Abstract

The regionalization of the cerebral cortex proceeds gradually from early embryonic stages under the control of transcription factors that are expressed in gradients. Two phases can be distinguished at the beginning of cortical development: the genesis of a precocious and transient structure, the preplate, which is followed by development of the cortical plate within the preplate. Cellular indices of early regionalization have not yet been described either in the preplate or in the early cortical plate. In the present study, we identify two regions, lateral and dorsal, in the mouse cortex embryo, which differ strongly in the functional properties of their early neurons. By using culture experiments and grafts on organotypic slices, we show that the earliest neurons in the dorsal cortex extend axons before and more rapidly than the earliest neurons in the lateral cortex. In contrast to the lateral cortex, the dorsal cortex differentiates neurons migrating along axons in vitro. These cells express markers of the GABAergic lineage. Early differences between the two regions suggest that the dorsal part of the cortex generates early neurons with particular intrinsic properties that may in turn specifically influence the later development of the cortical plate in this domain.

Introduction

Recent studies on the expression domains of transcription factors have shown that the proliferative neuroepithelium of the pallium can be subdivided into four regions: the embryonic ventral, lateral, dorsal and medial pallium (Puelles et al., 2000; Yun et al., 2001). Within the dorsal pallium in which the neocortex differentiates, genes encoding transcription factors, cell adhesion or attractive molecules show graded patterns of expression before birth (Frantz et al., 1994; Gulisano et al., 1996; Yoshida et al., 1997; Inoue et al., 1998; Nakagawa et al., 1999; Rubenstein et al., 1999; Liu et al., 2000). The adult cerebral cortex is divided into areas with distinct anatomical and functional organization. There is increasing evidence for a genetic control of the process of cortical arealization during embryonic development. Experimental evidence suggests that complementary gradients of transcription factors, in particular Emx2 and Pax6, cooperate to determine the location and extension of cortical areas (Bishop et al., 2000; Mallamaci et al., 2000). The early patterning of genes controlling area identities apparently involves FGF8, a morphogen secreted by the anterior neural ridge (Fukuchi-Shimogori and Grove, 2001). In addition, the Emx2 gene has recently been identified as a direct transcriptional target of Bmp and Wnt signaling proteins in the dorsal telencephalon (Theil et al., 2002).

Transplantation experiments have shown that parts of the pallium identified by the expression of specific markers are committed to regional fates as early as stage E12.5 (embryonic day 12.5) in the rat (Horton and Levitt, 1988; Barbe and Levitt, 1991, 1992; Arimatsu et al., 1992, 1999) and E11.5 in the mouse (Cohen-Tannoudji et al., 1994; Gitton et al., 1999). LAMP, a marker of the limbic cortex, is expressed at E16 in the rat. However, at this stage it shows a decreasing latero-dorsal gradient of expression in the presumptive limbic cortex (Horton and Levitt, 1988) that could be related to the latero-rostral to medio-caudal gradient of dorsal pallium development.

In rodents, the latero-rostral to medio-caudal gradient of neurogenesis is believed to control most early events in neurogenesis, including developmental changes of cell-cycle dynamics (Schmahl, 1983; Bayer and Altman, 1991; Reznikov and van der Kooy, 1995; Polleux et al., 1997), maturation of the cortical plate and later events, such as the development of callosal projections or synaptogenesis (Voigt et al., 1993; Ozaki and Wahlsten, 1998). Accordingly, labeling experiments suggest that the first cortical axons to reach the internal capsule originate from the lateral and anterior cortex (De Carlos and O’Leary, 1992).

By examining the expression patterns of several transcription and guidance factors in the telencephalon of E12.5 mouse embryos, we have observed that the slope of their latero-medial gradients of expression is accentuated in a region located 400–500 μm dorsally to the lateral ventricular angle. The lateral cortical domain that extends between this change in slope and the ventricular angle expresses Cadherin6 at E12.5. By comparing the properties of neurons generated in this lateral domain with those of neurons generated more dorsally, we have observed that dorsal cortex axons grow earlier and further than lateral cortex axons and that dorsal axons are much more fasciculated than lateral axons. The lateral and dorsal parts of the cortex, moreover, differ in their capacity to produce tangentially migrating neurons and to differentiate GABAergic cells in vitro. These strong differences are opposite to those expected from the lateral to medial and rostral to caudal gradient of development of the dorsal pallium. They suggest that early neurogenetic events occurring before the differentiation of the cortical plate might contribute to differentiate dorsal and lateral domains into the developing cortical plate.

Materials and Methods

Animals

Embryos were produced in the laboratory from crosses between OF1 mice from IFFA Credo (Lyon, France), or from crosses between OF1 and Rosa 26 or GFPU transgenic mice (Jackson Laboratories, Bar Harbour, ME) that ubiquitously express the GFP and the β-galactosidase transgenes, respectively (Zambrowicz et al., 1997; Hadjantonakis et al., 1998). The day of vaginal plug detection was considered as E0.5.

In Situ Hybridization

Olfactory bulbs were removed and the occipital cortex was opened to allow easy fluid movements throughout the whole telencephalic vesicle of fixed brains. Whole brains were hybridized overnight at 70°C with digoxigenin-UTP labeled riboprobes (2 μg/ml) for Emx1, Emx2 (gifts of E. Boncinelli), Pax6 (gift of P. Gruss), Cadherin6 (gift of R.-M. Mège) and Slit1 (gift of M. Tessier-Lavigne) following a previously described protocol (Wilkinson and Nieto, 1993). The anti-digoxigenin alkaline-phosphatase coupled antibody used at a final concentration of 1/2000 was washed for at least 1 day before revelation with NBT/BCIP. Reacted brains were cryo-protected in phosphate-buffered saline (PBS) 30% sucrose, frozen and sectioned at 14 μm in the frontal plane with a cryostat. The sections were again treated with NBT/BCIP to increase the labeling intensity. Bright field images were acquired using a Spot 3.0 camera (Optilab SA, France). Paraformaldhehyde fixed explants were acetylated for 10 min and then hybridized overnight at 70°C with digoxigenin-UTP labeled riboprobes (2 mg/ml) for Dlx1 (Smith Fernandez et al., 1998) and Mash1 (gift of F. Guillemot) using the same protocol as for whole brains.

