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IntroductionIt is well recognized that both radial and tangential migrationcontribute significantly to neuronal diversity within thetelencephalon (reviewed by Marin and Rubenstein, 2003).Projection neurons originate in the ventricular zone of thecortical anlage (pallium) and migrate radially to form thedeveloping cortex, while GABAergic interneurons originate inrestricted regions of the ventricular zone of the subcorticaltelencephalon (subpallium) and migrate tangentially to reachtheir final destination, in defined telencephalic regions. Withinthe subpallium, the medial ganglionic eminence (MGE)appears to be the major source of cortical interneurons(Anderson et al., 2001; Jimenez et al., 2002; Marin et al.,2000; Wichterle et al., 2001). Furthermore, it has been shownin vivo that neurons that derive from the lateral ganglioniceminence (LGE) and the caudal ganglionic eminence (CGE)contribute to the generation of projection neurons in thestriatum and GABAergic interneurons in the cortex,respectively (Nery et al., 2002; Wichterle et al., 2001).Although there is an overlap in the structures populated byneurons originating from all three ganglionic eminences(GEs), the nuclei derived from the progeny of each eminenceare distinct and can vary in their neurochemical profiles (Neryet al., 2002). As a further source of cellular diversity, the

anterior entopeduncular area, which is located between theMGE and the hypothalamus, contributes to the generation ofoligodendrocytes in the cortex and hippocampus (He et al.,2001). Therefore, interneurons and oligodendrocytes appear tofollow a mainly ventral-to-dorsal migratory route beforereaching their target tissues, and it is not known whether thesecells can also migrate in a dorsal-to-ventral direction. Theidentification of other migratory pathways, such as thegangliothalamic body connecting the GEs with the thalamus(Letinic and Kostovic, 1997; Rakic and Sidman, 1969), andthe rostral migratory stream between the LGE and theolfactory bulb (Altman, 1969; Lois and Alvarez-Buylla, 1994;Luskin, 1993), makes it plausible that the GEs generateneurons directed to other embryonic brain regions besides thedorsal telencephalon.

As suggested by various studies, directional migration canbe achieved by short-range guidance cues and by long-rangediffusible gradients (Tessier-Lavigne and Goodman, 1996).Contact guidance relies on a permissive environment, onwhich gradients of diffusible guidance molecules can besuperimposed, to achieve directional migration. However, thedistribution and molecular nature of short- and long-rangemolecules, and the substratum that interneurons use in theirmigration towards the cortex, are poorly understood.Motogenic factors, such as hepatocyte growth factor and the

Cells migrate via diverse pathways and in different modesto reach their final destinations during development.Tangential migration has been shown to contributesignificantly to the generation of neuronal diversity in themammalian telencephalon. GABAergic interneurons arethe best-characterized neurons that migrate tangentially,from the ventral telencephalon, dorsally into the cortex.However, the molecular mechanisms and nature of thesemigratory pathways are only just beginning to beunravelled. In this study we have first identified a noveldorsal-to-ventral migratory route, in which cells migratefrom the interganglionic sulcus, located in the basaltelencephalon between the lateral and medial ganglioniceminences, towards the pre-optic area and anteriorhypothalamus in the diencephalon. Next, with the helpof transplantations and gain-of-function studies in

organotypic cultures, we have shown that COUP-TFI andCOUP-TFII are expressed in distinct and non-overlappingmigratory routes. Ectopic expression of COUP-TFs inducesan increased rate of cell migration and cell dispersal,suggesting roles in cellular adhesion and migrationprocesses. Moreover, cells follow a distinct migratory path,dorsal versus ventral, which is dependent on the expressionof COUP-TFI or COUP-TFII, suggesting an intrinsic roleof COUP-TFs in guiding migrating neurons towards theirtarget regions. Therefore, we propose that COUP-TFs aredirectly involved in tangential cell migration in thedeveloping brain, through the regulation of short- andlong-range guidance cues.

Key words: COUP-TFs, Cell migration, Forebrain, Mouse

Summary

The COUP-TF nuclear receptors regulate cell migration in themammalian basal forebrainMarco Tripodi*, Alessandro Filosa*, Maria Armentano and Michèle Studer†

TIGEM (Telethon Institute of Genetics and Medicine), Via P. Castellino 111, 80131 Napoli, Italy*These authors contributed equally to the work†Author for correspondence (e-mail: studer@tigem.it)

Accepted 13 October 2004

Development 131, 6119-6129Published by The Company of Biologists 2004doi:10.1242/dev.01530

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neurotrophin molecules BDNF and NT4, have been shown toinfluence the numbers of cells migrating away from thesubpallium (Polleux et al., 2002; Powell et al., 2001).Guidance molecules with repulsive and attractive activitiesare likely to be involved in guiding migrating cells from thesubpallium to the pallium, and within the subpallium (Marinet al., 2003; Wichterle et al., 2003). For the substratum, onereport has shown that blocking the function of the TAG1adhesion molecule results in a marked reduction inGABAergic neurons in the cortex (Denaxa et al., 2001).However, in Tag1 mutant mice, there is no major alterationin the tangential migration of interneurons (Marin andRubenstein, 2003), and treatment of cortical slices withphosphoinositide-specific phospholipase C (PI-PLC), anenzyme that cleaves GPI-anchored proteins, such as TAG1,does not alter interneuron migration (Tanaka et al., 2003).Therefore, nothing is known about the nature of short-rangeguidance contact cues involved in tangential migration.

