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Development 139, 3136-3141 (2012) doi:10.1242/dev.078394© 2012. Published by The Company of Biologists Ltd

INTRODUCTIONNeuronal migration is a fundamental process in the developmentof the central nervous system because neurons eventually dwell inregions distinct from their origin. From ventricular zones, neuronsand/or neuronal progenitors navigate along diverse courses, radiallyand tangentially, to their final destination and integrate into specificbrain circuits (Corbin et al., 2001; Hatten, 2002; Marín andRubenstein, 2001; Parnavelas, 2000). A concerted and/orsequentially regulated migration of both excitatory and inhibitoryneurons is essential for the emergence of their proper connectivityand brain functions. Unlike in the telencephalon, where neuronalmigration has been well elucidated, in the mesencephalon this vitalevent has been understudied and key factors remain to be defined.Dopaminergic (DA) neurons located in the three anatomicallydefined areas of the ventral mesencephalon (VM) – the substantianigra (SN), ventral tegmental area (VTA) and retrorubral field(RRF) – are involved in controlling diverse brain functions,including motor control and cognition, emotion and rewardbehaviors (Björklund and Dunnett, 2007; Damier et al., 1999; Dinget al., 2011; Lennington et al., 2011; Schultz, 2001; Seeman et al.,1993; Smidt and Burbach, 2007). The migration routes of DAneurons are not well understood and the related literature iscontradictory (Hanaway et al., 1971; Kawano et al., 1995), whilethe route of GABA neurons to the VM is unknown.

Here, we study the migratory trajectories of DA and GABAneurons and find that proper migration of GABA neurons to theirfinal location in the VM is dependent on the pre-existing DAneuron palisade. These results provide several new conceptsregarding functional interactions between DA and GABA neurons

necessary for their proper migration and final connectivity that maytranslate into novel understanding of potential etiology as well astherapeutic development for many neurological diseases.

MATERIALS AND METHODSAnimalsTimed pregnant CD1 mice were purchased from Charles RiverLaboratories. Colonies of GAD65-GFP and ak/ak mice were maintained inour institutional animal facility. Day of plug discovery was designatedembryonic day (E) 0. Animal experiments were in full compliance with theNIH Guide for the Care and Use of Laboratory Animals and wereapproved by the McLean Institutional Animal Care and Use Committee.

BrdU labeling and immunohistochemistryA single BrdU injection (50 g/g body weight) was administered to pregnantdams carrying E10 or E11 embryos. Embryonic brains were removed after2 hours, at E13, E15, E17 and postnatal (P) day 0 stages, immersed in zincfixative (BD Pharmingen) for 24 hours and processed for paraffin waxhistology. BrdU immunohistochemistry was performed on 10-m sectionswith a mouse monoclonal anti-BrdU antibody (1:75, BD Pharmingen). Otherantibodies used were anti-TH (1:200, Millipore), anti-Otx2 (1:200,Neuromics), anti-GAD65/67 (Gad2/1 – Mouse Genome Informatics) (1:400,Millipore), anti-Lmx1b (1:100; Drs Carmen Birchmeier and Thomas Muller,Max-Delbrück-Center for Molecular Medicine, Berlin, Germany), anti-Foxa2 (1:100, Santa Cruz), anti-Lmx1a (1:100, Millipore), anti-Ki67 (1:30,Sigma), anti-Pax6 (1:30, Sigma), anti-DAT (Slc6a3 – Mouse GenomeInformatics) (1:200, Millipore), anti-Helt (1:30, Sigma) and anti-calbindin(1:100, Swant). BrdU+ cells in the red nucleus and BrdU+ GAD65/67+ co-labeled cells in the VM were counted using ImageJ software (NIH).

Explant culturesBasal plate (BP) and VM explants were dissected from mesencephalic slicesof E15 wild-type (WT) and ak/ak embryos. Explants were plated in Matrigel(BD Biosciences) at a distance of 600 m, overlaid with Neurobasal medium(1�, Invitrogen/Life Technologies) and co-cultured for 36 hours. Explantswere fixed in 4% paraformaldehyde, stained with Hoechst (Sigma) andimaged. For quantification, BP explants were subdivided into proximal (P)and distal (D) quadrants. The areas occupied by migrating cells in eachquadrant were determined using ImageJ. The P/D ratio was calculated andused as a measure of chemoattraction in each case.