Culture of Explants

Embryonic brains at stages E11.5–E13.5 were dissected in L15 medium with 50 UI/50 μg of penicillin/streptomycin. Meninges were carefully removed from the dorsal telencephalon. Cortical explants from E11.5, E12.5 and E13.5 embryos were dissected from the dorsal and lateral cortex (see Fig. 2A) with tungsten needles. Rostro-caudal strips of cortex corresponding to the lateral and dorsal cortex were divided into small squares ∼150 × 150 μm in size, deposited on polylysine/laminin coated glass coverslips and cultured 2, 3 or 7 days in Dulbecco’s modified Eagle medium (DMEM)/F12, N2, B27 medium. Latero-dorsal strips of cortex dissected from E12.5 and E13.5 embryos were deposited on polylysine/ laminin-coated glass coverslips and cultured for 1 or 2 days in the same medium.

DiI Injections and Cortical Grafts in Forebrain Slices

Embryonic brains at stages E12.5–E14.5 were dissected in L15 medium with penicillin/streptomycin and embedded in 4% agarose (Sigma, Type VII) in L15 medium. Frontal sections 250 μm thick were cut using a tissue slicer (Campden Instruments, UK). The slices were collected in L15 medium and transferred on floating Biopore membranes (Millipore, Bedford, MA). They were cultured in DMEM/F12 medium supplemented with 2 mM l-glutamine, 33 mM d-glucose, 3 mM sodium bicarbonate, 10 mM HEPES buffer (pH 7.4), 50 UI/50 μg penicillin/streptomycin, 10% N2 and B27 complement (DMEM/F12, N2, B27), all from Gibco BRL. Glass micropipettes with a tip aperture ∼20 mm in diameter, filled with 5% DiI (Molecular Probes) in dimethylformamide were used to make a single small injection in the cortex of floating forebrain slices. After 2 days in vitro, the slices were fixed by immersion in 4% paraformaldehyde and analyzed. Cortical explants from E12.5 and E13.5 GFPU transgenic embryos, 150 × 150 μm in size, were dissected from the dorsal cortex, lateral cortex or the intervening region, as described above. Explants were grafted into E12.5 or E13.5 forebrain slices at the cortico-striatal frontier or in the dorsal cortex and grafted slices were cultured for 2 days. After fixation in 4% paraformaldehyde, the GFP was revealed by immunohistochemistry. Grafted slices immunostained with both anti-GFP and anti-GABA antibodies were fixed for 10 min in 4% paraformaldehyde and 0.2% glutaraldehyde and then post-fixed overnight in 4% para-formaldehyde.

Immunohistochemistry

Slices and cultures were fixed as described above. For GABA immuno-staining, we added 0.2% glutaraldehyde to the fixative. If diamino-benzidine (DAB) was the final reaction product, sections or cultures were pretreated for 1 h in PBS with 1% H2O2. For GABA, TUJ1 or GFP immunostaining, cultures or slices were preincubated for 1 h in PBS with 2 g/l gelatin and 0.2% Triton-X and incubated overnight with the primary antibody [rabbit anti-GABA serum (Sigma) 1/10 000 in PGT; mouse anti-TUJ1, 1/500 in PGT; mouse anti-GFP (Quantum) 1/200 in PGT]. The secondary antibodies [biotinylated or Cy3-conjugated or Alexa 488 coupled goat anti-mouse IgG (Jackson Laboratories, Bar Harbour, ME) and Cy3-conjugated or biotinylated goat anti rabbit IgG (Vector laboratories)] were diluted to 1/200 in PGT. The biotinylated antibodies were revealed using the Vectastain ABC Elite Kit (Vector Laboratories Inc.) and DAB as a chromagen. Sections and cultures were mounted in Mowiol and observed on a Zeiss Axioskop microscope (Zeiss GmbH, Germany). Grafted slices were observed using a confocal microscope (Leica DMRE TCS-SP2)

Results

Expression of Transcription Factors and Adhesive or Guidance Molecules Differ in the Lateral and Dorsal Cortical Domains

As previously described (Gulisano et al., 1996; Yoshida et al., 1997), the pallial expression of Emx1 and Emx2 showed a medio-dorsal/latero-ventral decreasing gradient in E12.5 brains, with a particularly weak expression level in the lateral cortex (Fig. 1AD). At E12.5, however, Emx2 showed a line of stronger expression at the upper side of the ventricular zone (VZ) in the dorsal cortex that was absent in the lateral cortex. This strong expression at the upper side of the dorsal cortical VZ was no longer observed at E13.5 (Fig. 1E). Pax6 was homogeneously expressed in the pallial VZ at E13.5 (Fig. 1H). At E12.5, however, it was more strongly expressed in the VZ of the lateral part of the cortex, particularly at its ventricular side, than in the VZ of the dorsal cortex (Fig. 1F,G).

Transcription factors control the expression of molecules implicated in adhesive or anti-adhesive interactions during axonal growth and cell migration. We therefore analyzed at E12.5 and E13.5 the pattern of expression of Cadherin6 and Slit1, already known for their regional expression in the embryonic cortex (Inoue et al., 1998; Nakagawa et al., 1999; Nguyen Ba-Charvet et al., 1999). These factors show regionalized expression within the cortical VZ. At E12.5, Slit1 was strongly expressed in the lateral part of the cortical VZ, whereas its expression decreased in the dorsal part of the cortical VZ (Fig. 1I,J). At E12.5, Cadherin6 was expressed in the VZ. In the cortex, its expression was restricted to a lateral sector (Fig. 1L,M). The regionalized expression of Slit1 in the cortical expression expanded dorsally in the cortical VZ (Fig. 1N).

Therefore, at E12.5 a lateral cortical region differed from a dorsal region in its expression level of Emx2 and Pax6, and in the VZ expression of Slit1 and Cadherin6. This lateral domain was enlarged dorsally in the caudal part of the telencephalic vesicle (cf. Fig. 1D,G,J,M and C,F,I,L). It extended from the lateral ventricular angle to a transition zone located ∼400 μm dorsally. In the parietal cortex (see Fig. 2A), the latero-medial extent of this region varied from 300 μm rostrally to 500 μm caudally. Because preplate neurons and early cortical plate neurons are generated at E12.5 in mouse, we set out to investigate whether the properties of early cortical neurons in these two cortical domains differed.