The nuclear orphan receptor COUP-TFI favours migrationof neurons through an integrin-dependent mechanism in cellcultures (Adam et al., 2000). Overexpression of COUP-TFI inretinoic-acid-treated neuronal aggregates coated with differentcell substrata, such as polylysine, laminin or fibronectin,results in a higher rate of migration compared with controls,whereas overexpression of a mutated form of COUP-TFI hasno effects on cell migration. Moreover, increased levels ofvitronectin, an extracellular matrix (ECM) molecule involvedin neurite extension, motor neuron differentiation and possiblyneuronal migration, are detected both in aggregate andmonolayer cell cultures after overexpression of COUP-TFI.This, and the data that show that COUP-TFI stimulatestranscription from the vitronectin promoter in vitro, stronglysuggests a role of COUP-TFI in the regulation of ECMsynthesis during the process of neuronal migration. AlthoughCOUP-TFI has been shown to be involved in corticalregionalization and axon pathfinding in the developingtelencephalon (Zhou et al., 1999; Zhou et al., 2001), its role inpromoting cell migration in vivo has not been established sofar.

Here, we show that COUP-TFI and COUP-TFII areexpressed in migrating cells in specific pathways in the basaltelencephalon of mouse embryos at a stage when interneuronmigration is robust. Moreover, expression of COUP-TFI in thecortex co-localizes with the GABAergic marker calbindin,suggesting that a subpopulation of COUP-TFI-positive cellsin the cortex are GABAergic interneurons. With the help oftransplantation and tracing techniques in organotypic slicecultures, we first show the presence of a dorsal-to-ventralmigratory pathway that originates in the region of theinterganglionic sulcus. We then demonstrate that COUP-TFIis expressed in the basal telencephalon in neurons not onlymigrating dorsally towards the cortex, but also ventrallytowards the pre-optic area (POa) and the hypothalamus.Finally, by ectopic expression of COUP-TFI and COUP-TFIIin the GEs, we show an increased rate of migrating cells.While COUP-TFI appears to control dorsal and ventralmigration in the basal telencephalon, COUP-TFII is morespecific in modulating tangential migration into theintermediate zone of the cortex, suggesting distinct functionsof COUP-TFs in regulating cell migration in the developingforebrain.

Materials and methodsGeneration of polyclonal antibodies of COUP-TFI andCOUP-TFIITo eliminate any possibility of crossreactions between the antibodiesagainst COUP-TFI and COUP-TFII, and to identify the lessconserved, more exposed and immune-reactive regions of theproteins, we performed an in-silico analysis using Blastp,PredAccServer (Protein Hydropathic Profile) and Antigenic(following Kolasker-Tangaonkar algorithm) software. GST-fusionproteins were produced using the pGEX cloning vector, in which theN-termini of COUP-TFI and COUP-TFII, comprising the first 203amino acids, were cloned into the EcoRI-SphI (for COUP-TFI) andthe MslI-SphI (for COUP-TFII) restriction sites of pGEX. The fusionproteins were grown in BL21 cells, and purified by affinitychromatography on glutathione-coated columns before being injectedinto rabbits. The immunization protocol consisted of five rounds ofinjections before the final bleeding, and it was carried out by Primmsrl, Milan, Italy. The specificities of the antibodies were tested byblocking them with their respective immunogens, and using them incontrol frozen sections. No signals were detected (data not shown).

Slice culture experimentsOrganotypic slice cultures of embryonic mouse telencephalon wereprepared as previously described (Anderson et al., 1997). Brain slices250 µm thick were used for the grafting experiments (see below), fortracing with the fluorescent carbocyanide dye 1,1′-dioctadecyl3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; MolecularProbes), and for electroporation and lipofectamine injections (seebelow).

Grafting experimentsFor the grafting experiments, the MGE, the LGE, or the region at theMGE/LGE border (MLGE) were removed from the donor brain slicesderived from a green fluorescent protein (GFP)-expressing transgenicembryo (Hadjantonakis et al., 1998) and transferred to the sameposition in a host wild-type brain slice. The chimaeric slices wereexamined 1 hour after the transplant under a stereoscope with a GFPfilter, to confirm that the transplants were successfully and correctlyintegrated into the host; slices where the grafts did not integrate, orintegrated in an incorrect location, were not used for this study. Themedium was changed every day, and after about 60 hours the sliceswere fixed in 4% paraformaldehyde.

Electroporation and lipofectamine-mediated injectionFor the fate map, a GFP-expressing vector was electroporated into theGEs of embryonic day (E) 13.5 brain slices using the same methodand conditions as described previously (Stuhmer et al., 2002). For thegain-of-function approach, we used the PolyFect Transfection Kit(Qiagen, Hilden, Germany), containing 250 ng/µl of the expressionvectors (CMV-GFP or CMV-COUP-TFs-IRES-GFP). Full-lengthmouse COUP-TFI and COUP-TFII (kindly donated by M. Tsai,Baylor College, USA) were cloned into the BamHI-XhoI sites of thepIRES-hrGFP-2a expression vector (Stratagene, California, USA).We injected the constructs into the desired region of the brain slices,under microscopic guidance with a micro-syringe (Hamilton, Reno,Nevada, USA) held by a micromanipulator system using a 33-gaugeblunt-ended needle (Hamilton). The slices were then cultured for 60hours, fixed in 4% paraformaldehyde, and either observed by confocalmicroscopy (Leica DMIRE2) or embedded in OCT for furthersectioning in a cryostat.

