Early regionalisation of the neocortex and the medial ganglionic eminence

Brain Research Bulletin 66 (2005) 402–409

Early regionalisation of the neocortex and the medial ganglionic eminence Arnaud Bellion, Christine M´etin ∗ INSERM U616, Hˆopital Piti´e-Salpˆetri`ere, 47 Bld de l’Hˆopital, 75651 Paris C´edex 13, France Available online 10 August 2005

Abstract The two major functional classes of neurons that build the cerebral cortex are generated in two distinct parts of the telencephalon. Excitatory long distance projecting neurons are produced dorsally in the pallium, whereas local inhibitory interneurons are mainly produced in the medial ridge of the ventral telencephalon. These two parts of the telencephalon are molecularly regionalized from early embryonic stages, but cellular indices of regionalisation are observed only at later stages of development. We have looked for cellular indices of regionalisation in the cortical anlage at early embryonic stages, when the first efferent cortical neurons are generated. Similarly, we have looked for functional regionalisation of the medial ganglionic eminence at the same stages, when the future cortical interneurones are generated. Here, we summarize data showing that two regions in the mouse cortex embryo, the lateral and dorsal cortex, differ strongly in their early neurogenesis. Moreover, the two domains differ in their capacity to produce GABAergic neurons in vitro; this capacity is only observed in the dorsal cortex. The differentiation of the two domains appears to be independent of the laterorostral to mediocaudal gradient of maturation of the cortex. In the basal telencephalon too, the capacity to differentiate GABAergic neurons is not uniformly distributed across the medial ganglionic eminence. The neurogenesis of future cortical interneurons is seen to be highly active in a small area located in the rostral MGE, at mid dorso-ventral level. © 2005 Elsevier Inc. All rights reserved. Keywords: Cortex; Efferent projection; GABA interneuron; MGE; Organotypic slice; Explant; Graft

1. Introduction The two major functional classes of neurons in the cerebral cortex are the glutamatergic excitatory neurons and the GABAergic inhibitory interneurons. Glutamatergic neurons are generated in the ventricular zone of the dorsal telencephalon [19], whereas many GABAergic cortical interneurons originate ventrally in the subpallium, outside the cortical anlage ([31,43] for reviews in rodents; [2] in ferret). Differences in origin of the two main functional classes of cortical neurons illustrate in telencephalon the relationship linking the positional identity and the phenotypic specification of neuronal precursors [11]. Dorsal cortical progenitors and future cortical interneurons differentiate in territories that express distinct and specific sets of transcription factors. GABAer-

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0361-9230/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2005.07.010

gic interneurons differentiate in the ventral telencephalon that expresses Gsh2, Mash1, Dlx and Lhx families, whereas Pax6, Tbr1, Emx and Ngn families are expressed dorsally [28,46,58]. Genes expressed in the dorsal and ventral telencephalic territories cross react, in particular Pax6 with Gsh2 and Mash1 with Ngn1/2. Their repressive interactions determine the positioning of the limit between the dorsal and ventral territories [17,38,50,58]. A palisade of radial glial cells that restricts the migrations between the dorsal and ventral territories differentiates at this pallium–subpallium boundary [13]. Gain of function experiments have shown that the expression of GADs, the GABA synthesis enzymes, is controlled by Dlx genes [51] that are expressed in the ventral telencephalon under the control of Mash1, a bHLH transcription factor [12,17]. Nevertheless, observations in mouse invalidated for ventrally expressed transcription factors [1] and in vitro experiments [21,23] suggest that progenitors in the dorsal telencephalon can also generate GABAergic neurons.