Lateral and Dorsal Cortex Explants Differ in their Axonal Outgrowth Properties and in their Capacity to Release GABAergic Cells In Vitro

Dorsal Cortex Explants Extend Longer Axons than Lateral Cortex Explants

We first analyzed the axonal outgrowth properties of early post-mitotic cortical neurons belonging to the lateral and dorsal cortical domains identified at E12.5. In E12.5 telencephalic vesicles, we dissected squares of pallium ∼800 × 800 μm2 in surface area, beginning at the ventricular angle. Each square was divided into three rostro-caudal strips, 200–300 μm wide (see Fig. 2A). Lateral (respectively dorsal) cortical explants were prepared by recutting the ventral-most (respectively dorsal-most) strip into smaller pieces, 150 × 150 μm2 in size. Explants were also prepared at E11.5, before the arrival of ganglionic eminence (GE) cells within the cortical wall (Anderson et al., 1997; Tamamaki et al., 1997; Lavdas et al., 1999) and at E13.5 when gene expression patterns no longer differed in the lateral and dorsal cortex. Explants were cultured on a laminin-coated substrate. After 2–3 days in culture, dorsal explants extended more and longer axons than lateral explants, which were surrounded by short and/or sparse axons (Fig. 2). In addition, dorsal axons extended 1 day before ventral axons. At E11.5, E12.5 and E13.5, we observed this difference in axon length between the dorsal and lateral explants. Cortical explants nevertheless matured, as attested by the progressively faster and more robust axonal outgrowth. After 7 days in culture, differences between dorsal and lateral explants were no longer visible (not illustrated). To visualize the shift in axonal outgrowth properties between the lateral and dorsal cortical domains, we cultured dorso-ventrally oriented strips of E13.5 neocortex on polylysine/laminin coated coverslips for 1 (Fig. 2E2) or 2 days (Fig. 2E1,E3). A dense axonal outgrowth was only observed around the dorsal part of explants. In addition, the bands of cortex showed shape modifications in the lateral domain that suggested distinct adhesive interactions in the two regions (Fig. 2E2,E3).

Overall, these experiments show that the earliest efferent projections from the dorsal and from the neighboring lateral cortex differ in their axonal growth rates and also in their adhesive properties.

GABAergic Cells Differentiate around Dorsal Cortical Explants

An additional striking feature distinguished isolated explants of dorsal and lateral cortex after 2 days in vitro at E11.5, E12.5 and E13.5. Numerous TUJ1-immunopositive cells were observed along efferent axons surrounding the dorsal explants (Fig. 2C1). Very few TUJ1-positive cells were observed around the lateral explants (Fig. 2C2). Dorso-ventrally oriented cortical strips were surrounded by TUJ1-positive cells only in their dorsal domain (Fig. 2E2). The lateral and dorsal cortex therefore differed in their ability to produce or release precocious neurons that migrated along efferent axons.

A large proportion of neurons that surrounded the dorsal explants became GABA-immunoreactive after 2–3 days in vitro (Fig. 3, cf. A1 and B1). These neurons had a rather simple bipolar morphology (Fig. 3C,C′). Only a small number of these bipolar GABAergic cells were observed around lateral cortical explants after several days in vitro. This suggests that the capacity to produce the ‘smooth’ bipolar GABAergic cells in vitro was mostly a property of the dorsal cortex. These cells differed from a second type of strongly GABA-immunopositive cells, showing neurites covered with multiple short branches (Fig. 3A2′), that were observed in E12.5 lateral explants from the beginning of the culture time (Fig. 3, cf. A2 and B2). These cells were very rare in E11.5 lateral cortical explants and absent from E11.5 and E12.5 dorsal cortical explants, but were observed in both lateral and dorsal E13.5 cortical explants. They do not seem to differentiate in vitro as they were not observed in E11.5 explants after 7 days in culture (not illustrated). This suggests that they might invade the cortex by tangential migration, starting from ventro-lateral regions.

We then checked whether neurons that migrated around E12.5 dorsal cortex explants among efferent axons expressed the same markers as cells of the GABAergic lineage originating in the ventral telencephalon, in particular Dlx1 and Mash1 (Schuurmans and Guillemot, 2002). These cells indeed expressed Dlx1 and Mash1 at low levels (Fig. 3D1,E1). The few cells that surrounded lateral cortex explants did not express Dlx1, whereas cells expressing Dlx1 at high levels were scattered within lateral explants (Fig. 3D2,D2′). In contrast, Mash1 was not expressed at high levels in lateral explants (Fig. 3E2,E2′). One or two cells expressing Dlx1 at high levels were also occasionally observed within dorsal explants (not illustrated).

Therefore, the early neurons that migrate from dorsal explants among efferent axons in vitro express markers of the ventral GABAergic lineage weakly, in particular Dlx1 and Mash1.

Axonal Outgrowth Properties of Dorsal and Lateral Cortical Neurons in Organotypic Slices

Labeling of Corticofugal Axons in Organotypic Slices

The axonal growth from the dorsal and lateral parts of the neocortex was analyzed in organotypic slices at both E12.5 and E13.5. Small injections of DiI performed in the cortex at E12.5 labeled axons that were mostly orientated ventrally (Fig. 4). At this stage, axons labeled from dorsal or lateral sites showed similar ranges of lengths. At E13.5, injections at dorsal, lateral and intervening sites labeled axons that reached the cortico-striatal junction and stopped there. The axons from the dorsal region were thus significantly longer than those from either intervening or lateral sites. In addition, the axons labeled from dorsal or lateral sites exhibited strong differences in fasciculation, suggestive of differences in adhesion properties. Dorsal injections labeled fasciculated axons, whereas lateral injections labeled no fasciculated axons (Fig. 4C1–C3). In E12.5 slices, however, these differences in adhesive properties were not as clearly defined.

We had previously shown that early corticofugal axons wait for ∼24 h in the lateral part of the GE before pursuing their journey (Métin and Godement, 1996). Differences in lengths of axons originating in the dorsal and lateral cortex could therefore result from differences in distances between cell bodies and the region where growth cones wait. To assess whether dorsal and lateral cortical axons extending in organotypic slices differ in their intrinsic growth rate, we examined the axonal outgrowth from grafts of dorsal or lateral cortex placed into the same region in organotypic slices.

Axonal Outgrowth from Grafted Cortical Explants

Small explants of E12.5 or E13.5 GFP-expressing cortex from the dorsal, lateral or intervening zones were grafted close to the cortico-striatal junction in isochronic telencephalic slices. After 2 days in culture, explants extended axons into the host slice. Short, dorsally oriented (toward the cortex) GFP-positive axons were commonly observed around all types of grafts (Fig. 5AE). In contrast, explants of dorsal and lateral E13.5 cortex differed in the aspect of the ventrally oriented GFP-positive axons. The proportion of dorsal explants extending axons was three times higher (see Table 1) than that of lateral explants, and dorsal E13.5 explants grew more axons than the lateral or intermediate explants. Dorsal explants extended long, fasciculated ventral axons, whereas lateral and intermediate explants grew short, unfasciculated ventral axons (Fig. 5BD). This result therefore suggested that the differences in axonal length between the dorsal and lateral cortical neurons result from intrinsic differences in their growth rates.