Heterochronic microtransplantsBP tissue obtained from E15 GAD65-GFP mesencephalic slices wasinserted into ak/ak mesencephalon using fine tungsten needles under ahigh-magnification stereomicroscope. For some of these ak/ak slices (withtransplanted BP), the VM was discarded and substituted by VM from WT

1Angiogenesis and Brain Development Laboratory, Division of Basic Neuroscience,McLean Hospital/Harvard Medical School, 115 Mill Street, Belmont, MA 02478,USA. 2Molecular Neurobiology Laboratory and Program in Neuroscience, McLeanHospital/Harvard Medical School, 115 Mill Street, Belmont, MA 02478, USA.3Institute of Life Science and College of Veterinary Medicine, Gyeongsang NationalUniversity, Jinju 660-701, Korea. 4Institute of Experimental Medicine, Department ofGene Technology and Developmental Neurobiology, Laboratory of Molecular Biologyand Genetics, 1083 Budapest, Hungary.

*These authors contributed equally to this work‡Authors for correspondence (avasudevan@mclean.harvard.edu;kskim@mclean.harvard.edu)

Accepted 11 June 2012

SUMMARYNeuronal migration, a key event during brain development, remains largely unexplored in the mesencephalon, where dopaminergic(DA) and GABA neurons constitute two major neuronal populations. Here we study the migrational trajectories of DA and GABAneurons and show that they occupy ventral mesencephalic territory in a temporally and spatially specific manner. Our results fromthe Pitx3-deficient aphakia mouse suggest that pre-existing DA neurons modulate GABA neuronal migration to their finaldestination, providing novel insights and fresh perspectives concerning neuronal migration and connectivity in the mesencephalonin normal as well as diseased brains.

KEY WORDS: Midbrain, Neuronal migration, Parkinson’s disease

Dopaminergic neurons modulate GABA neuron migration inthe embryonic midbrainAnju Vasudevan1,*,‡, Chungkil Won1,2,3,*, Suyan Li1, Ferenc Erdélyi4, Gábor Szabó4 and Kwang-Soo Kim2,‡

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slices. Slices were transferred to polycarbonate membrane filters(Invitrogen) in sterile six-well plates containing Neurobasal medium andcultured for 48 hours. Slices were fixed and TH immunohistochemistryperformed. The number of GFP+ cells that migrated to the VM wascounted in all slices and average values obtained.

StatisticsStatistical significance of differences between groups was analyzed by two-tailed Student’s t-test (Prism, GraphPad software). Results were expressedas mean ± s.d. and statistical significance reported at P<0.05.

RESULTS AND DISCUSSIONNeuronal migration silhouette in themesencephalonBrdU birthdating studies have been widely used to study neuronalmigration in the developing neocortex (López-Bendito et al.,2008; Mathis et al., 2010; Ori-McKenney and Vallee, 2011;Soriano and Del Rio, 1991; Supèr et al., 2000; Wines-Samuelsonet al., 2005). Because BrdU is integrated into the DNA of S-phaseprogenitor cells, it serves as a stable marker for cells born around