Immunohistochemistry and immunofluorescenceThe brains were sectioned and treated for immunostaining accordingto standard procedures. Combined immunohistochemistry and in-situhybridization on cryosections were carried out as described previously(Hirsch et al., 1998). The times of incubation with the primary

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antibodies ranged from 90 minutes (anti-COUP-TFs, anti-reelin) toovernight. The following antibodies were used: rabbit α-COUP-TFI(1:500), rabbit α-COUP-TFII (1:1000), mouse α-reelin (clone G10,1:500; kind gift of A. Goffinet), mouse α-calbindin (clone CB-955,Sigma, 1:500), mouse α-GABA (clone GB-69, Sigma, 1:1000) andmouse α-β-tubulin type III (clone SDL.3D10, Sigma, 1:400). Sectionswere alternatively incubated in a biotinylated secondary antibody(Vector, 1:200) and processed by the ABC histochemical method(Vector) or with a fluorescent secondary antibody (Molecularprobes 1:400; Alexafluor 488 α-rabbit, Alexafluor 594 α-rabbit,Alexafluor 594 α-mouse) for 1 hour at room temperature. Theimmunohistochemistry-treated sections were dried, dehydratedand mounted on a coverslip with Permount (Fisher); theimmunofluorescence-treated sections were mounted with Vectashield(Vector). The GFP was detected by its endogenous fluorescence.

Cell countingIn the grafting experiments, the cells were counted on images obtainedfrom 6 µm sections of the slices that were acquired with a CCDcamera attached to a conventional microscope. To count the cells, astandardized box (a 40× enlargement of the slice) was used; for eachtransplant, three sections were evaluated, and for each section, thecells in two different regions were counted. In the gain-of-functionexperiments, an image of the whole slice was obtained by confocalmicroscopy, after reconstruction of all of the confocal planes. TheGFP-positive cells that migrated towards the cortex or towards thePOa were counted on this reconstructed image, and normalized forthe total number of GFP-positive cells [e.g. cortex/(cortex + basalganglia), or POa/(POa + basal ganglia)]. The data from control andoverexpressing cells within the same slice were compared, and pairedStudent’s t-tests were used for the analysis.

ResultsCOUP-TFI and COUP-TFII are expressed in distinctdomains in the developing telencephalonTo analyse the expression of COUP-TFs at a cellular level inthe developing telencephalon, we generated polyclonalantibodies against COUP-TFI and COUP-TFII. Because of thehigh sequence homology between COUP-TFI and COUP-TFIIand the other members of the steroid/thyroid superfamily, wechose the N-termini of the proteins, as these show the highestsequence divergence (see Materials and methods for moredetails). Western blotting and immunohistochemistrydemonstrated no crossreactivities between the two members ofthe family (data not shown).

To study the expression profile of both COUP-TFsduring the period of neural migration, we performedimmunohistochemistry on E13.5 mouse forebrains, whentangential migration is well pronounced. At this stage, COUP-TFI expression was localized to all layers of the neocortex, tothe piriform cortex, to the ventricular and subventricular zonesof the LGE, to the ventricular zone of the MGE, to the POa,and in scattered cells in various regions of the basaltelencephalon (Fig. 1A). By contrast, COUP-TFII expressionwas mainly restricted to the marginal zone of the neocortex andto the piriform cortex and POa of the basal telencephalon.Scattered COUP-TFII-positive cells were also identified in theLGE, at the cortico-striatal boundary, and in the intermediatezone of the neocortex (arrowheads in Fig. 1A′). Expressionof COUP-TFI and COUP-TFII was also seen in theinterganglionic region where the MGE and LGE are fused, butbefore the appearance of the CGE (asterisk in Fig. 1A,A′). Forconvenience, we will define this area as the ‘MLGE border’

Fig. 1. COUP-TFs are expressed in migrating cells. Adjacentcoronal sections of an E13.5 mouse brain at a medio-caudal levelimmunostained with anti-COUP-TFI (A) and COUP-TFIIantibodies (A′). The asterisks in A and A′ indicate expression in theinterganglionic region (the ‘MLGE’). The insets show anenlargement of this region after double labelling of COUP-TFs(COUP-TFI mRNA/COUP-TFII antibody in A; COUP-TFIantibody/COUP-TFII mRNA in A′). The arrow in A indicates theexpression boundary of COUP-TFI at the level of the medialcortex, whereas in the same region COUP-TFII is expressed in theputative cortical hem region (ch in A′). An arrow in A′ indicatesexpression in the marginal zone of the ncx, and the arrowheadsshow expression at the cortico-striatal boundary and in theintermediate zone of the ncx. (B-B′′′) Two different enlargementsof the regions indicated in A and A′. Most COUP-TF-positive cellsshow a polarized soma (arrows in B-B′′′), indicative of migratingcells. (C-C′) Expression of COUP-TFs in the POa region. Note thatCOUP-TFI is mostly restricted in the ventricular zone and in twostreams of migrating cells, as indicated by the arrows. Highexpression of COUP-TFII is mainly localized in the mantle zoneand in a dorsally migrating stream (arrow in C′). ch, cortical hemregion; cp, choroid plexus; LGE, lateral ganglionic eminence;MGE, medial ganglionic eminence; mz, mantle zone; ncx,neocortex; pcx, piriform cortex; POa, pre-optic area; VZ,ventricular zone.

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henceforth. Double labelling for COUP-TFI and COUP-TFIIand COUP-TFI and COUP-TFII highlighted an increasedexpression of COUP-TFs in this region (insets in Fig. 1A,A′).