To assess the influence of local environment on axonal outgrowth, we placed grafts of E12.5 dorsal or lateral cortex in the dorsal cortex of E13.5 forebrain coronal slices. After 2 days in culture, >80% of dorsal grafts (n = 42) extended fasciculated axons at the surface of the host cortex (Fig. 5H,J) compared to 15% of lateral grafts (n = 38). Lateral grafts were either surrounded by very short axons (Fig. 5F) or extended small tufts of axons that did not form long fascicles as observed around dorsal grafts (Fig. 5G,I).

These experiments show that in the early embryonic cortex, dorsal axons grow faster than lateral axons. Dorsal axons also form long fascicles, whereas lateral axons do not.

Cells Migrating from Lateral and Dorsal Cortical Explants Differ in their Distribution and GABAergic Differentiation

Numerous cells migrated from the GFP-expressing cortical explants grafted either at the cortico-striatal junction or in the dorsal cortex. When grafts were performed at the cortico-striatal junction, isolated cells were observed dorsally within the cortex and ventrally within the ventral pallium or subpallium. The number of cells migrating dorsally into the cortex was much higher in E13.5 grafts than in E12.5 grafts. However, we observed that cells originating in E12.5 dorsal explants migrated toward the cortex of host slices (Fig. 5E). Accordingly, DiI injections performed in the dorsal cortex of E12.5 forebrain slices labeled a large number of dorsally migrating cells, when tangentially migrating cells from extrinsic origin were still rare in the dorsal cortex (see Fig. 4D).

When E12.5 dorsal or lateral cortical explants were placed in the dorsal cortex of E13.5 forebrain slices, a large number of isolated GFP-positive cells migrated over long distances in the host slices (Fig. 5FH). The number of GFP-positive cells that distributed far from explants after 2 days in vitro was higher in slices grafted with lateral explants than in slices grafted with dorsal explants (Table 2). In addition, cells originating in dorsal explants distributed at the surface of cortical plate, whereas cells originating in lateral explants distributed both in the cortical plate and in the intermediate zone. These numerous long-distance tangentially migrating cells released by lateral explants into host slices did not migrate out of isolated lateral explants in vitro (see Fig. 2C2).

We then analyzed the GABAergic phenotype of cells that migrated from lateral and dorsal explants. Surprisingly, a minority of cells that migrated over long distances in the host were GABA-immunopositive after 2 days in vitro. Their proportion was higher in slices grafted with lateral cortex explants than in slices grafted with dorsal cortex explants (see Table 2 and Fig. 6A,B). In slices grafted with dorsal cortex explants, however, we observed that numerous GFP-expressing cells surrounded explants. These GFP-positive cells were more numerous around dorsal than lateral explants and, in addition, the proportion of GABA-positive cells was significantly higher in cells surrounding dorsal explants than in cells surrounding lateral explants (Table 2 and Fig. 6C).

These results show that the dorsal cortex differs from the lateral cortex in its capacity to differentiate short-distance-migrating GABAergic cells and that lateral cortex explants can release many more long-distance-migrating cells when grafted in organotypic slices than dorsal cortex explants.

Discussion

In the present study, we show that the dorsal part of the cortex differentiates early neurons that markedly differ in their axonal outgrowth properties from early neurons produced in the lateral part of the cortex. These differences are opposite to those expected from the rostro-lateral to caudal-dorsal maturation gradient observed in the cortical plate. In addition, the cells that migrate tangentially from these two cortical regions differ in their migratory pathways and their capacity to differentiate as GABAergic cells in vitro and in organotypic slices. These early regional differences observed at E12.5 are maintained at E13.5, beyond the time when several transcription factors show transient differences in their expression levels between the lateral and dorsal parts of the cortex.

Early Efferent Projections from the Dorsal and Lateral Cortex

We have observed that the earliest efferent axons from the dorsal cortex elongate faster than those originating from the lateral cortex. Differences in the rate of elongation of early cortical axons have been reported previously. In the ferret, for example, layer V axons develop faster than layer VI axons (Clasca et al., 1995). In E17–P0 rat embryos, efferent neurons in the medial cortex have been shown to grow faster than efferent neurons in the dorso-lateral cortex (Mouveroux et al., 2000).

Differences in the rate of elongation between lateral and dorsal early efferent cortical axons were not suspected previously. Developing axons have been analyzed on fixed brains injected with carbocyanine dyes (DeCarlos and O’Leary, 1992; Miller et al., 1993) and it was observed that early cortical axons accumulate for ∼1 day in the lateral part of the lateral ganglionic eminence (LGE) and then pursue their migration into the basal telencephalon (Métin and Godement, 1996). During this pause, both fast and slow corticofugal axons can reach the LGE. As previously proposed for sensory ganglia neurons (Davies, 1994), the existence of fast and slow pioneer projections might synchronize the arrival of the first axons from the dorsal and lateral cortex to the LGE, their common intermediate target (Métin and Godement, 1996). The two projections would therefore be able to interact during the same period of time with the same set of local cues. Alternatively, the early differentiation of two pioneer projections, differing in their growth rates and adhesion properties, might contribute to distinguishing two neighboring cortical domains. Together with mechanisms diversifying the molecular properties of target cells in neighboring cortical regions, the early differentiation of efferent projections might facilitate specific interactions with distinct sets of afferent projections (Molnar et al., 1998) and participate in the establishment of specific thalamo-cortical connections. For example, two molecules involved in the targeting of thalamic axons, LAMP and Ephrin-A5, are differentially expressed in the infragranular layers of the developing limbic and somatosensory cortex (Pimenta et al., 1995; Gao et al., 1998; Mann et al., 1998).

The early neurons that extend fast axons in the dorsal cortex and slower axons in the lateral cortex remain to be identified. The early axons might arise either from the preplate/subplate or from layer VI. In organotypic slices, fast-growing axons appeared to originate in the preplate in the dorsal cortex, whereas slow-growing axons originated in the preplate or lateral subplate in the lateral cortex. Early neurons in the subplate can extend long efferent axons (De Carlos and O’Leary, 1992; McConnell et al., 1994). An earlier study (Meyer et al., 1998) described preplate pioneer neurons with long axons. The fast early efferent projection from the dorsal cortex might therefore arise from a specific population of preplate/subplate neurons able to extend long projections. This early population could be restricted to the dorsal cortex and the early efferent projections from the lateral cortex could be established by an early population of layer VI neurons. Alternatively, preplate neurons able to extend early axons could differentiate in both the lateral and dorsal cortex, but lateral preplate neurons would then strongly differ from dorsal preplate neurons by their axonal outgrowth properties.

