Surmeier, 2011). Because DARPP32 labels both axon types,

we next generated Fzd3/

;D2-EGFP and Fzd3/

;M4-EGFPmice to assess whether or not both striatopallidal and striatoni-gral pathways are affected. MSN axons accumulated in very close

proximity to the GP in Fzd3/;D2-EGFP and Fzd3/;M4-EGFP mice but never entered this structure, confirming theDARPP32 data and showing that both striatonigral and striato-pallidal axons are affected (Fig. 3JM).

In addition to a failure to grow into the GP, we noted thatfewer EGFP axon bundles appeared to arrive at the rostral bor-der of GP in E17.5 Fzd3/;D2-EGFPand Fzd3/;M4-EGFP

mice, compared with wild-type controls(Fig. 3 L,M). Quantifi-cation of axonal EGFP signals at the GP at E17.5 confirmed thisobservation (relative axonal intensity at GP: Fzd3/;D2-EGFP100 3.3, Fzd3/;D2-EGFP 81.1 2.8; Fzd3/;M4-EGFP100 3.9, Fzd3/;M4-EGFP76.0 4.1;n 3 mice per geno-type;p 0.01). Thus, our results indicate that, inFzd3/mice,(1) fewer MSN axons project fromthe striatumto the GP, and (2)no MSN axons enter the GP.

To further characterize these MSN axon defects, we geneti-cally ablatedFzd3from (1) striatonigral or striatopallidal MSNsby crossing conditionalFzd3fl/flmice(Chai et al., 2014) withIsl1-Cre (Srinivas et al., 2001) or D2-Cre (Gong et al., 2007)mice,respectively, or (2) GP neurons by crossing Fzd3fl/fl mice with

Nkx2-1-Cremice(Xu et al., 2008). Isl1-Cre;ROSA26-EYFPandD2-Cre;ROSA26-EYFPmice revealed robust recombination inthe early embryonic striatum but not in the GP (Fig. 5A, B,E).MSN axons entered and crossed the GP in Isl1-Cre;Fzd3fl/flandD2-Cre;Fzd3fl/flmice (Fig. 5C, D, F,G). However, the number ofaxons arriving at the GP was reduced in Isl1-Cre;Fzd3fl/fl andD2-Cre;Fzd3fl/flmice compared with littermate controls (relativeaxonal intensity at GP: Isl1-Cre;Fzd3/, 100 5.1; Isl1-Cre;Fzd3fl/fl, 78.2 3.8;D2-Cre;Fzd3/, 100 4.5;D2-Cre;Fzd3fl/fl,81.8 3.2;n 3 per genotype;p 0.01).Nkx2-1-Cre;ROSA26-EYFPmice revealed robust recombination in the GP and MGEbut not in the early embryonic striatum, except for a small num-ber of interneurons (Fig. 5H). MSN axons entered and crossedtheGPinNkx2-1-Cre;Fzd3fl/flmice(Fig. 5 I,J), and the number of

axons arriving at the GP was similar inNkx2-1-Cre;Fzd3/ andNkx2-1-Cre;Fzd3fl/flmice (relative axonal intensity at GP:Nkx2-1-Cre;Fzd3/, 100 5.1;Nkx2-1-Cre;Fzd3fl/fl, 99.8 4.7;n 3per genotype;p 0.8351). Together, these data support a cell-autonomous role for Fzd3 in MSNs during the growth of MSNaxons toward the GP and a noncell-autonomous role in theirsubsequent entry into the GP.

Frizzled3 dictates the AP guidance of striatal axonsA decrease in the number of MSN axons arriving at the rostralborder of the GP in Fzd3/, Isl1-Cre;Fzd3fl/fl, or D2-Cre;Fzd3fl/fl mice could simply reflect a reduction in MSN axonoutgrowth. However, analysis of axon outgrowth in dissoci-

ated MSN cultures derived from E15.5Fzd3

/

;D2-EGFPorFzd3/;M4-EGFPmice, and littermate controls did not re-veal a difference in axon outgrowth between wild-type andmutant cultures (data not shown). Frizzleds have been shownto regulate AP guidance of axons in the brainstem and spinalcord. Therefore, another explanation for the decrease in thenumber of striatal axons, reaching the GP is a defect in axonalAP guidance in the striatum. To test this model, we analyzedtheorientation of MSNcellbodies andaxons in thestriatum ofwild-type and Fzd3/, Isl1-Cre;Fzd3fl/fl, or D2-Cre;Fzd3fl/fl

embryos. In wild-type embryos, many DARPP32 MSN cellbodies were oriented toward the GP with their axons runningposteriorly in a specific mediallateral plane within the stria-

tum. MSN cell bodies at the same anatomical location in (con-ditional) knock-out mice were oriented more randomly, andtheir axons were misrouted along the lateralmedial and