Higher magnifications of the region between the MLGEborder and POa showed that the scattered COUP-TF-positivecells were grouped into streams of cells with polarized bodies,indicative of migrating neurons (Fig. 1B-B′′′). Although bothproteins were localized in the same area, double detection ofCOUP-TFI and COUP-TFII indicated that expression in thesepopulations overlapped only partially (data not shown). In thePOa region, COUP-TFs presented complementary expression(Fig. 1C,C′). While COUP-TFI was expressed at high levels inthe ventricular zone and in streams of cells oriented dorsallyand ventrally (arrows in Fig. 1C), COUP-TFII was mainlyexpressed in the mantle zone and in cells dorsal to the POaregion (arrow in Fig. 1C′). An additional complementaryexpression was evident at the level of the caudomedial wall ofthe telencephalon. While cortical COUP-TFI expressionstopped abruptly at the level of the hem region (Fig. 1A),COUP-TFII was specifically expressed in this region and in thechoroid plexus (Fig. 1A′).

In summary, COUP-TFI and COUP-TFII showed distinctand mostly non-overlapping expression profiles in thedeveloping telencephalon at E13.5. Furthermore, COUP-TFsappeared to be expressed in migrating cells at the stages inwhich several tangential migratory pathways within thedeveloping telencephalon are well pronounced.

COUP-TFs are detected in specific and non-overlapping cellular populations in the developingcortexTo characterize the types of cells in which COUP-TFs are

expressed, we performed a series of doubleimmunofluorescence studies for both COUP-TFs and cell-type-specific markers (Fig. 2).

The expression data described previouslysuggested that COUP-TFs are expressed inmigrating neurons. To test this hypothesis, weused an antibody against calbindin that labelsa subpopulation of GABAergic interneurons.At E13.5, the majority of calbindin-positivecells co-labelled with COUP-TFI-positivecells in the neocortex and piriform cortex (Fig.

2A,B), but not with COUP-TFII-positive cells (Fig. 2D,E).Similar results were obtained with an anti-GABA antibody(data not shown). Thus, in the marginal zone of the cortex,GABAergic neurons are positive for COUP-TFI and negativefor COUP-TFII. To assess whether COUP-TFII-positive cellsin the marginal zone were rather Cajal-Retzius cells, we usedan antibody against reelin. All COUP-TFII-positive cells co-expressed reelin (Fig. 2F), whereas no COUP-TFI-positivecells were positive for reelin in the cortex (Fig. 2C). At E13.5and E15.5 co-localization of COUP-TFII and reelin was alsodetected in the caudomedial wall of the telencephalic vesicles(Fig. 2G,H), which has been recently described as one of thesources of Cajal-Retzius cells (Takiguchi-Hayashi et al., 2004).This is not the case for COUP-TFI, in which no co-expressionof COUP-TFI and reelin has ever been detected in this region(Fig. 2I).

Taken together, these data strongly suggest that in the cortexCOUP-TFI is expressed in GABAergic interneurons, whereasCOUP-TFII is expressed in Cajal-Retzius cells.

COUP-TFI, and not COUP-TFII, is expressed inneurons migrating tangentially from the basaltelencephalon to the cortexWe have previously shown that some COUP-TFI-positive cellsin the cortex are GABAergic interneurons. It is now wellestablished that the GEs are the major source of mostneocortical interneurons (reviewed by Corbin et al., 2001;Marin and Rubenstein, 2001). Therefore, to assess whetherCOUP-TFI-positive cells located in the basal telencephalon areinterneurons migrating to the cortex, a series of grafts from theGEs of E13.5 GFP-expressing transgenic brain slices werehomotopically transplanted into wild-type E13.5 brains (n=27)

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Fig. 2. COUP-TFI is expressed in GABAergicinterneurons, while COUP-TFII is expressed inCajal-Retzius cells. Double immunofluorescenceof COUP-TFs (green) and cell-type-specificmarkers (red) in coronal sections of E13.5 (A-G) and E15.5 (H,I) mouse brains. COUP-TFI(A,B), but not COUP-TFII (D,E), co-labels withcalbindin in the MZ of the ncx (A,D) and of thepcx (B,E). COUP-TFII (F,G,H), but not COUP-TFI (C,I) co-labels with reelin in the MZ of theneocortex (C,F) and in the caudomedial corticalwall (G-I). The inset in G is a schematic diagramof the section, and indicates the position of panels(G-I). The arrows indicate double-positive cells.Note that in H, all the cells are double positive,while in I none of the cells are double positive.MZ, marginal zone; ncx, neocortex; pcx, piriformcortex.

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Fig. 3. COUP-TFI-positive cells in the MZ of the cortexoriginate from the basal telencephalon. (A) A graftingexperiment performed at E13.5, in which dorsallymigrating cells are analysed. The box delineates the areashown in C-H. (B) Example of an MLGE transplant thatshows GFP-positive cells migrating dorsally into thectx, laterally into the pcx, and ventrally into thePOa/hypo. (C-H) Double immunofluorescence for GFP(green) and COUP-TFs (red) shows that GFP-positivecells originating from the graft are double positive forCOUP-TFI in the MZ (arrowheads in C-E) and in theventricle-oriented cells deriving from the MZ (arrows inC-E). Arrows in insets show labelling of cells in theintermediate zone (IZ). (F-H) Doubleimmunofluorescence of GFP (green) and COUP-TFII(red) shows no co-localization of these two proteins. ctx,cortex; MZ, marginal zone; pcx, piriform cortex;POa/hypo, pre-opic area/anterior hypothalamus.