Early Differentiation of Two Cortical Regions

Lateral and dorsal cortical regions that differed in their axonal outgrowth properties were initially identified at E12.5 by their differences in expression of Pax6 and Emx2 and by the expression of Slit1 and Cadherin6 in the VZ. However, none of these genes showed a regionalized expression in postmitotic cells and therefore they could not be used as markers of lateral and dorsal cortical neurons. In addition, gene patterns no longer identified a lateral sector at E13.5, whereas strong differences in axonal outgrowth were still observed between the lateral and dorsal cortex.

Pax6 regulates the expression of R-cadherin (Stoykova et al., 1997), which, together with Cadherin6, is strongly expressed in the lateral cortex at E12.5. Accordingly, we observed that the adhesive properties of lateral cortex explants differed from those of dorsal cortex explants in vitro. Emx2 and Pax6 transcription factors are involved in controlling the establishment of regional differences in cell proliferation and cell differentiation (Yoshida et al., 1997; Gotz et al., 1998; Warren et al., 1999; O’Leary and Nakagawa, 2002). Latero-dorsal differences in the cell-cycle length have been observed in the cortical VZ (Schmahl, 1983; Takahashi et al., 1995; Bellion and Métin, unpublished results) and changes in G1 phase length between the lateral and dorsal cortex have been described at E12.5 and E13.5 in mouse embryos (Miyama et al., 1997). Because the same transcription factors may regulate both cell-cycle length and cell fate determination (Nieto et al., 2001; Ohnuma et al., 2001), these regional differences in proliferation rate in the cortical VZ at an early embryonic stage might be related to the differentiation of early neurons with fast-growing axons and/or to the differentiation of early tangentially migrating cells in the dorsal cortex.

The later fate of cortical regions with lateral or dorsal axonal outgrowth properties is unclear, since no molecular marker that is expressed regionally at E12.5/E13.5 in postmitotic cortical cells has been described. Dorsal explants were dissected in the presumptive dorsal pallium (Puelles et al., 2000; Yun et al., 2001). Lateral explants were dissected in a pallial domain dorsal to the ventricular angle and that faintly expressed Emx1. This domain lies above the piriform cortex and could belong to the dorsal pallium. It might prefigure the limbic subdivision of the neocortex that is specified from an early embryonic stage — E13/E14 in the rat embryo (Horton and Levitt, 1988; Barbe and Levitt, 1991).

Differentiation of Tangentially Migrating and GABAergic Cells in the Dorsal Cortex

Dorsal and lateral explants differed in their capacity to produce or release tangentially migrating neurons when cultured on a laminin substrate. Dorsal explants prepared at stages E11.5/ E12.5, when very few GE cells have reached the dorsal cortex (Anderson et al., 1997; Tamamaki et al., 1997; Lavdas et al., 1999), produced tangentially migrating cells. A much lower number of tangentially migrating neurons was observed around lateral cortical explants, though they are expected to contain many more GE cells than dorsal explants at this stage. Neurons observed around isolated dorsal cortical explants are therefore very unlikely to have originated in the GE. They appeared to be produced within the dorsal cortical neuroepithelium. Cells in cultured cortical explants that strongly expressed Dlx1 could originate in the GE. Indeed, they were much more frequent in lateral explants than in dorsal explants at E12.5. Because these cells were never observed around explants cultured on laminin, they seem unable to migrate out of isolated cortical explants in vitro.

In contrast, cells migrating around dorsal explants cultured on laminin weakly expressed the ventral telencephalic markers Dlx1 and Mash1 (Anderson et al., 1997; Casarosa et al., 1999). This property correlates with the progressive acquisition of a GABAergic phenotype by these cells in vitro. Indeed, genetic studies, in particular using gain of function strategies, have shown that ventral telencephalic markers such as Dlx and Mash1 actually identify the GABAergic lineage (Anderson et al., 1999; Fode et al., 2000; Parras et al., 2002; Schuurmans and Guillemot, 2002). It has been reported that cells in the dorsal part of the early embryonic cortex weakly express Mash1 (Fode et al., 2000). Our results suggest that a subpopulation of cortical cells expressing Mash1 at a weak level could give rise to GABAergic cells during normal corticogenesis in the mouse. This conclusion is in agreement with studies showing that mutations affecting the basal forebrain reduce, but do not eliminate, the population of cortical interneurons (Anderson et al., 1997; Casarosa et al., 1999; Sussel et al., 1999). A recent study in the human fetal cortex has shown that a proportion of GABAergic interneurons is generated within the cortical proliferative zone (Letinic et al., 2002). These cells express Dlx1/2 and Mash1 and contribute to more than a half of the GABAergic interneurons in the cortex of human fetuses. Our results suggest that the genetic program that controls the genesis of these Dlx2- and Mash1-expressing cortical interneurons could also operate in the mouse cortex.

By comparing the distribution of cells around isolated or grafted cortical explants, it appears that lateral cortex explants produce cells able to migrate long distances in host slices, but unable to leave isolated explants. In contrast, dorsal cortex explants mostly produce short-distance-migrating cells that can leave both isolated and grafted explants. Cells that migrate from lateral explants might originate in the GE. In slices, they distribute widely within the cortical plate and intermediate zone and their distribution resembles that of labeled cells from the GE in organotypic slices (Anderson et al., 1997; Tamamaki et al., 1997; Lavdas et al., 1999). We observed that a large fraction of migrating cells were not GABAergic. Accordingly, in cells that migrate tangentially from the basal telencephalon, the proportion of GABAergic cells was recently evaluated at 35–40% (Polleux et al., 2002). Dorsal explants also released a small population of long-distance, tangentially migrating cells that showed similarities with cells released by lateral explants. We cannot exclude the possibility that a few GE cells had already reached the dorsal part of cortex at E12.5. Alternatively, these long-distance-migrating cells could also originate in the cortex. Long-distance, tangentially migrating cells have been labeled following adenovirus infections of the cortex in organotypic slices (Chapouton et al., 1999). Nevertheless, a large proportion of cells that left dorsal cortex explants distributed close to their explants of origin and were able to migrate both in vitro and in host slices. Half of them, at least, differentiated into GABAergic cells. In addition to probably originating in the dorsal cortex, these cells therefore differ in their remarkable migration properties from cells released by lateral explants. Their functional characteristics could rely on the expression of a particular combination of transcription factors, including Mash1. As they mostly differentiate into GABAergic cells, they could significantly contribute to the precocious population of GABAergic neurons in the dorsal part of the mouse cortex.