Figure5. Conditional ablation of Frizzled3in thestriatumand GP.A,SchematicofacoronalsectionindicatingthelocationofBJ.Cx,Cortex;STR,striatum. B, E,H, Immunohistochemistry

for EYFP on E13.5 coronal sections inD2-Cre;Rosa26-EYFP,Isl1-Cre;Rosa26-EYFP, andNkx2-1-Cre;Rosa26-EYFPmice. D2-Cre;Rosa26-EYFPand Isl1-Cre;Rosa26-EYFPmiceshowspecificlabel-ing of the STR (B,E).Nkx2-1-Cre;Rosa26-EYFPmice show labeling of the GP (dotted line as

determinedby Nkx2-1immunolabeling; datanot shown). EYFP cellsintheSTRareinterneu-rons.C,D,F,G,I,J, Immunohistochemistry for DARPP32 (red) in coronal sections from E18.5mice.SectionsarecounterstainedwithDAPI(blue).SpecificablationofFzd3inMSNs(in D2-Cre;Fzd3fl/flor Isl1-Cre;Fzd3fl/flmice)reducesMSNaxongrowthtowardGPbutdoesnotaffectentryof axons into the GP. Ablation of Fzd3 in the GP (in Nkx2-1-Cre;Fzd3fl/flmice) does not affectstriatalaxongrowthtoward orintothe GP.n 3 mice analyzedfor each genotype.Scale bars:B, E, 375m;H, 400m; C, D, F, G, 600m; I,J, 900m.



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rostralcaudal axes (i.e., no longer con-fined to their normal posterior direc-tion) (Fig. 6AC). The only knownligands for Fzd3 are Wnts, and gradientsof Wnt proteins can guide longitud-inal axon tracts along the AP axis inthe brainstem and spinal cord in a

Fzd3-dependent manner (Zou, 2012).Whether similar mechanisms functionin the forebrain is unknown. SeveralWnts are expressed in the embryonicstriatum, but only Wnt5a and Wnt5bdisplay an anterior low, posterior highexpression pattern consistent with a rolein AP guidance (Fig. 6 D, E) (Fenster-maker et al., 2010). Therefore, we deter-mined whether or not Wnt5a andWnt5b can direct the growth of striatalaxons using a previously establishedcollagen matrix assay (Schmidt et al.,

2012). When confronted with cell ag-gregates releasing Wnt5a or Wnt5b, ax-ons emanating from striatal explantsdisplayed biased growth toward the cells(Fig. 6F). Quantification of P/D ratiosin these cultures confirmed the axon attrac-tive effect of Wnt5a and Wnt5b (con, P/Dratio,1.050.05; n14;Wnt5a,P/D ratio,1.45 0.11; n 10; Wnt5b, P/D ratio,1.38 0.09;n 14,p 0.001, KruskalWallistest). Previouswork identifiesFzd3asan axonal receptor for instructive Wnt5agradients during axon pathfinding (Fen-stermaker et al., 2010). To determine

whether, similar to Fzd3, Wnt5a is requiredin vivo for striatalaxon guidance, Wnt5a/

mice (Yamaguchi et al., 1999) were ana-lyzed. DARPP32 axons entered the GP inWnt5a/miceanddidreach the SN. How-ever, innervationof theSN wasdramaticallyreduced in the absence of Wnt5a, and thenumber of axons that arrived at the GP wasdecreased (Fig. 6GJ). Although DARPP32expression in the striatum was reduced,quantification showed that a smaller subsetof axons arrived at the GP in Wnt5a/

mice compared with wild-type mice (rela-

tive axonal intensity at GP normalized toneuronal DARPP32 expression: Wnt5a/,1003.3; Wnt5a/, 71.2 3.9; n 3mice per genotype; p 0.01). Similar toFzd3/ mice, many DARPP32 axons inWnt5a/mice were no longer confined totheir normal posterior trajectory in thestriatum (Fig. 6K). Analysis of mice defi-cient for Ryk (Halford et al., 2000), anotherWnt5a receptor, did not reveal obvious de-fects (Fig. 6K). These results, together withWnt expression analysis and data from col-lagen matrix assays, suggest that Fzd3 and

Wnt5a cooperate to attract MSN axonsintotheir normal posterior direction in thestria-tum toward the GP.

Figure6. Frizzled3 andWnt5aregulateAP guidanceof striatal axons.A,Top,Theembryonicbrainwiththestriatumand

its projections in green. Red box represents po sition of bottom panels. Dotted line indicates baseline for qua ntification in

B,C, andK. Bottom, Immunohistochemistry for DARPP32 (green) in sagittal sections from E17.5 Fzd3/ andFzd3/

mice obtained from similar mediallateral levels. InFzd3/mice, most MSN cell bodies and axons are oriented poste-

riorly (white arrows), whereas in Fzd3/ mice MSNs project their axons more randomly (white arrows). MSN axons

project in an abnormal lateralmedial direction and are therefore for the most part not visible. B , Quantification of the

angles between baseline (0, black dotted line inA) and the trajectory of the axon segment just prox imal to the cell body.Inset, Axon orientation plots for a few rando mly selected axons. Number of mice analyzed: E17.5 Fzd3/,n 4; E17.5