Fig. 4. Dorsal-to-ventral migration within the basal telencephalon. (A) A grafting experiment performed at E13.5, in which ventrally migratingcells are analysed. The box delineates the area shown in C-E′′. (B) Example of a graft in which GFP-positive cells have reached the POa.Arrows in C show co-localization of COUP-TFI (red) with GFP (green), whereas no co-labelling with COUP-TFII (red) has been identified(D) within the migratory cells. (E-E′′) Arrows in (E′′) indicate that migrating cells are neurons by double labelling of GFP (green) with Tuj1(red). (E′′) is a merge of panels (E) and (E′). (F) Double immunofluorescence of COUP-TFI (green) and GABA (red) shows that some COUP-TFI-positive cells (arrows) are GABAergic in the ventral migratory stream. (G) By electroporation of a GFP-expressing construct into theMLGE region, GFP-positive cells migrate ventrally, and these cells express COUP-TFI (arrows in H). Similarly, by inserting a DiI crystal intothe MLGE region, fluorescent cells are detected ventrally, in the POa/hypothalamic region (I). These cells co-express COUP-TFI (J). The arrowindicates a leading process. Note that the diffuse red signal in J is the result of the DiI signal after fixation.

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(Fig. 3A); expression of COUP-TFI and COUP-TFII wassubsequently monitored in adjacent sections of the same slices.An example of an MLGE graft is shown in Fig. 3B, whichdemonstrates the fate of fluorescent cells after 60 hours ofincubation, at which point GFP-positive cells were distributedin the mantle layer of the basal telencephalon and in variouslayers of the cortex. High magnification of the lateral andmedial cortex showed that GFP-positive cells were present inthe marginal and intermediate zones (Fig. 3C,F). Interestingly,some GFP-positive cells were also present in deeper layers,with a radial orientation towards the surface of the cortex(arrows in Fig. 3C-E). These cells were previously reported tomigrate from the marginal zone to the ventricular zone beforemoving radially, to take up their positions in the cortical anlage(Nadarajah et al., 2002). After double labelling with anti-COUP-TFI, 75% of the GFP-positive cells (n=250) were alsopositive for COUP-TFI, including the ventricle-directed cells(Fig. 3E). Adjacent sections of the same grafted brain slicesshowed no co-localization of COUP-TFII and GFP (Fig. 3H).This is in agreement with the observation that COUP-TFII-positive cells in the cortex are non-GABAergic, and thereforedo not originate from the basal telencephalon. Thus, our dataconfirm that COUP-TFI in the cortex is expressed inGABAergic interneurons originating from the subpallium,whereas COUP-TFII expression has a pallial origin.

COUP-TFI is expressed in neurons migratingtangentially towards ventral regions of thetelencephalonIn the transplantation experiments described above, weobserved that when the grafted region included the MLGEregion, a significant number of neurons also migrated towardsthe piriform cortex and towards more ventral regions, such as

the POa and the anterior hypothalamus (Fig. 3B). In somegrafts, GFP-positive cells were also identified in the POa (Fig.4B), suggesting that cells deriving from the interganglionicregion migrate in various directions: dorsally, laterally andventrally. By double labelling GFP-positive cells with anti-COUP-TFI and anti-COUP-TFII antibodies, we found double-positive cells for COUP-TFI, but not for COUP-TFII, in thisventral migratory path (Fig. 4C,D). This suggests that asubpopulation of COUP-TFI-positive cells located in thePOa/anterior hypothalamus might originate from the GEs.Furthermore, some of these migrating cells were neurons, asdemonstrated by the double staining of GFP with the neuronalmarker Tuj1 (Fig. 4E-E′′). Finally, a subpopulation of COUP-TFI-positive cells in the ventral stream was positive for GABA(Fig. 4F), suggesting that some ventrally migrating cells areGABAergic interneurons.

To validate the origin of this ventral stream, we used twofurther experimental approaches. We first electroporated aGFP expression vector into the MLGE region of E13.5 brainslices and followed the fate of the electroporated cells after60 hours of incubation (Fig. 4G). GFP-positive cells locatedventrally with respect to the injected side showed a leadingprocess and co-labelled with COUP-TFI, but not with COUP-TFII (Fig. 4H, and data not shown). Similarly, upon placinga crystal of DiI in the MLGE region (Fig. 4I), fluorescent cellswere subsequently identified in more ventral regions, andthese cells were positive for COUP-TFI (Fig. 4J). Themorphology of the DiI/COUP-TFI-positive cells fits wellwith the morphology of tangentially migrating cells observedby other authors (Nadarajah and Parnavelas, 2002). Indeed,the nuclei of these cells were oriented in the sense of themigration and had a process lagging behind the soma (arrowin Fig. 4J).

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Fig. 5. Cells originating from the caudalganglionic eminence can migrateventrally. (A) Summary of the type ofgraft and the results obtained after 60hours of incubation. (B) A caudalganglionic eminence (CGE) transplantshows GFP-positive cells migratingventrally in two streams: a medialstream and a lateral stream. The arrowsindicate that a few cells have reachedthe anterior hypothalamus. (C-F) Double immunofluorescence of GFP(green) and COUP-TFI (C,D) or COUP-TFII (red); E,F) on adjacent sections ofthe graft shown in B. Note that all GFP-positive cells have a leading processoriented ventrally (arrows), but that onlyCOUP-TFI is double positive for GFP(insets in C,D). ant hypo, anteriorhypothalamus; LS, lateral stream; MS,medial stream.