In conclusion, we have presented evidence showing that at early developmental stages — E12.5/E13.5 in the mouse — the cortex already contains two domains with marked differences in the functional properties of their early neurons. These precocious neurons might play important roles in controlling the establishment of later cortical structures.

The authors thank Patricia Gaspar and Boris Barbour for helpful comments at different stages of the manuscript preparation, Sheela Vyas for revising the English and Rosette Goiame for technical support. This work was supported by EU grant Bio4CT960146 to M.W. and grant No. 5934 from Association pour la Recherche contre le Cancer (ARC) to C.M.

Table 1

Axonal outgrowth from GFP-expressing cortical explants grafted at the cortico-striatal frontier of forebrain slices

 Embryonic stages 
 E12.5 graft in E12.5 slice E13.5 graft in E13.5 slice 
Quantitative analysis of the percentage of cortical explants of dorsal, lateral or intervening origin that extended axon bundles after grafting at the cortico-striatal frontier of forebrain slices. The mean number of axon bundles per graft is indicated in each situation. The longest ventrally and dorsally oriented axons were measured in each graft and mean values calculated. 
aSignificantly different from axon lengths in E13.5 lateral and intervening grafts (P < 0.001, Student’s t-test in two cases). 
bSignificantly different from the percentage in lateral explants (Student’s t-test, P < 0.02). 
cSignificantly different from the result in lateral and intervening explants (Student’s t-test, P < 0.0001 in the two cases). 
dSignificantly greater than E12.5 equivalent case (Student’s t-test, P < 0.01). 
Origin of cortical grafts lateral intermediate dorsal lateral intermediate dorsal 
Number of grafted slices  18  28  16  45  39  39 
Grafts growing ventral axon bundles (%)  17  57  56b  27  38  79c,d 
Mean number of bundles per graft  1.6 ± 0.6  1.9 ± 1.2  2 ± 0.9  1.2 ± 0.4  1.7 ± 0.7  3.1 ± 1.8c 
Maximum length of axons (μm)       
    Ventrally oriented axons 270 ± 110 270 ± 100 320 ± 110 260 ± 100 300 ± 110 410 ± 120a 
    Dorsally oriented axons 250 ± 50 230 ± 70 200 310 ± 160 310 ± 140 240 ± 60 
 Embryonic stages 
 E12.5 graft in E12.5 slice E13.5 graft in E13.5 slice 
Quantitative analysis of the percentage of cortical explants of dorsal, lateral or intervening origin that extended axon bundles after grafting at the cortico-striatal frontier of forebrain slices. The mean number of axon bundles per graft is indicated in each situation. The longest ventrally and dorsally oriented axons were measured in each graft and mean values calculated. 
aSignificantly different from axon lengths in E13.5 lateral and intervening grafts (P < 0.001, Student’s t-test in two cases). 
bSignificantly different from the percentage in lateral explants (Student’s t-test, P < 0.02). 
cSignificantly different from the result in lateral and intervening explants (Student’s t-test, P < 0.0001 in the two cases). 
dSignificantly greater than E12.5 equivalent case (Student’s t-test, P < 0.01). 
Origin of cortical grafts lateral intermediate dorsal lateral intermediate dorsal 
Number of grafted slices  18  28  16  45  39  39 
Grafts growing ventral axon bundles (%)  17  57  56b  27  38  79c,d 
Mean number of bundles per graft  1.6 ± 0.6  1.9 ± 1.2  2 ± 0.9  1.2 ± 0.4  1.7 ± 0.7  3.1 ± 1.8c 
Maximum length of axons (μm)       
    Ventrally oriented axons 270 ± 110 270 ± 100 320 ± 110 260 ± 100 300 ± 110 410 ± 120a 
    Dorsally oriented axons 250 ± 50 230 ± 70 200 310 ± 160 310 ± 140 240 ± 60 
Table 2

Percentage of GABA-immunopositive, GFP-expressing cells that migrated from E12.5 dorsal or lateral cortical explants grafted in the dorsal cortex of E13.5 forebrain slices

 Total no. of cells Close to explant Long-distance migrating 
   Percentage in category  Percentage in category 
  No. of cells in the category (% of total) GABA-negative Uncertain GABA-positive No. of cells in the category (% of total) GABA-negative Uncertain GABA-positive 
Evaluation of GABA immunostaining in GFP-immunopositive cells using confocal microscopy (40× objective). Only cells surrounded by GABA-immunopositive cells or fibers of the host were analyzed. Cells were counted as ‘GABA-positive’ if cell body and processes were strongly GABA-immunopositive. In ‘GABA-negative’ cells, no red labeling could be observed, even with increased contrast. Cells with faint cell-body staining were classified as ‘uncertain’. Cells were classified as ‘long-distance’ migrating cells if >200 μm away from the explants. Dorsal explants released a higher proportion of short-distance, tangentially migrating cells, whereas lateral explants released a higher proportion of long-distance, tangentially migrating cells. Differences were significant (χ2-test, P = 0.001). The proportion of GABAergic cells was higher in cells that distributed around dorsal explants than in cells close to lateral explants (χ2-test, P = 0.05). The proportions of GABAergic cells in long-distance-migrating cells released by dorsal or lateral explants did not significantly differ. 
Dorsal cortex (11 grafts) 93 50 (54%) 20 30 50 43 (46%) 30 40 30 
Lateral cortex (9 grafts) 109 31 (28%) 45 23 32 77 (72%) 33 23 44 
 Total no. of cells Close to explant Long-distance migrating 
   Percentage in category  Percentage in category 
  No. of cells in the category (% of total) GABA-negative Uncertain GABA-positive No. of cells in the category (% of total) GABA-negative Uncertain GABA-positive 
Evaluation of GABA immunostaining in GFP-immunopositive cells using confocal microscopy (40× objective). Only cells surrounded by GABA-immunopositive cells or fibers of the host were analyzed. Cells were counted as ‘GABA-positive’ if cell body and processes were strongly GABA-immunopositive. In ‘GABA-negative’ cells, no red labeling could be observed, even with increased contrast. Cells with faint cell-body staining were classified as ‘uncertain’. Cells were classified as ‘long-distance’ migrating cells if >200 μm away from the explants. Dorsal explants released a higher proportion of short-distance, tangentially migrating cells, whereas lateral explants released a higher proportion of long-distance, tangentially migrating cells. Differences were significant (χ2-test, P = 0.001). The proportion of GABAergic cells was higher in cells that distributed around dorsal explants than in cells close to lateral explants (χ2-test, P = 0.05). The proportions of GABAergic cells in long-distance-migrating cells released by dorsal or lateral explants did not significantly differ. 
Dorsal cortex (11 grafts) 93 50 (54%) 20 30 50 43 (46%) 30 40 30 
Lateral cortex (9 grafts) 109 31 (28%) 45 23 32 77 (72%) 33 23 44 
Figure 1.