Fzd3/,n 4. C, Quantification of MSN axon orientation in E18.5D2-Cre;Fzd3fl/florIsl1-Cre;Fzd3fl/flmice and littermate

controlsasinB. n 3miceforthedifferentgroups.D, Insitu hybridizationforWnt5a oncoronalsectionsfromE15.5mouse

brain. Images containing the anterior and posterior striatum (STR) are shown. Dotted lines outline the striatum. Insets,

Higher magnification of ISH signals in the striatum.Wnt5aexpression displays an anterior low, pos terior high gradient in

the striatum and surrounding regions (cortex, LGE). Sections incubated with Wnt5a sense probes do not exhibit specific

labeling. LV, Lateral ventricle. E, In situhybridization for Wnt5bon a sagittal section from E14.5 mouse brain. Image

obtained fromwww.genepaint.org(EH3735)(Visel et al., 2004).Wnt5bis expressed in an anterior low, posterior high

gradient in the striatum (STR). F, Collagen matrix cultures of E14.5 striatal explants cultured adjacent to HEK293 cells

(arrowhead) secreting either control protein, Wnt5a (data not shown) or Wnt5b. Con, n 14 explants; Wnt5a,n 10;

Wnt5b, n 14. GJ, Immunohistochemistry for DARPP32 (green) in coronal sections from E17.5 Wnt5a/ and

Wnt5a/miceobtainedattheleveloftheSTRandGP( G, I)ortheSN(H,J).SectionsarecounterstainedwithDAPI(blue).

K, Quantification of the angles as in B. Number of mice analyzed: E17.5Wnt5a/,n 3; E17.5Wnt5a/,n 3; E17.5

Ryk/

,n

3. Scale bars:A, 20 m;D, 90 m;D, insets, 30 m;E, 1 mm;F, 250 m;GJ, 100 m.



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Non cell-autonomous role for Frizzled3 during entry of MSNaxons into the GPMSN axons that do reach the GP inFzd3/ mice fail to enterthis nucleus. The observation that selective ablation of Fzd3from MSNs in Isl1-Cre;Fzd3fl/fl or D2-Cre;Fzd3fl/fl embryosdoes not cause MSN axon stalling at the GP hints at a noncell-autonomous rolefor Fzd3in GP entry. To further confirmthis model, we performed in vitro transplantation experi-ments. Striatal explants were dissected from wild-type orFzd3/ embryos and grafted into the striatum of wild-typeand mutant hemislice cultures adjacent to the GP, at the onsetof striatalaxon pathfinding(Fig. 7A). Although thissetup doesnot allow analysis of striatal AP guidance given the close prox-

imity of the striatal explants to the GP, it can be used to mon-itor axon growth into the GP. As the majority of MSNs aregenerated from E13 onwards(Passante et al., 2008), explants

and hemislices were dissected at E14.5and cultured for 3 din vitro(DIV). Im-munohistochemistry for DARPP32 inDIV3 hemislices revealed prominentstriatal projections emanating from thestriatum toward synaptic targets, suchas the SN, as observedin vivo, confirm-

ing the physiological relevance of theculture system (Fig. 7B). Explants werelabeled with the lipophilic dye DiI andthe GP, in the hemislices, was visualizedusing whole-mount immunohistoche-mistry for Nkx2-1. At DIV3, wild-typegrafts send out DiI-labeled axons intothe endogenous striatum and the GP. Inaddition, a subset of DiI-labeled axonsalready extended beyond the GP towardthe midbrain (Fig. 7CF,K). Intrigu-ingly, axons derived from Fzd3/

grafts extended into and beyond the GP

in wild-type hemislices (Fig. 7G, H, K).The irregular trajectories of individualDiI-labeled wild-type and knock-outaxons, which are clearly distinct fromendogenous axon tracts, suggestthat ax-ons emanating from the grafts do notsimply follow endogenous axons butrather engage in active pathfinding. Instriking contrast to the ability of wild-type and Fzd3/ striatal axons tonavigate the GP in wild-type hemisli-ces, axonal projections from wild-typegrafts stalled at the border of the GP inhemislices derived fromFzd3/ mice,

resembling the defects observed inFzd3/ micein vivo(Fig. 7IK). Thesedata together with the intact growth ofMSN axons into the GP in Isl1-Cre;Fzd3fl/florD2-Cre;Fzd3fl/flembryos(Fig.5) support a non cell-autonomous rolefor Fzd3 during the entry of MSN axonsinto the GP.