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Finally, to assess whether ventrally directed migration alsooriginates from more caudal levels, we grafted the CGE of aGFP E13.5 mouse brain to a wild-type brain of the same stage(n=5) (Fig. 5A). After 60 hours of incubation, GFP-positivecells migrated out of the graft into two main streams: a medialstream directed towards the third ventricle, and a lateral streamdirected laterally and ventrally into the hypothalamic region(Fig. 5B). Both streams contained single neurons with shortand polarized leading processes indicative of migrating cells(Fig. 5C-F). Adjacent sections of the same brain slices showedco-labelling of GFP with COUP-TFI (Fig. 5C,D), whereas noCOUP-TFII-positive cells were co-localized with GFP-positivecells (Fig. 5E,F).

Taken together, these data show that cells located in the basaltelencephalon migrate tangentially not only dorsally into thedeveloping neocortex, as previously described, but alsoventrally into the basal diencephalon, including the pre-opticand hypothalamic regions. Furthermore, our results show thatCOUP-TFI, but not COUP-TFII, is expressed in thesepopulations of migrating neurons, suggesting a functional roleof COUP-TFI in the patterning of tangential migration.

Ectopic expression of COUP-TFI in the basaltelencephalon promotes cells to migrate in bothdorsal and ventral directionsAt this stage, we have demonstrated that a subpopulation ofCOUP-TFI-positive cells in the basal telencephalon are cellsthat are in the process of migrating; however, this does notdemonstrate that COUP-TFI is directly involved in migration.To address this issue, we set up a functional assay in E13.5brain slice cultures, in which we ectopically expressed COUP-TFI in the GEs at different dorso-ventral and antero-posteriorlocations. The migrating cells were followed after 60 hours ofincubation. On one side, and as a control, we injected aconstruct expressing GFP under the control of a CMVpromoter, while on the contralateral side, we injected aconstruct expressing COUP-TFI followed by an IRES-GFPsequence. In this way, we could follow the fate of the injectedcells by following GFP fluorescence in the same slices (Fig.6A). The histogram in Fig. 6B shows the average value of GFP-positive cells in the cortex and POa, expressed as a percentageof the total number of positive cells (for control 2838 cells, andfor COUP-TFI 2318 cells; n=25; see also Materials and

Fig. 6. COUP-TFI modulatesdorsal and ventral migration inbrain slices. (A) Summary of thetype of experiments performedon E13.5 mouse brain slices. Onone side, a GFP-expressingvector is injected as a control,while on the contralateral side aninjection of a COUP-TFI-IRES-GFP expressing vector shows anincreased rate of migrating cells.(B) The average value of theGFP/GFPtot ratio (blackcolumns) or the GFP-COUP-TFI/GFPtot ratio (white columns)of all of the injected slices,calculated independently in thecortex or ventrally in the POa(see also Materials andmethods). Bars indicate standarderrors of the mean. In theCOUP-TFI injected side, 72±4%of the GFP-positive cells migrateinto the cortex, versus 24±4% ofthe GFP-positive cells in thecontrol side. For the POa ectopicexpression of COUP-TFI,46±3% of the GFP-positive cellswere induced to migrate, versus17±3% in the control situation.Significant P-values areindicated (for the cortexP=0.0008, for the POa P=0.004).Example of a control (C) and aCOUP-TFI overexpressing (D)brain slice after 60 hours ofincubation. The vectors werefocally injected into the MLGE region. Note that in the control situation (C), a few cells were found in the cortex (C′), while the majorityremain compacted in the injection point. (D) After injection of high levels of COUP-TFI into the MLGE region, the GFP-positive cells aremore dispersed (arrowheads) around the injection point, and a more pronounced contingent of cells migrates in the dorsal and ventraldirections. MZ, marginal zone; Cx, cortex.

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methods for details). In all the cases examined, the COUP-TFI-injected sides showed a statistically significant increase in thenumber of cells in the cortex, compared with the control side(Fig. 6B-D). Ventral migration was observed only in caudalsections in which the constructs were focally injected into thesubventricular zone of the MLGE region (Fig. 6A). In controls,24% and 17% of GFP-positive cells were scored on average inthe cortex and in the POa, respectively (Fig. 6B), whereas thenon-migrating cells were maintained in compact clusters nextto the injection site. The injection of a CMV-COUP-TFI-IRES-GFP construct into the contralateral side of the same slicesresulted in GFP-positive cells that were more dispersed in themantle layer (arrowheads in Fig. 6D), and that were still in theprocess of migrating dorsally towards the marginal zone of thecortex and ventrally towards the POa (arrows in Fig. 6D).Moreover, a higher number of GFP-positive cells were scoredin the cortex and in the POa (72% and 46%, respectively),compared with the control side. Thus, in the presence of higherlevels of COUP-TFI, injected cells showed significantlyincreased spreading and migrating, compared with the normalsituation (Fig. 6B), suggesting that COUP-TFI modulates cellmigration in the basal telencephalon.

Ectopic expression of COUP-TFII in the ganglioniceminences induces a higher rate of migration intothe intermediate zone of the cortexNext, we tested whether COUP-TFII was also able to promotecell migration in the basal telencephalon. We generated anadditional construct in which the COUP-TFII-IRES-GFPcassette was under the regulation of the CMV promoter.The control CMV-GFP- and CMV-COUP-TFII-IRES-GFP-