Fig. 1. Neuronal migrational profiles in the mesencephalon. (A-G)E10 BrdU-labeled progenitors in the mesencephalon at 2 hours (A),E13 (B), E15 (C), E17 (D) and P0 (E). Blue arrows in D point to ventral and perpendicular migration that depletes the red nucleus of BrdU-labeled cells (white asterisks, D-F) to form the anatomical architecture (arrowheads, D,E) of VTA and SN. Yellow arrows in E show dorsally andventrally segregated cells. (F)The anatomical architecture of SN and VTA confirmed by co-labeling of BrdU (red) and TH (green). (G)A 20�magnification of F showing that some BrdU+ cells are TH+ (white arrows), whereas others are TH– (blue arrows). (H-K)GABA neurons areabsent from the VM in coronal (H-J) and sagittal (K) slices (30m) from E13 GAD65-GFP embryos. White asterisk indicates GABA neurons inthe basal plate (BP) (H,J) and pink arrows indicate possible ventral migration in a magnified BP (I). (J)No overlap of TH neurons (red) withGABA neurons was seen. (K)Dashed circle indicates the absence of GABA neurons from VM. (L-O)GABA neurons appear in VM in coronal (L-N) and sagittal (O) slices from E17 GAD65-GFP embryos. White asterisk indicates the GABA neuron stream from BP to VM (L), as magnified inM (pink arrows). GABA neurons and TH neurons are in intimate contact (white arrows, N). (O)Dashed circle indicates GABA neurons in theVM. (P,Q)GFP profiles of E17 mesencephalon collected from a second GAD65-GFP transgenic line (GAD65_Ncol/gfp/1e_6e) also showed asimilar picture. (P)White arrows indicate GABA neurons descending to VM and integrating with TH+ DA neurons (red). The boxed region in Pis magnified in Q and TH-GABA neuron contact is indicated (arrows). (R-U)Birthdating experiment in GAD65-GFP (R,T) and CD1 (S,U) mice.Many of the BrdU+ cells that migrated into VM by E17 were GFP+ (R) or stained positively for GAD65/67 (S). VM regions of R and S aremagnified in T and U and co-expressing cell nuclei appear yellow (arrows). n5. aq, aqueduct; SN, substantia nigra; VTA, ventral tegmentumarea; VM, ventral mesencephalon; SC, superior colliculus. Scale bars: 100m in A-H,J-L,N-S; 50m in I,M,T,U.

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the time of injection. First, we performed a thorough andsystematic BrdU birthdating study to understand mesencephalicneuronal migration in CD1 mice. We labeled neuronalprogenitors born at E10 with a single BrdU pulse and followedtheir migration in the mesencephalon until P0 (Fig. 1A-G). BrdU-labeled cells first spread out uniformly in the mesencephalonfrom E10-15 (Fig. 1A-C). However, a major change in neuronalmigration was observed at E17 (Fig. 1D), by which time neuronshad migrated both ventrally and perpendicular to the aqueduct toform the distinct anatomical architecture of the boat-shaped SNand VTA (Fig. 1D). The red nucleus area was significantlydepleted of E10-labeled neuronal progenitors by thisperpendicular migration. By P0, most of the neurons hadsegregated to both dorsal and ventral mesencephalon (Fig. 1E).The boat-shaped architecture was confirmed by tyrosinehydroxylase (TH) staining (Fig. 1F,G). E11-labeled neuronalprogenitors followed the same route as E10-labeled neuronalprogenitors and formed the distinct anatomical architecture of SNand VTA in VM (supplementary material Fig. S1A). Our resultscorroborate those of previous studies indicating that neurons ofthe SN and VTA in the mouse are generated on or before E12(Bayer et al., 1995). E13-labeled neuronal progenitors migrated

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predominantly to the dorsal mesencephalon and E15-labeledprogenitors gave rise to a limited number of lateral neurons(supplementary material Fig. S1B,C).

Although many BrdU+ neurons in SN and VTA at P0 weredopaminergic/TH+ (Fig. 1F,G), there were also many TH– neurons.Given that DA and GABA neurons constitute two major neuronalpopulations in the ventral midbrain, these neurons might be GABAneurons. Although GAD mRNA expression starting at E10.5 hasbeen reported in BP, alar plate and dorsal mesencephalon (Guimeraet al., 2006; Katarova et al., 2000), and the number, frequency andtopography of GABA neurons in SN and VTA regions of the adultbrain have been characterized (Korotkova et al., 2004; Nair-Robertset al., 2008; Olson and Nestler, 2007), it is not known when and howGABA neurons become admixed with ventral mesencephalic DAneurons during development. Are GABA neurons of the VM bornelsewhere, then come to reside there to form the final connectivity?To address these fundamental questions we used the GAD65-GFPmouse model, in which GABA neurons can be clearly visualized(López-Bendito et al., 2004). Strikingly, we found that whereas atE13 the VM was completely devoid of GABA neurons (Fig. 1H-K),by E17 GABA neurons were substantially intermingled with DAneurons (Fig. 1L-Q). At E13, many BP GFP+ neurons were oriented