Axon entry into the GP is independentfrom reciprocal axon-axon interactionsOne mechanism through which Fzd3could noncell-autonomously regulate

striatal pathfinding at theGP is by controlling thedevelopmentofother axon tracts on which striatal axons depend for guidance.For example, interactions between reciprocal axon projectionsregulate pathfinding in the embryonic subpallium and midbrain(Deck et al., 2013;Schmidt et al., 2014). During early embryonicstages, striatal axons at the GP grow in close proximity to dopa-minergic and thalamocortical axons en route to the striatum andcortex, respectively (Fig. 8AD) (Uemura et al., 2007). Otherprojections, such as corticothalamic and cortifugal axons, arriveat the GP after striatal axons have entered this nucleus (Uemuraet al., 2007;Leyva-Daz and Lopez-Bendito, 2013). In Fzd3/

mice, dopaminergic and thalamocortical pathways are disruptedand fail to enter the subpallium (Wang et al., 2002; Fenstermaker

et al., 2010). To evaluate a model in which striatal axons usereciprocal dopaminergic or thalamocortical projections to tra-verse the GP, we performedin vivogenetic ablation studies. DTA

Figure 7. Frizzled3 noncell-autonomously regulates the entry of striatal axons into the GP.A, Schematic illustrating the

transplantationofFzd3/ (WT)or Fzd3/explantsintoWTor Fzd3/hemislicesfollowedbystriatalpathwayanalysisusingDiI labeling. STR, Striatum.B, E14.5 hemislice cultured for 3 DIV and subjected to whole-mount immunostaining for DARPP32(green).Cx,Cortex.DottedlinesindicateboundariesofhemisliceandGP. CJ,ExamplesofWTandFzd3/explantstransplantedinto hemislices and analyzed at DIV3. DiI labeling in red and immunostaining for Nkx2-1, to label the GP, in green. C, Dotted lineoutlines hemislice. D,G,I, Dotted line outlines the GP. DiI-labeled striatal axons from WT explants fail to enter the GP in KOhemislices(I,J). K, Quantification of thenumber of hemislicesshowingDiI-labeledprojections fromtransplanted explants rostralto the GP, in the GP, or caudal to the GP. **p 0.01 (KruskalWallis test). ***p 0.001 (KruskalWallis test). Data representmean SEM. Scale bars: B, 100m; C, 2.2 mm; D, G, I, 75m; E, F, H,J, 20m.



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mice, whichconditionally express subunitA of the diphtheria toxin, were crossedwithPitx3-Creor Gastrulation Brain Ho-meobox 2 (Gbx2)-CreERT2 mice to ablatethe midbrain dopamine system and prin-cipal nuclei of the thalamus, respectively(Chen et al., 2009; Smidt et al., 2012).

E17.5 Pitx3-Cre;DTA embryos displayed aloss of midbrain dopamine neurons andtheir axon projections in the midbrainand forebrain, including those in the me-dial forebrain bundle, as visualized by im-munohistochemistry for TH(Fig. 8 E, G).However, DARPP32 MSN axons werepresent andprojected into andbeyond theGP in Pitx3-Cre;DTA mice (Fig. 8F, H).Similarly, inGbx2-CreERT2;DTA mice, inwhich thalamocortical axons from princi-pal thalamic nuclei were ablated, naviga-tion of the GP by DARPP32MSN axons

was comparable with control (Fig. 8IL).Thus, MSN axons do not rely on reciprocaldopaminergic and thalamalocortical axonsfor navigation into and beyond the GP.

The permissive effect of the GP onstriatal axons is dependent on Frizzled3An alternative explanation for the accu-mulation of axons at the GP in Fzd3/

mice is that Fzd3 deficiency affects the GPand thereby striatal axon pathfinding atthis nucleus. Complex target-mediatedmolecular mechanisms regulate the entryof axons at other (intermediate) targets,

such as the spinal cord midline (Derijck etal., 2010; Dickson and Zou, 2010; Che-dotal, 2011;Izzi and Charron, 2011). Therefore, we first exam-ined whether the GP releases instructive cues for striatal axons.Striatal and GP explants were cocultured in collagen matrices(Schmidt et al., 2014). Axons emanating from wild-type stri-atal explants showed preferred growth toward wild-type GPexplants, indicating that this nucleus secretes attractive or per-missive cues for striatal axons (Fig. 9A, E). Interestingly,Fzd3/ striatal explants also displayed biased growth towardwild-type GP explants. In contrast, quantification showed thatwild-type andFzd3/ striatal explants do not display biasedgrowth towardFzd3/GP (Fig. 9BE). Thus,Fzd3deficiency

causes loss of GP-derived permissive signals but leaves theability of striatal axons to sense these signals in wild-type GPtissue intact. It is unlikely that Fzd3 directly promotes theexpression of permissive cues in GP cells, or represses inhibi-tory ones, becausein situhybridization forFzd3showedFzd3expression in the embryonic striatum, but no labeling in themid-embryonic GP (Fig. 9FH). In addition, we found nodifference in the ability of striatal axons to enter the GP in

Nkx2-1-Cre ;Fzd3fl/fl mice, in which Fzd3 is genetically ablatedfrom most GP neurons (Fig. 5). Together, these data argueagainst a direct role for Fzd3 in the regulation of molecularcues produced by GP neurons.