Expression of Emx1, Emx2, Pax6, Slit1 and Cadherin6 in the cortex at embryonic stages E12.5 (AD,F,G,I,J,L,M) and E13.5 (E,H,K,N). In situ hybridization was performed on 14 μm thick frontal cryostat sections. Two rostro-caudal levels are illustrated at E12.5: anterior sections corresponding to the beginning of the thalamus and posterior sections to the level of the caudal GE. E12.5 sections show a decreasing dorso-lateral gradient of Emx1 expression (A,B). At E12.5, Emx2 (C,D) and Pax6 (F,G) show opposite latero-dorsal gradients of expression. At E13.5, the gradient of Emx2 expression is strongly attenuated (E), whereas the Pax6 gradient is no longer detectable (H). At E12.5, expression of Slit1 (I,J) and Cadherin6 (L,M) in the ventricular zone (VZ) is restricted to a latero-ventral sector of the cortex. In E13.5 embryos, the VZ expression of Slit1 is fainter but reaches the dorsal cortex. In comparison, the dorsal expansion of the VZ expression of Cadherin6 is moderate and leaves the most dorsal part of the cortex unlabeled. Scale bars = 200 μm.

Figure 1.

Expression of Emx1, Emx2, Pax6, Slit1 and Cadherin6 in the cortex at embryonic stages E12.5 (AD,F,G,I,J,L,M) and E13.5 (E,H,K,N). In situ hybridization was performed on 14 μm thick frontal cryostat sections. Two rostro-caudal levels are illustrated at E12.5: anterior sections corresponding to the beginning of the thalamus and posterior sections to the level of the caudal GE. E12.5 sections show a decreasing dorso-lateral gradient of Emx1 expression (A,B). At E12.5, Emx2 (C,D) and Pax6 (F,G) show opposite latero-dorsal gradients of expression. At E13.5, the gradient of Emx2 expression is strongly attenuated (E), whereas the Pax6 gradient is no longer detectable (H). At E12.5, expression of Slit1 (I,J) and Cadherin6 (L,M) in the ventricular zone (VZ) is restricted to a latero-ventral sector of the cortex. In E13.5 embryos, the VZ expression of Slit1 is fainter but reaches the dorsal cortex. In comparison, the dorsal expansion of the VZ expression of Cadherin6 is moderate and leaves the most dorsal part of the cortex unlabeled. Scale bars = 200 μm.

Figure 2.

Axonal outgrowth and cell migration around explants of lateral and dorsal cortex. The diagram in (A) shows the cortical regions that were used to prepare dorsal and lateral explants at E12.5. The thick open rectangle represents the region used to prepare latero-dorsally oriented explants. Explants prepared from the dorsal or lateral cortex at E11.5 (B), E12.5 (C), or E13.5 (D,E) were cultured for several days on a laminin substrate and reacted with TUJ1 antibodies. After 2 or 3 days in vitro, they show striking differences in their density of axonal processes and in the number of long-distance-migratory neurons. Latero-dorsal bands of E13.5 parietal cortex cultured for 2 days (E1,E3) show long and dense axons only around their dorsal part. Similarly, cells were seen to migrate out of the dorsal part of the E13.5 explants that were cultured for 1 day (E2). Scale bars = 200 μm.

Figure 2.

Axonal outgrowth and cell migration around explants of lateral and dorsal cortex. The diagram in (A) shows the cortical regions that were used to prepare dorsal and lateral explants at E12.5. The thick open rectangle represents the region used to prepare latero-dorsally oriented explants. Explants prepared from the dorsal or lateral cortex at E11.5 (B), E12.5 (C), or E13.5 (D,E) were cultured for several days on a laminin substrate and reacted with TUJ1 antibodies. After 2 or 3 days in vitro, they show striking differences in their density of axonal processes and in the number of long-distance-migratory neurons. Latero-dorsal bands of E13.5 parietal cortex cultured for 2 days (E1,E3) show long and dense axons only around their dorsal part. Similarly, cells were seen to migrate out of the dorsal part of the E13.5 explants that were cultured for 1 day (E2). Scale bars = 200 μm.

Figure 3.

GABAergic phenotype and expression of Dlx1 and Mash1 in cells migrating around dorsal (A1,B1,C,D1,E1) and lateral (A2,B2,D2,E2) cortical explants cultured for 2 (A,C,D,E) or 3 (B) days. The long-distance-migratory neurons observed around dorsal explants of E12.5 (B1) and E13.5 (C,C′) cortex were GABA-immunopositive after 2 or 3 days in vitro (cf. A1 and B1). Strongly GABA-immunoreactive cells with small expansions on their neurites were observed in E12.5 lateral explants (A2, A2′, B2) from the beginning of the culture (not shown). (C′) and (A2′) are high-magnification views of GABAergic cells in (C) and (A2), respectively. A large proportion of cells that migrated around dorsal cortex explants expressed Dlx1 (D1) and Mash1 (E1) weakly (open arrowheads point to one labeled cell in each case). Expression of Dlx1 and Mash1 around lateral explants (D2,E2) and within the thickness of the same explants (D2′,E2′) show that a few cells scattered within explants strongly express Dlx1 (D2′, black arrowheads) and a few cells around or within explants weakly express Mash1 (E2,E2′, open arrowheads). Scale bars = 50 μm.

Figure 3.