Fig. 2. Abnormal neuronal migration anddistribution of neurons in ak/akmesencephalon. (A-M)The migrational profileof E11-labeled neuronal progenitors wasanalyzed in WT (A-C,G) and ak/ak mutant (D-F,H-M) mice at E17. White asterisks indicateareas free of BrdU-labeled cells in WTmesencephalon (A-C,G) and blue asterisksindicate abnormal cell clusters in the rednucleus of the ak/ak mutant (D-F,H).(G,H)Higher magnifications of C and Fdisplaying failed perpendicular migration(arrowheads) in the ak/ak mutant. (I-M)Stalledcells in the ak/ak mutant are double positive forBrdU/Otx2 (arrowheads, I), BrdU/Lmx1b (J,K)and BrdU/Foxa2 (L,M). The boxed regions in Jand L are magnified in K and M, respectively,and white arrows indicate double-positive cells.(N)Quantification of E11 BrdU-labeled cellsdistributed in the red nucleus (bothhemispheres) of WT and ak/ak mutant (meandensity of BrdU+ cells ± s.d.). A significantincrease in BrdU+ cells was observed in theak/ak mutant. *P<0.0001; n5. Scale bars:100m in A-J,L; 50m in K,M.

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ventrally (Fig. 1H,I) and by E17 a cohort of GFP+ neurons werepositioned in stream-like routes to the VM (Fig. 1L,M,P,Q). Further,high-magnification images showed how GABA neurons of the VMare in close physical contact with DA neurons (Fig. 1N,P,Q).Birthdating experiments revealed that many E11-labeled neuronalprogenitors migrated to contribute to GABA neurons of the VM byE17 (Fig. 1R-U).

Abnormal neuronal migration in the ak/akmesencephalonThis novel finding that DA and GABA neurons occupy separateterritories in the E13 mesencephalon (Fig. 1J) prompted us tohypothesize that the early-formed DA neuron palisade might havea role in GABA neuron migration into the VM at laterdevelopmental stages. In line with this possibility, the direction andentry of the GABA neuron stream were oriented towards VMterritory (Fig. 1H,I), leading to intimate contact with DA neurons(Fig. 1L-Q). To address our hypothesis and to further investigatemesencephalic neuron migration, we postulated that the Pitx3-deficient aphakia (ak) mouse might provide an ideal animal modelwith a defective DA neuron architecture as Pitx3 is one of thecrucial regulators of mesencephalic DA neuron development (Dinget al., 2011; Kim et al., 2007; Nunes et al., 2003; Smidt et al., 2004;van den Munckhof et al., 2003) and there is selective and early lossof A9 DA neurons in the SN of ak/ak mice (Hwang et al., 2005;Smidt et al., 2004; van den Munckhof et al., 2003).

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Since expression of both Pitx3 and Th begins at E11, weconsistently studied the migration of E11-labeled neuronal progenitorsin both WT and ak/ak mice (Fig. 2). Mesencephalic sections wereanalyzed at E17 with BrdU and TH markers (Fig. 2A-H). Strikingly,in the ak/ak mutant, BrdU+ cells were scattered aberrantly in BPregions and failed in their perpendicular migration to the SN. Thedistinct anatomical architecture of the boat-shaped SN and VTAoutlined by BrdU+ cell migration did not form in the ak/ak mutant(Fig. 2D), in contrast to WT (Fig. 2A; supplementary material Fig.S1A). BrdU and TH double labeling revealed many E11-labeled cellsdisplaying a dopaminergic phenotype after arriving at their finaldestination in both VTA and SN regions in WT mesencephalon (Fig.2A-C,G), whereas in the ak/akmutant the cells were unable to reachthe SN and display their full dopaminergic phenotype (Fig. 2D-F,H).Instead, these E11-labeled cells appeared to be stuck or trailing in themiddle of their migratory trajectory and distributed abnormally in thered nucleus area in the ak/ak mutant (Fig. 2F,H).

To further investigate the abnormally migrating cells, we testedwhether the stalled E11-labeled cells in the ak/ak red nucleus areacontain DA progenitor cells. Many cells were positive for Otx2(Fig. 2I), a marker for DA progenitors (Chung et al., 2009; Vernayet al., 2005), but never expressed the TH marker of DA neurons(Fig. 2E,F,H), indicating impaired differentiation. The stalled cellswere also positive for the markers Lmx1b (Fig. 2J,K) and Foxa2(Fig. 2L,M), confirming their DA progenitor identity. They werepositive for the proliferating progenitor markers Lmx1a, Ki67 and