As an alternative model, we examined whether Fzd3 con-

trols the cellular make-up of the GP and thereby indirectly thepermissive effect of this nucleus. Immunohistochemistry wasperformed for different molecular markers known to be ex-

pressed in and around the GP (Fig. 10A). Immunostaining forNkx2-1 revealed that Nkx2-1 GP neurons occupied a largerarea in Fzd3/ mice, but that their number was unchanged(E17.5 Fzd3/ 481.7 1.2 Nkx2-1 cells, Fzd3/ 480.7 1.3; n 3 per genotype; p 0.607) (Fig. 10 B, C). A similarresult was obtained by using in situ hybridization for two otherGP markers,Sema3EandNetrin-1.Sema3E, andNetrin-1ex-pression was detected but occupied an expanded area (Fig.10DG). Expression of OL-pc, a marker for MSN neurons andaxons, was unaffected in the striatum ofFzd3/ embryos, butas expected OL-pc striatal axons did not project into the GP(Fig. 10H, I). Intriguingly, double immunolabeling forNkx2-1 and Islet1 (Isl1) revealed an invasion of Isl1 cells intothe GP ofFzd3/mice (Fig. 10J, K). Isl1 is expressed in stria-tonigral MSNs and in corridor cells, a population of guide-post cells for thalamocortical axons located in between the GPand MGE (Lopez-Bendito et al., 2006). Double labeling forIsl1 and Ctip2 confirmed the presence of ectopic Isl1 cells inthe Fzd3/ GP (Fig. 10 L,M). At early embryonic stages,Ctip2 labels the GP, striatum, and corridor cells, but at laterstages (E15.5 onwards) becomes restricted to the striatum andcorridor cells. Interestingly, at E15.5 ectopic Ctip2 cells wereobserved in theE15.5 GP ofFzd3/ but not control mice (Fig.10 L,M, inset). To determine whether the ectopic Isl1;

Ctip2

cells in the Fzd3

/

GP represent MSNs or corridorcells, we performed immunostaining for Foxp1, which is ex-pressed in MSNs but not in corridor cells. Immunolabeling

Figure8. Striatal axonsdo not requiredopaminergicor thalamocorticalprojectionsto enterthe GP.AD, Immunohistochem-istry for EGFP (green) and TH (red) in coronal sections of E13.5 or E15.5M4-EGFPembryos. Arrowhead indicates dopaminergicaxons.Dottedline indicatesGP. Striatal and dopaminergicaxons growin closeproximityduring earlydevelopment.Cx, Cortex; ic,internal capsule; STR, striatum; Thal, thalamus. EH, Immunohistochemistry for TH (E, G) or DARPP32 (F, H) in sagittal cryosec-tionsof E17.5 WT;DTA or Pitx3-Cre;DTA mice.Sectionsare counterstained withDAPI (blue). Arrowheadsindicate medial forebrain(E)andstriatonigral(F,H)bundles.Hip,Hippocampus;SN,substantianigra. IL,Immunohistochemistryforneurofilament(NF;I,K) or DARPP32 (J,L) in coronal vibratome sections of E17.5WT;DTAorGbx2-CreERT2;DTAmice. Sections are counterstained withDAPI (blue).I, Arrowhead indicates thalamocortical pathway. Whereas dopaminergic and thalamocortical pathways are lost inPitx3-Cre;DTA and Gbx2-CreERT2;DTA mice,respectively,DARPP32-positivestriatalprojectionsareintact.Numberofmiceanalyzed:E13.5andE15.5M4-EGFP, n2;E17.5 WT;DTA or Pitx3-Cre;DTA, n4;E17.5 WT;DTA or Gbx2-CreERT2;DTAmice, n3.Scalebars:A, B, 100m; EH, 200m; I, K, 560m;J, L, 250m.



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revealed no Foxp1cells in the GP ofFzd3/ mice. This sug-gests that corridor cells are misplaced into the GP in theFzd3/ mice (Fig. 10N, O). Corridor cells express repulsiveaxon guidance proteins, such as Slit1, ephrinA5, and class 3semaphorins (Sema3s), that guide thalamocortical axons in

the subpallium (Garel and Lopez-Bendito, 2014). The ectopicexpression of these cues in the GP, due to mislocalization ofcorridor cells, could counteract the normally attractive effectof the GP on MSN axons. To test this model, we determinedwhether repulsive guidance molecules are ectopically ex-pressed in theFzd3/GP and assessed whether corridor cellsaffect MSN axon growth. In situ hybridization revealed thatcues, such as ephrinA5, Sema3A, and Sema3F, are absent fromwild-type GP but expressed inFzd3/ GP(Fig. 11AD; datanot shown). In addition, cocultures of corridor and striatalexplants showed that striatal axons are repelled by corridorcells (Fig. 11 E, F). Together, our results are consistent with amodel in which mislocalization of corridor cells into the GP

due to Fzd3 deficiency causes loss of the permissive effect ofthe GP on MSN axons due to the ectopic expression of axonrepulsive cues.