expressing constructs were injected into the subventricularzone of the striatum on opposite sides of E13.5 brain slices(n=10) (Fig. 7A). The histogram in Fig. 7B shows that in thepresence of ectopic expression of COUP-TFII, the averagenumber of GFP-positive cells found in the cortex (60%, of atotal number of 467 cells) was significantly higher than in thecontrol situation (32%, of a total number of 875 cells). Anexample of an injection performed in the whole striatum isshown in Fig. 7C,D. As expected, a high number of GFP-positive cells migrated out of the injection point into themarginal and intermediate zones of the neocortex. Afterinjection of the COUP-TFII-expressing construct, astatistically significant increase in the GFP-positive cells wasfound uniquely in the intermediate layer of the neocortex, andno GFP-positive cells were ever identified in the marginal zone(arrowheads and asterisk in Fig. 7D, respectively). However,and by contrast to previous experiments, no GFP-positive cellswere ever located ventrally in the POa region, even if wefocally injected the construct into the MLGE region (data notshown). Thus, ectopic expression of COUP-TFII in the basaltelencephalon promotes migrating neurons to undertake apreferential route into the intermediate zone of the neocortexby avoiding the marginal zone, suggesting a cell-autonomousproperty of COUP-TFII in guiding cortical neurons into aspecific layer of the cortex.

DiscussionInterneurons can migrate dorso-ventrally from thetelencephalon to the diencephalonCell migration within the telencephalon can be subdivided

Development 131 (24) Research article

Fig. 7. COUP-TFII regulates migration intothe intermediate zone of the neocortex.(A) Summary of the type of experimentsperformed on E13.5 mouse brain slices. Therespective vectors are depicted to the left ofeach part of the diagram. (B) The averagevalues of the GFP/GFPtot ratio (black column)and the COUP-TFII-GFP/GFPtot ratio (whitecolumn) in the cortex of all of the injectedslices. Bars indicate standard errors of themean. In the COUP-TFII injected side,60±4% of the injected cells migrate into thecortex, versus 25±5% of the GFP-positivecells in the control side. Examples of control(C) and COUP-TFII mis-expressing (D) brainslices in which the vectors have been injectedinto the Str. (C) Arrows indicate GFP-positivecells in the MZ, whereas arrowheads indicatethe IZ migratory stream. (D) Arrowheadsindicate a higher amount of GFP-positivemigrating cells in the IZ of the neocortex,whereas no cells are seen in the MZ(asterisk). Dotted line shows the putativeboundary between neocortex and striatum.All the cells external to the dotted line havebeen counted as cells located in the cortex.IZ, intermediate zone; MZ, marginal zone;Str, striatum.

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broadly into radial, and non-radial or tangential. While radialmigration uses the radial glia as a scaffold upon which corticalneurons migrate to the developing cortical plate, GABAergicinterneurons originate in the GEs and use tangential migrationto migrate over long distances to reach their destinations in thecortex (Marin and Rubenstein, 2003; Rakic, 1990).

In this report, we have identified a novel migratory path,which to our knowledge has never been reported before. Usinggrafting experiments, DNA electroporation and tracerinjections, we have shown that cells located in theinterganglionic region, where the MGE and LGE fuse together,have the capability of migrating ventrally towards the pre-opticand hypothalamic regions of the diencephalon (Figs 3-5). Asthis path originates from the interganglionic region at specificcaudal levels, it is very likely that it has been missed in otherstudies using similar approaches (Anderson et al., 2001;Jimenez et al., 2002; Lavdas et al., 1999; Wichterle et al.,2001). The novelty of this path is twofold: first, cells migratetangentially from dorsal to ventral, along the subventricularzone of the MGE, i.e. in opposite directions with respect to theMGE-derived cortical interneurons; and second, these cellsexit the basal telencephalon and migrate into the basaldiencephalon. These cells are different from the gonadotropin-releasing hormone neurons, which originate in the olfactoryplacode and migrate along the vomeronasal nerve fibres untilthey reach the POa and hypothalamus (Wray et al., 1994;Yoshida et al., 1995). Migration of GABAergic neurons fromthe GEs to the dorsal thalamus has been observed in humanbrains; however, rodent embryos did not reveal a similarmigratory pathway (Letinic and Rakic, 2001). With the help ofretroviral cell tracing, Marin and collaborators (Marin et al.,2000) have shown that cells emanating from the MGE andthe adjacent POa/anterior entopeduncular area express thetranscription factor Nkx2.1 and migrate to the developingstriatum, where they differentiate into local circuit neurons. Itwould be interesting to find out whether Nkx2.1 is also involvedin the opposite migratory path described in this report.

Furthermore, we have shown that cells originating from theCGE migrate via different streams into the basal diencephalon,and in particular into the hypothalamus (Fig. 5B). As opposedto the MLGE transplant, in which the stream of ventrallymigrating cells is quite compact, cells migrating out of theCGE take a medial and latero-ventral route. We do not knowthe fate of these ventrally migrating cells. In-vivo ultrasound-guided labelling or transplantation (Nery et al., 2002;Wichterle et al., 2001), in which total cortices are manipulatedin toto and characterized at later stages, would help us tounderstand the final destinies of these migrating cells.

Is the basal telencephalon only chemorepulsive?Transplantation experiments and co-culture assays have shownthat the POa and hypothalamic regions prevent interneuronsfrom migrating in a ventral direction by secreting repulsivemolecules that are responsible for the dorsal migration ofinterneurons towards the cortex (Marin et al., 2003; Wichterleet al., 2003). However, the cues mediating this action have notbeen identified yet. Here, we show that cells originating frommore dorsal and caudal regions are capable of migratingventrally towards the POa/anterior hypothalamus. How do wereconcile these different results? First, the previous studiesshowed that the majority of interneurons that originated from

the MGE avoided migrating in ventral directions. This is whatis expected for a circumscribed graft in a section where the twoGEs are well separated (Marin et al., 2003) (data not shown).However, the dorsal-to-ventral cell migration that we describein this report originates from a restricted region, theinterganglionic sulcus, at more caudal levels. It is plausible thatin order to follow the migration that originates in the MGE andLGE, this region was not included in other studies. Therefore,we hypothesize that cells originating from different rostro-caudal levels can have different responses to the sameenvironment by expressing position-dependent receptors.Accordingly, COUP-TFs are not expressed in ventrallymigrating cells at more rostral levels, where the two GEs arewell separated (data not shown). Thus, the POa andhypothalamus can have different roles, repulsion versusattraction, according to the type of cells they encounter.