GABAergic phenotype and expression of Dlx1 and Mash1 in cells migrating around dorsal (A1,B1,C,D1,E1) and lateral (A2,B2,D2,E2) cortical explants cultured for 2 (A,C,D,E) or 3 (B) days. The long-distance-migratory neurons observed around dorsal explants of E12.5 (B1) and E13.5 (C,C′) cortex were GABA-immunopositive after 2 or 3 days in vitro (cf. A1 and B1). Strongly GABA-immunoreactive cells with small expansions on their neurites were observed in E12.5 lateral explants (A2, A2′, B2) from the beginning of the culture (not shown). (C′) and (A2′) are high-magnification views of GABAergic cells in (C) and (A2), respectively. A large proportion of cells that migrated around dorsal cortex explants expressed Dlx1 (D1) and Mash1 (E1) weakly (open arrowheads point to one labeled cell in each case). Expression of Dlx1 and Mash1 around lateral explants (D2,E2) and within the thickness of the same explants (D2′,E2′) show that a few cells scattered within explants strongly express Dlx1 (D2′, black arrowheads) and a few cells around or within explants weakly express Mash1 (E2,E2′, open arrowheads). Scale bars = 50 μm.

Figure 4.

Axonal outgrowth in cultured forebrain slices. DiI crystals were placed at dorsal, intervening and lateral sites in the cortex of telencephalic slices before culturing them for 2 days. The characteristic patterns of labeling observed in E12.5 (A) and E13.5 (B) frontal slices according to injection site [dorsal (A1,B1), lateral (A3,B3), intervening (A2,B2)] are schematized on the left. N is the number of slices injected at each level. The range of axon lengths (mm) and the percentage of slices with labeled axons (in brackets) are indicated in each case. Labeled axons are shown in photomicrographs of E13.5 slices: axons labeled from dorsal sites (C1) form long fascicles growing ventrally, whereas ventrally oriented axons labeled from lateral sites (C3) are shorter and unfasciculated. DiI labeled cells (D) are observed both dorsally and ventrally to the injection site. Their labeling appears either punctuate (open arrowhead) or ‘Golgi-like’ (white arrowhead). Scale bars = 100 μm (C1–C3) and 50 μm (D).

Figure 4.

Axonal outgrowth in cultured forebrain slices. DiI crystals were placed at dorsal, intervening and lateral sites in the cortex of telencephalic slices before culturing them for 2 days. The characteristic patterns of labeling observed in E12.5 (A) and E13.5 (B) frontal slices according to injection site [dorsal (A1,B1), lateral (A3,B3), intervening (A2,B2)] are schematized on the left. N is the number of slices injected at each level. The range of axon lengths (mm) and the percentage of slices with labeled axons (in brackets) are indicated in each case. Labeled axons are shown in photomicrographs of E13.5 slices: axons labeled from dorsal sites (C1) form long fascicles growing ventrally, whereas ventrally oriented axons labeled from lateral sites (C3) are shorter and unfasciculated. DiI labeled cells (D) are observed both dorsally and ventrally to the injection site. Their labeling appears either punctuate (open arrowhead) or ‘Golgi-like’ (white arrowhead). Scale bars = 100 μm (C1–C3) and 50 μm (D).

Figure 5.

Axonal outgrowth and cell migration in grafted slices. Small explants of E12.5 lateral (1), dorsal (3) or intervening (2) GFP-expressing cortex were placed at the cortico-striatal frontier (AE) or within the dorsal cortex (FJ) in E13.5 telencephalic slices. After 2 days in culture, GFP-expressing cells were visualized by immunostaining. Lateral explants grafted at the cortico-striatal frontier grew short and unfasciculated ventrally oriented axons (B), whereas dorsal explants grew long and fasciculated ventrally oriented axons (D). Axons leaving explants of the intervening zone (C, intermediate) resemble those leaving lateral explants. GFP-positive cells were found in the host slice at long distances from the graft (E, black arrowheads). In all cases, short axons are oriented dorsally and few cells migrated ventrally. Lateral cortical explants grafted in the dorsal cortex extend either short (F) or longer (G) axons that only formed fascicles if their density was high (I). Explants of dorsal cortex grew long axons on the pial surface of slices (H) that formed fascicles even at low density (J). CX, cortex; LGE, lateral ganglionic eminence. Scale bars = 200 μm (BD), 150 μm (FH) and 40 μm (I,J).

Figure 5.

Axonal outgrowth and cell migration in grafted slices. Small explants of E12.5 lateral (1), dorsal (3) or intervening (2) GFP-expressing cortex were placed at the cortico-striatal frontier (AE) or within the dorsal cortex (FJ) in E13.5 telencephalic slices. After 2 days in culture, GFP-expressing cells were visualized by immunostaining. Lateral explants grafted at the cortico-striatal frontier grew short and unfasciculated ventrally oriented axons (B), whereas dorsal explants grew long and fasciculated ventrally oriented axons (D). Axons leaving explants of the intervening zone (C, intermediate) resemble those leaving lateral explants. GFP-positive cells were found in the host slice at long distances from the graft (E, black arrowheads). In all cases, short axons are oriented dorsally and few cells migrated ventrally. Lateral cortical explants grafted in the dorsal cortex extend either short (F) or longer (G) axons that only formed fascicles if their density was high (I). Explants of dorsal cortex grew long axons on the pial surface of slices (H) that formed fascicles even at low density (J). CX, cortex; LGE, lateral ganglionic eminence. Scale bars = 200 μm (BD), 150 μm (FH) and 40 μm (I,J).

Figure 6.

GABA expression in cells migrating tangentially from cortical explants into host slices. Slices grafted with GFP-expressing lateral (A1–A3) or dorsal (B1–B3,C1–C3) cortical explants were immunostained with GABA antibodies (A2,B2,C2) after 2 days in culture. A few GFP-positive cells migrating tangentially into host slices from lateral cortical explants were immunopositive for GABA (white arrowhead in A2,A3). GABA-immunopositive cells migrating at the pial surface of slices grafted with dorsal cortical explants were rarely observed after 2 days in vitro (B1–B3), whereas they were much more numerous close to grafted explants (white arrowheads in C2,C3). Open arrowheads indicate cells whose double labeling was difficult to evaluate. Scale bar = 40 μm.

Figure 6.

GABA expression in cells migrating tangentially from cortical explants into host slices. Slices grafted with GFP-expressing lateral (A1–A3) or dorsal (B1–B3,C1–C3) cortical explants were immunostained with GABA antibodies (A2,B2,C2) after 2 days in culture. A few GFP-positive cells migrating tangentially into host slices from lateral cortical explants were immunopositive for GABA (white arrowhead in A2,A3). GABA-immunopositive cells migrating at the pial surface of slices grafted with dorsal cortical explants were rarely observed after 2 days in vitro (B1–B3), whereas they were much more numerous close to grafted explants (white arrowheads in C2,C3). Open arrowheads indicate cells whose double labeling was difficult to evaluate. Scale bar = 40 μm.

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