DiscussionStriatal axon pathways mediate complexphysiological functions and are a knowntherapeutic target, underscoringthe needtodefine how these connections are estab-lished. MSN axon projections are remark-ably complex and their development is

contingent on the completion of severalhighly stereotypic events, such as AP exten-sion and intermediate target navigation.The molecular programs regulating thesedisparate developmental processes remainmostly unknown. Here, we determine theembryonic ontogeny of the two mainstriatal pathways (striatonigral and striato-pallidal) and identify a role for Fzd3 in un-characterized aspects of striatal pathwayformation (i.e., AP guidance in the stri-atum and axon entry into the GP). Further,our results link Fzd3 to corridor cell

development.

Frizzled3 and Wnt5a control APguidance of striatal axonsFrizzleds can pattern axonal connec-tions along the AP axis different species(Zou, 2012; Aviles et al., 2013; Ackley,2014). In mice, these molecules providedirectional guidance for ascending anddescending axons along the AP axis ofthe spinal cord and brainstem (Ly-uksyutova et al., 2003;Wolf et al., 2008;Fenstermaker et al., 2010;Shafer et al.,2011;Hua et al., 2014). Until now, it wasunknown whether AP guidance of lon-gitudinal axon tracts originating in theforebrain also requires Frizzleds. Here,we show that indeed Fzd3 is required forAP guidance of striatal axons in theforebrainin vivo. InFzd3/mice, MSNaxons are misrouted along the lateralmedial and rostralcaudal axes of the

striatum (i.e., no longer confined to their normal posteriortrajectory). As a result, fewer MSN axons arrive at the rostralborder of the GP (Fig. 11G). In the mouse brainstem andspinal cord, Fzd3-mediated AP guidance is controlled by com-plex Wnt protein gradients that bind Fzd3 to repel or attractaxons into the appropriate direction (Lyuksyutova et al., 2003;Fenstermaker et al., 2010). Our data show expression of twoWnt family members, Wnt5aandWnt5b, in an anterior low,posterior high pattern in the striatum and reveal chemoattrac-tive effects of both Wnts on striatal axons in vitro. In addition,Wnt5a/ mice display defects in AP guidance of MSN axonssimilar to those observed inFzd3/mice. These observationstogether with previous work identifying Fzd3 as an axonalreceptor for instructive Wnt5a gradients during axon path-finding (Fenstermaker et al., 2010) are consistent with a modelin which Fzd3 and Wnt5a cooperate to attract MSN axonsposteriorly in the developing striatum (Fig. 11G). More gen-

erally, these data are, to our knowledge, the first to identify acue, Fzd3, that dictates AP axon guidance within a large brainnucleus, the striatum.

Figure9. GP-mediatedchemoattractionofstriatalaxonsislostin Frizzled3/mice.AD,CollagenmatrixcoculturesofE14.5Fzd3/ (WT) orFzd3/ (KO) striatal (STR) and GP explants maintained for 3 DIV and immunostained for III-tubulin. GPexplants are on the left and dotted line indicates STR explants.E, Quantification of the P/D ratios of striatal explants in collagenmatrixculturesasshownin AD.P/D1.0indicatesattraction.wtSTRversuswtGP, n 18;wtSTRversuskoGP, n29;koSTRversuskoGP, n 11;koSTRversuswtGP, n 28.*p 0.05(KruskalWallistest).ReddottedlineindicatesP/D 1.0. FH, In

situhybridization forFzd3in coronal sections of E15.5 and E17.5 wild-type mice. Dotted line indicates GP, which does not showspecific Fzd3 signal. No specific staining is detected following incubationwith Fzd3 sense probes(H).Cx, Cortex. Scale bars:AD,180m; F, 580m; G, H, 650m.



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Entry of striatal axons into the GP noncell-autonomously requires Frizzled3In the mouse, the GP is a final target forstriatopallidal axons and an intermediatetarget for striatonigral axons (LoopuijtandvanderKooy,1985).Weshowthat,inFzd3/ mice, MSN axons are present

andthe outgrowthofFzd3/

MSN axonsin vitro is intact. However, both striato-pallidal and striatonigral axons stall at therostal border of the GP failing to enter thisstructure (Fig. 11G). Our data show thatthe GP releases permissive cues for striatalaxons and provide support for a model inwhich Fzd3 deficiency induces loss of thispermissive natureof theGP, thereby caus-ing axon stalling at this nucleus. First, inin vitrotransplant studies, Fzd3/ MSNaxons successfully navigate the wild-typeGP, but wild-type axons stall at the

Fzd3

/

GP, as observedin vivo. Second,genetic ablation of Fzd3 in striatopallidaland striatonigral MSNs inD2-Cre;Fzd3fl/fl

and Isl1-Cre;Fzd3fl/fl mice, respectively,does not prevent MSN axons from enter-ing the GP. Third, axons derived fromwild-type and Fzd3/ striatal explantsgrow toward wild-type but not knock-outGP explantsin vitro. Finally, genetic abla-tion of axonal populations that traversethe GP at early embryonic stages (i.e., do-paminergic and thalamocortical axons)and that may serve as a scaffold for MSNaxons through reciprocal axonaxon in-

teractions, does not influence the entry ofMSN axons into the GP.