A novel role for COUP-TFs in neuronal migration inthe developing forebrainCOUP-TFs are orphan nuclear receptors of the steroid/thyroidsuperfamily shown to be involved in neurogenesis, axogenesisand neural differentiation (Park et al., 2003). In the cortex,COUP-TFI is mainly known for its role in regionalization andin guidance of thalamocortical projections (Zhou et al., 1999;Zhou et al., 2001), whereas COUP-TFII is a fundamentalplayer in angiogenesis and heart development (Pereira et al.,1999). In this report, we have provided the first insights intothe functional roles of COUP-TFI and COUP-TFII in neuronalmigration in vivo. In a previous report, it was shown thatCOUP-TFI could regulate cell adhesion mechanisms requiredfor the differentiation of embryonal carcinoma cells (Adam etal., 2000). Because of the substrates on which cells wereplated, it was suggested that COUP-TFI could modify thesynthesis of ECM molecules, making cells autonomous formigration and spreading. Our expression data in vivo and ourfunctional data in organotypic cultures support these results ina context in which cell migration assumes a fundamental rolein achieving neuronal diversity. By using these differentapproaches, we have first shown expression of COUP-TFs inhighly motile neurons, such as GABAergic and reelin-positivecells (Fig. 2); we have then confirmed the co-localization ofCOUP-TFI in migrating neurons in a series of graftingexperiments (Figs 3-5); and finally, we have directlydemonstrated a role of COUP-TFs in modulating cellmigration in a gain-of-function approach (Figs 6, 7). Our dataalso demonstrate that COUP-TFs can control directionalmigration. Indeed, cells expressing higher levels of COUP-TFs(and thus GFP) not only tend to spread and lose their aspect ofaggregates, but also follow specific migratory pathways (dorsaland/or ventral) that are intrinsic to cells expressing COUP-TFs(Fig. 6, Fig. 7D).

Therefore, we hypothesize a dual role for COUP-TFs. First,they should regulate short-range cues, such as ECM and/oradhesive molecules, and in this way modulate the neighbouringenvironment. Changes in a permissive environment wouldallow random dispersion of migratory cells. Indeed, in theabsence of COUP-TFI, GABAergic cells do migrate into thecortex, although in a less organised fashion than in the normalsituation (M.S., unpublished). Therefore, we favour thehypothesis that COUP-TFI is involved in modulating cellmigration, more than inducing cells to initiate migration.

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Second, we propose that COUP-TFs control diffusibleguidance molecules, and/or their receptors, involved in cellmigration. Indeed, ectopic expression of COUP-TFI led to thecorrect directional migration of the targeted cells, i.e. dorsalinto the cortex and ventral into the POa (Fig. 6D), suggestingthat cells over- or mis-expressing COUP-TFI were able torespond to COUP-TFI-dependent guidance cues. Morestriking is the behaviour of MGE-migrating cells after mis-expression of COUP-TFII. In this case, neurons that werecommitted to migrate into the marginal and intermediatelayers of the neocortex (see control in Fig. 7C) migrated athigher rates solely into the intermediate layer, avoidingspecifically the marginal layer (Fig. 7D), suggesting thatCOUP-TFII regulates guidance cues required for migrationinto the intermediate zone of the neocortex. This behaviour isin accordance with the COUP-TFII expression pattern and thegrafting experiments described in this study. Indeed, COUP-TFII-positive cells in the marginal zone are Cajal-Retziuscells, as seen by the co-expression with reelin, and it is nowwell established that Cajal-Retzius cells originate exclusivelyfrom the pallium (Hevner et al., 2003; Meyer et al., 1999;Takiguchi-Hayashi et al., 2004).

ConclusionsIn summary, our data show a novel role for the COUP-TFnuclear receptors in tangential migration in the developingforebrain. COUP-TFI has previously been reported to berequired in axon guidance in the telencephalon andromboencephalon (Qiu et al., 1997; Zhou et al., 1999). Here,we show that COUP-TFs are involved in modulating the ratesand directions of tangentially migrating cells from the basaltelencephalon to the cortex, and from the basal telencephalonto the basal diencephalon. Interestingly, the cells follow amigratory path that is dependent on the expression of COUP-TFI and COUP-TFII, suggesting an intrinsic role for COUP-TFs in guiding migrating cells towards their target region.Finally, we speculate that COUP-TFs are regulators of a seriesof factors, such as short-range, non-diffusible contact guidancecues and long-range, diffusible guidance cues.

We thank A. Nagy for the GFP-expressing mice, A. Goffinet forthe anti-reelin antibody and M. Tsai for the COUP-TFI and COUP-TFII full-length cDNAs. We are grateful to G. Diez-Roux, P.Malatesta and G. Fishell for helpful comments on the manuscript, andto the whole Studer laboratory for fruitful discussions. This work wassupported by the Telethon Foundation and by the Fondo per gliInvestimenti della Ricerca di Base (FIRB RBNE01WY7P;RBAU01RW82 and RBNE01BBYW).

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