Fig. 3. Impaired GABA neuron development inak/ak mesencephalon. (A-H)GAD65/67 and THlabeling at E17 in WT (A-C,G) and ak/ak (D-F,H)mouse mesencephalon. Higher magnification of VMfrom C and F show intimate association of GABAneurons and DA neurons in WT (co-label in yellow,arrows in G) and decreased GAD65/67 label andlimited physical contact with TH neurons in the ak/akmutant (blue arrows, H). (I-O)WT BP explants(outlined) show robust cell migration towards WT VM(I-K,O), whereas WT BP explants fail to migratetowards ak/ak VM (L-O) in proximal quadrants. WT BPexplants from I and L are magnified in J and M,respectively. (K,N)Hoechst staining of WT BP explantsfrom I and L, respectively. (O)Quantification ofchemoattraction expressed as a ratio of the quadrant(dotted lines) facing the VM explants (proximal, P)relative to the area occupied by cells in the oppositequadrant (distal, D). *P<0.01; n25; error barsindicate s.d. Scale bars: 100m in A-H,J,K,M,N;200m in I,L.

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Pax6 and negative for DAT, a marker of immature postmitotic DAprogenitors (supplementary material Fig. S2A-E). In addition,many BrdU+ cells were observed along perpendicular migrationroutes in a short-pulse experiment at E17 (supplementary materialFig. S2F), confirming the presence of abnormally proliferatingprogenitors in the ak/ak mesencephalon at late embryonic stages.The mean density of BrdU+ cells in the red nucleus of the ak/akmutant was significantly higher than in WT (Fig. 2N).

Taken together, our data provide strong evidence of theperpendicular migration of DA neurons to the VM and that this issignificantly disturbed in the ak/ak mutant. Thus, in the absence ofPitx3, DA neuronal migration is impaired, contributing to severeloss of A9 DA neurons in the SN. Interestingly, we also found thatmigration of E13-labeled neuronal progenitors was similarlyaffected in the ak/ak mutant (supplementary material Fig. S3).

Pre-existing DA neurons modulate GABA neuronmigration to ventral mesencephalonGABA neuron development was also significantly affected in theak/ak mesencephalon. By E17, in WT mouse embryos GABAneurons had settled together in close physical contact with THneurons (Fig. 3A-C,G). This profile was substantially altered in theak/ak mutant, leading to significantly limited contact of theseneurons in the VM (Fig. 3D-F,H). GABA neurogenesis wasunaffected in the ak/akmutant (supplementary material Fig. S4). The

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stalled cells on the route of perpendicular migration or in the VMwere not apoptotic (supplementary material Fig. S5), and so weexamined whether the decreased GABA neuron profile in the ak/akmutant by late embryonic stages was due to impaired migration. Tosearch for cellular sources of guidance cues in the VM for migratoryBP neurons, explants of VM were confronted with explants of BPfrom WT mice. BP neurons were markedly attracted towards VM(Fig. 3I-K,O). BP explants from WT mice, by contrast, showed noattraction to VM from the ak/ak mutant (Fig. 3L-O).

Birthdating studies indicated that E11-labeled neuronalprogenitors contributed to GABA neurons of VM in WTembryos and many BrdU+ GAD65/67+ co-labeled cells wereobserved (Fig. 4A,C). In the ak/ak mutant, E11-labeled neuronalprogenitors did not contribute significantly to GABA neurons ofthe VM, as illustrated by the significant decrease in BrdUGAD65/67 co-labeling (Fig. 4B,D) and mean density of BrdU+

GAD65/67+ cells in the VM area (Fig. 4E). The stalled cellsalong the perpendicular migration routes expressed the GABAneuron progenitor markers Helt (Fig. 4F,G) and calbindin (Fig.4H-J). Heterochronic microtransplants were also performed tounderstand mesencephalic GABA neuron migration. When a BPexplant from a GAD65-GFP mouse was transplanted into ak/akmesencephalon, GFP+ cells appeared to be stalled around thetransplantation site and were unable to migrate and integrate intoVM (Fig. 4K,M,O). By contrast, when ak/ak VM was substituted