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Within the cortical plate, projection cortical neurons develop specific phenotypes according to their location in the tangential and radial planes [32]. These phenotypes are tightly linked to the positional identity of precursors in the dorsal proliferative neuroepithelium (area location) and to their time of neurogenesis (laminar position). Because cortical projections progressively establish and refine during embryonic and perinatal development, the phenotype of projecting neurons is fully expressed late in development. At early embryonic stages, the cortical plate appears as a homogeneous structure, that follows a rostro-lateral to caudomedial gradient of development ([7], see Fig. 1). At this time, the areal identity of cortical progenitors and precursors is defined by the expression of specific molecular markers, in particular transcription factors ([42,47,49] for reviews). Genetic studies from the groups of O’Leary and of Mallamaci have shown that two developmental genes, Emx2 and Pax6, that are expressed in opposite and complementary gradients in the proliferative zone of the pallium before and during the cortical neurogenesis, play an important role in controlling the arealization process [9,10,29,39]. The purpose of our study was to determine neurons born at the beginning of the corticogenesis in regions that differ

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in their expression level of Emx2 and Pax6, exhibit differences in their phenotypes [8]. Results of these studies have revealed an early regionalisation of the cortical plate and shown that the functional properties of lateral and dorsal cortical cells differ from the beginning of the corticogenesis. Differences can be revealed in vitro using appropriate assays. These studies have moreover shown that progenitors in the dorsal pallium can produce GABAergic neurons both in vitro and in organotypic slices. However, cortical regions close to the pallium–subpallium boundary do not seem to be able to differentiate into GABAergic neurons. This result prompted us to examine whether the capacity to generate long distance tangentially migrating cells is a property distributed across the entire medial ganglionic eminence of the subpallium (MGE) or whether it is restricted to specific areas in this embryonic zone. The results of our in vitro and ex vivo experiments are summarized and discussed below.

2. Methods 2.1. Animals Embryos ubiquitously expressing either the GFP or the ß-galactosidase transgene were produced in the laboratory from crosses between OF1 and Rosa 26 [60] or OF1 and GFPU transgenic mice (Jackson Laboratories, Bar Harbour, ME), respectively. The day of vaginal plug detection was considered as embryonic day 0.5 (E 0.5). 2.2. In situ hybridization Brains were fixed in paraformaldehyde (PAF) 4%. Whole telencephalic vesicles were hybridized overnight at 70 ◦ C with digoxigenin-UTP labeled riboprobes (2 ␮g/ml) for Emx2 (gift of E. Boncinelli), Pax6 (gift of P. Gruss) and Cadherin6 (gift of R.M. M`ege) following the protocol described in [56]. The anti-digoxigenin alkaline-phosphatase coupled antibody was used at a final concentration of 1/2000. Reacted brains were cryoprotected in PBS 30% sucrose and sectioned in the frontal plane with a cryostat. Sections 14 ␮m in thickness were treated again with NBT/BCIP to increase the labeling intensity.

Fig. 1. A unique sequence of development is observed in the mouse cortical plate. Scheme on the left shows the caudal half of an E13.5 mouse telencephalon (redrawn from Bayer and Altman). The sequence of development schematized on the right is observed everywhere in the dorsal pallium (P), with a temporal delay between the lateral and dorsal regions, and between the rostral and caudal regions, according to the classical latero-medial and rostro-caudal gradient of proliferation and maturation in the cortex. The first post-mitotic cells accumulate at the pial surface in the preplate (1, PP). Cortical cells generated thereafter migrate within the PP and form the cortical plate (2, CP) that divides the PP in two layers, the marginal zone (MZ) and the subplate (SP). Soon after leaving the VZ, early post-mitotic cortical neurons extend an efferent axon in the intermediate zone that is oriented toward the ventral subpallium (see scheme on the left). Neurons in the PP, SP or layer VI could contribute to the early efferent projection [15,33,35].

2.3. Immunostaining For GFP, ß-III tubulin (TUJ1), or glutamate immunostainings, explants and slices were fixed in PAF 4%. For GABA immunostaining, explants and slices were fixed for 10 min in 4% PAF, 0.2% glutaraldehyde and then post fixed overnight in 4% PAF. If DAB was the final reaction product, sections or cultures were pretreated for 1 h in PBS with 1% H2 O. Cultures or slices were pre-incubated for 1 h in PBS with 2 g/l gelatin 0.2% Triton-X (PGT). They were then incubated overnight with the primary antibody diluted in PGT (antiGABA rabbit serum Sigma, 1/5000; anti-glutamate rabbit