How does Fzd3 control the permissivenature of the GP? A plausible explanationwould be that Fzd3 deficiency causes de-creased expression of axon attractants orenhanced expression of axon inhibitorycues in GPcells. However, the lack ofFzd3expression in the embryonic GP and thenormal trajectory of striatal axons in

Nkx2-1-Cre;Fzd3fl/flmice, in which Fzd3 isinactivated in a major subset of GP cells(Nobrega-Pereira et al., 2010), argue

against direct regulation of guidance cueexpression by Fzd3 in GP cells. Indeed,expression of several guidance cues, suchas Netrin-1, Sema3E, and Sfrp4, was un-changed in the GP ofFzd3/ embryos.Further, striatal axons enter the GPin knock-out mice lacking Netrin-1,Sema3E, or Sfrp4 (Chauvet et al., 2007;Ehrman et al., 2013;F.M. and R.J.P., unpublished observations).

We report that cells displaying a molecular signature of corri-dor cells (Isl1 and Ctip2, but Foxp1)(Lopez-Bendito et al.,2006;Bielle and Garel, 2013) are detected in the GP ofFzd3/

but not wild-type embryos. These data are in line with previous

work showing thalamocortical axon pathfinding defects and anabnormal distribution of cells expressing Ebf1, a marker forMSNs and corridor cells, inDlx5/6-Cre;Fzd3fl/flmice (Hua et al.,

2014; Qu et al., 2014). Corridor cells are guidepost cells forthalamocortical axons and are located in between the GP andMGE (Lopez-Bendito et al., 2006). Corridor cells originate in thelateral ganglionic eminence (LGE) and express molecular cues tosort and guide thalamocortical axons (Lopez-Bendito et al., 2006;

Bielle et al., 2011b; Garel and Lopez-Bendito, 2014; Leyva-Daz etal., 2014). These cues include several known axon repellents, in-cluding Slit1, Sema3A, and ephrinA5. It is therefore tempting to

Figure 10. Mislocalization of corridor cells in the GP ofFrizzled3/mice.A, Schematic of a coronal section indicating

the cortex (Cx), striatum (STR), GP, and corridor cells (Co). Listed at the right are proteins expressed by the indicated

structures. B,C, Immunohistochemistry for Nkx2-1 (red) in coronal sections from E13.5 embryos of the rostral part of the

GP. Sections are counterstained with DAPI (blue). The GP covers an expanded area in Fzd3/ embryos. DG, In situ

hybridization forSemaphorin 3E(D,E) andNetrin-1(F,G) in coronal sections from E13.5 embryos. Sema3E and Netrin-1

expression is intact in the GP ofFzd3/ embryos but covers an expanded region. H, I, Immunohistochemistry for

OL-protocadherin (OL-pc; green) in coronal sections from E13.5 embryo s of the caudal part of the GP. Arrowhead indicatesstriatal axons in GP.JO, Double immunostaining for Islet1 (Isl1; red) and Nkx2-1 (green) (J, K) and for Isl1 (red) and Ctip2

(green) (L,M), and immunohistochemistry for Foxp1 (gr een; DAPI in blue) (N, O), in coronal sections from E13.5Fzd3/

andFzd3/ embryos. L ,M, Insets, Ctip2 immunostaining (green) in the GP at E15.5 (blue; DAPI staining). Dotted lines

indicaterostralGP.TheGPin Fzd3/ embryosisinvadedbyectopicIsl1;Ctip2;Foxp1 corridorcells.Numberofmice

analyzed: E13.5Fzd3/,n 6; E15.5Fzd3/,n 3; E13.5Fzd3/,n 5; E15.5Fzd3/,n 3. Scale bars: B, C,

HM, 400 m;D,E, 85 m;F,G, 155 m;N,O, 145 m.



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speculate that axon repellents expressed by mislocalized corridorcells in theFzd3/GP disrupt molecular mechanisms that nor-mally would facilitate MSN axon target entry into the GP (Fig.

11G). Our observations are consistent with this model. First, ec-topic corridor cells in the GP ofFzd3/ mice express axon re-pellents, such as ephrinA5 and Sema3s. Second, MSN axons are

repelled by molecular cues released bycorridor cell explants in vitro. Therefore, apossible explanation for our observationsis that Fzd3 deficiency causes a mislocal-ization of corridor cells expressingaxon repellents in the GP rendering thisstructure nonpermissive for MSN axon

extension.Interestingly, recent studies analyzing

Fzd3/ mice report motor axon stallingat an important intermediate target in theperipheral nervous system, the plexus re-gion, and a failure of sympathetic axons toinnervate peripheral targets, such as theheart and kidneys (Armstrong et al., 2011;Hua et al., 2013). These phenotypes havebeen attributed to defects in axon-intrinsic mechanisms. These results cou-pled with our own findings in the CNSindicate that Fzd3 can regulate (interme-

diate) target innervation through cell-autonomous and noncell-autonomousmechanisms (i.e., by controlling axongrowth and guidance or by regulating thecellular composition of intermediate tar-gets, respectively).