Fig. 4. DA neurons modulate GABA neuronmigration to ventral mesencephalon. (A-E)E11-labeled neuronal progenitors were examined at E17 forBrdU and GAD65/67 markers in WT (A,C) and ak/akmutant (B,D). VM from A and B is magnified in C and D,respectively. White arrows show BrdU GAD65/67 co-labeling in WT mouse and blue arrows indicate the lackthereof in the ak/ak mutant. (E)BrdU+ GAD65/67+ cells inthe VM of WT and ak/ak mutant were quantified (meandensity of BrdU+ GAD65/67+ cells ± s.d.) and a significantreduction was observed in the mutant. *P<0.0001; n5;error bars indicate s.d. (F-J)The stalled cells in ak/akmesencephalon were Helt+ (F,G) and calbindin+ (H-J). Theboxed regions in F, H and I are magnified in G, I and J,respectively. Arrows indicate BrdU+ Helt+ (G) and BrdU+

calbindin+ (J) co-labeled cells. (K,L) Scheme oftransplantation of GAD65-GFP BP (green circle) and WTVM (pink shape) into ak/ak mesencephalon. Red crescentmarks the defective DA neuron architecture of the ak/akmesencephalon. (M,N)Blue arrows (M) indicate GFP+

cells close to site of transplantation (yellow dotted circle),white asterisk (M) indicates the lack of migration to ak/akVM, and white arrows (N) indicate significant migrationand integration into transplanted WT VM (white border).(O)Quantification of migrated GFP+ cells to ak/ak VM(K,M) and ak/ak VM substituted with WT VM (ak/ak-WT,L,N). *P<0.0001; n25; error bars indicate s.d.(P,Q)Model of mesencephalic DA (blue arrows) andGABA (green arrows) neuron migration in WT (P), whichinvolves ventral migration of VTA precursors (verticalarrow) and perpendicular migration of SN precursors andsome VTA precursors (perpendicular arrows). In the ak/akmutant (Q), perpendicular migration of DA neurons toVM is significantly affected (lower red cross) and cellscluster abnormally in the red nucleus. Then, GABAneurons also cannot migrate to VM (upper red cross). Aq,aqueduct; BP, basal plate; VM, ventral mesencephalon.Scale bars: 100m in A-D,F,H,M,N; 50m in G,I; 25min J.

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with a VM from WT mouse, GFP+ cells exited thetransplantation site and migrated robustly to integrate with DAneurons (Fig. 4L,N,O).

Together, these results strongly support our idea that the intactDA system of the VM guides the GABA neuron system to descendto VM and establish its connectivity with DA neurons.Furthermore, the reduction in GABA neurons observed in the ak/akmesencephalon was reflected in adult (4 month old) mice as well(supplementary material Fig. S6).

Our data provide novel insights into neuronal migration in theembryonic mouse mesencephalon and its relevance for final ventralmesencephalic neuronal population and connectivity. First, our datasupport a model for DA and GABA neuron migration in themesencephalon that depicts vertical migration of VTA precursorsand perpendicular migration of both SN and VTA precursors (Fig.4P). Second, our analysis of ak/ak mice indicates how A9 DAprogenitor cells show blocked perpendicular migration andaccumulate in the red nucleus area (Fig. 4Q). Perpendicularmigration is therefore essential to set up the proper anatomicalarchitecture of ventral mesencephalic structures. Third, we foundthat DA and GABA neurons occupy VM in a temporally sequentialmanner. Thus, at E13, the primary structure of DA neurons in theVM is completely devoid of GABA neurons, whereas by E17GABA neurons come to reside along with DA neurons.Remarkably, proper migration of GABA neurons to their finallocation is dependent on the complete DA neuron architecture inthe VM, strongly indicating an important interaction betweenGABA and DA neurons for final location and connectivity.

Given that major brain disorders such as schizophrenia, attentiondeficit hyperactivity disorder (ADHD) and Parkinson’s disease areconsidered to be caused by abnormal early brain development, thisstudy will serve as a gateway to a whole new field of explorationto identify substrates and mechanisms of neuronal migration in themesencephalon that might provide novel insights into theunderlying pathophysiology of these brain disorders.

AcknowledgementsWe thank Drs Carmen Birchmeier and Thomas Müller for the generous gift ofLmx1b antibody.

FundingSupported by a National Alliance for Research on Schizophrenia andDepression (NARSAD) Young Investigator Award to A.V. and National Institutesof Health grants [NS064386, NS073635 to A.V., MH48866, MH087903 andNS070577 to K.-S.K.]. Deposited in PMC for release after 12 months.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.078394/-/DC1

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