A role for Frizzled3 in corridorcell positioningCorridor cells migrate tangentially fromthe LGE to the MGE where they form aninstructive corridor for thalamocorticalaxons. The molecules that control corri-

dor cell migration or that confine thesecells to their final position are largely un-known (Bielle and Garel, 2013). Here, weshow that Fzd3 is required for restrainingcorridor cells to their normal position be-tween the GP and MGE. This effect ofFzd3 is most likely noncell-autonomousas analysis of Isl1-Cre;Fzd3fl/fl mice, inwhich Fzd3 is ablated in striatonigralMSNs (Ehrman et al., 2013; Lu et al.,2014) and corridor cells (Lopez-Benditoet al., 2006), does not revealdefects in stri-atal axon pathfinding nor in the location

of corridor cells (Fig. 5 F, G; data notshown). Fzd3 is known to regulate neuro-nal migration (Wada et al., 2006;Vivan-cos et al., 2009;Fenstermaker et al., 2010;Qu et al., 2010;Hua et al., 2013), and onemodel to explain our observations istherefore that Fzd3 deficiency triggers theaberrant migration of corridor cells fromthe LGE into the GP. Given the important

role of Slit2 in corridor cell migration(Bielle et al., 2011a) and expansion of

slit2 expression following fzd3a knockdown in zebrafish(Hofmeister et al., 2012), it is tempting to speculate that Fzd3

regulates Slit2 expression along the presumptive trajectory ofcorridor cells and thereby controls their distribution. Futurestudies will address this and other possible mechanisms.

Figure11. Corridorcellsinducestriatalaxonrepulsion.AD,DoublestainingcombiningimmunohistochemistryforNkx2-1(A,C)and insitu hybridizationfor ephrinA5(B,D)incoronalsectionsfromE13.5embryos. EphrinA5-positivecellsarepresentintheGPofFzd3/ but not wild-type embryos (indicated by arrowhead). Number of mice analyzed: E13.5 Fzd3/,n 3; E13.5Fzd3/,n 3.E, Collagen matrix cocultures of E14.5 striatal (STR) and corridor cell (Co) explants maintained for 2 DIV andimmunostained forIII-tubulin. Coexplants are at the left. Dotted line indicates STR explant. F, Left graph, Quantification of theP/DratiosofSTRexplantsincoculturesasinE.P/D 1.0indicatesrepulsion. n 34cocultures.Rightgraph,QuantificationofthenumberofSTRexplantsthatshowsymmetricalaxongrowthorgrowthtowardorawayfromcoexplants.Scalebars: AD,220m;E,160m.G, Schematicrepresentationof a coronalsectionof theembryonicmousebrainshowingthestriatum(STR)and GP.Inwild-type embryos, MSNs in the striatum send their axons posteriorly. These axons project into (striatopallidal pathway) orthrough (striatonigral pathway) the GP. An anterior (A) low, posterior (P) highWnt5a/Wnt5bgradient is present in the striatum,and MSN axons are attracted by Wnt5sin vitro.Fzd3/, andWnt5a/mice display aberrant caudal, lateral, and medial MSN

projections.Thesedata supporta modelin whichWnt5sguideFzd3-postive MSNaxons alongthe AP axis of theSTR.In Fzd3/

mice, all striatal axons accumulate at the GP and do not enter this structure. Corridor cells express axon repellents for MSN axonsandaremislocalizedintheGPofFzd3/mice.ThenormallyattractiveeffectoftheGPonMSNaxonsislostintheabsenceofFzd3.This suggests that patterning of corridor cells by Fzd3 is required to create permissive corridors for MSN axon growth. Cx, Cortex.



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Importantly, our data suggest that the proper positioning ofcorridor cells may not only be crucial for the development ofthalamocortical axons but also for the formation of permissiveterritories for other axonal populations, such as MSN axons. Inaddition, the contribution of Fzd3 to guidepost cell developmentis most likely not restricted to corridor cells. For example,Fzd3/ mice show defects in the distribution of putative calre-

tinin

guidepost cells in the midbrain (Hua et al., 2014)andknockdown offzd3ain zebrafish embryos perturbs the develop-ment of a population of glial guidepost cells at the midline of theforebrain (Hofmeister et al., 2012). Several different populationsof guidepostcellshave been defined in thebrain (Bielle and Garel,2013), and it will be interesting to determine whether, and if sohow, Fzd3 contributes to the development of these cells.

In conclusion, our study provides tools and a conceptual frame-work for further dissection of striatal circuit assembly. Further, ourdata identify cell autonomous and noncell-autonomous require-ments for Fzd3 in several fundamental and previously uncharacter-ized aspects of striatal pathway formation in vivo. More generally,our findings identify Fzd3 as the first cue that controls AP guidance

in a large brain nucleus and unveil a novel role for this transmem-brane protein in regulating (intermediate) target entry of CNS ax-ons. Further, they implicate Fzd3 in thepositioning of corridor cellsand indicate that an abnormal distribution of these cells may haveunexpected, widespread effects on the development of differentaxon tracts.

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