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serum Sigma, 1/10000; anti-TUJ1 mouse IgG Babco, 1/2000; anti-GFP mouse IgG Quantum, 1/200; anti-GFP rabbit serum Molecular Probes, 1/1000). The secondary antibodies were either biotinylated (Vector laboratories) or Cy3 conjugated or Alexa488 coupled (Jackson Laboratories, Bar Harbour, USA). The biotinylated antibodies were revealed using the Vectastain ABC Elite Kit (Vector Laboratories Inc., USA) and diaminobenzidine (DAB) as a chromagen. Grafted slices were observed using a confocal microscope (Leica, DMRE TCS-SP2). 2.4. Culture of forebrain slices and of explants Forebrain slices and cortical explants were prepared and cultured as explained in [8]. Ganglionic eminence (GE) explants were dissected at the ventricular surface of the lateral, medial or caudal GE using thin tungsten needles. Explants were about 200 ␮m × 200 ␮m × 200 ␮m in size and comprised both the ventricular and subventricular zones. Cortical explants expressing GFP were grafted in the cortex of wild-type forebrain slices. GE explants from ß-galactosidase

expressing embryos were grafted at the pallial–subpallial boundary of wild-type forebrain slices. Grafted slices were cultured for 2–3 days and fixed in PAF 4%. The GFP was revealed by immunohistochemistry and the ß-galactosidase was revealed with the X-gal reaction. DiI injections in forebrain slices were performed as explained in [8].

3. Results 3.1. Outgrowth properties of early efferent axons differ in the dorsal and lateral cortex At early embryonic stage, Pax6 and Emx2 show opposite rostro-caudal and latero-medial gradients of expression in the ventricular zone [39,40,53,57]. At E12.5 in mouse embryos, the lateral part of the cortical pallium express a high level of Pax6 and a low level of Emx2, whereas the opposite is observed in the dorsal part of the cortical pallium (Fig. 2). At the same stage, the expression levels of genes coding for

Fig. 2. Early regionalisation of the ventricular zone and cortical plate. (B–D) Patterns of Pax6, Emx2, and Cadh6 expression in the ventricular zone in E12.5 mouse embryo. On frontal cryostat sections taken at the level of the parietal cortex, the expression of Pax6 is stronger in the lateral cortex, in particular along the ventricle (B, ventrally to the arrow), whereas the expression of Emx2 is stronger in the dorsal cortex, in particular at the upper side of the VZ (C, dorsally to the arrow). The expression of Cadherin 6 (D) is restricted to the lateral cortex. Differences in gene expression levels in the dorsal and lateral cortex are summarized in A. (E–H) Early post-mitotic neurons in the dorsal and lateral cortex differ by their axonal outgrowth properties. DiI injections performed in the dorsal cortex of organotypic slices label longer and more fasciculated axons than injections in the lateral cortex (A, [8]). Differences in axonal outgrowth are still observed when dorsal (F1, G1, H1) and lateral (F2, G2, H2) explants are transplanted at the pallial–subpallial boundary of homochronic organotypic slices (F1, F2: transplants from transgenic embryos express the GFP), or cultured on a laminin substrate (G1, G2: TUJ1 immunostaining of explants), or dissociated (H1, H2: glutamate immunostaining). Long and thick axonal bundles are observed only in cultures of dorsal cortical cells (arrows in H1).

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adhesion factors and guidance molecules show significant differences in these two cortical regions ([18,25,44,48] and Fig. 2D). In mouse, pioneer efferent axons begin to extend between E11.5 and E12.5, immediately after the first postmitotic neurons have left the proliferative neuroepithelium [15,33–35]. Early neurons in the dorsal and lateral cortical pallium were then tested for their axonal outgrowth properties in organotypic slices and in culture experiments. In E12.5 and E13.5 organotypic slices, axons labeled from dorsal cortical regions were longer than axons labeled from the lateral cortex ([8], see scheme Fig. 2E). This difference in length is unexpected since it is opposite to the difference expected from the latero-medial gradient of cortical development. Indeed, according to this gradient of development, lateral axons would extend before dorsal axons and should therefore appear longer than dorsal axons if two types of axons were growing at the same rate. Labeling experiments were then suggestive of differences in the outgrowth rate of dorsal and lateral cortical axons. These differences were confirmed by in vitro experiments. In cultures of homochronic dorsal and lateral explants, axons developed firstly around dorsal explants. And dorsal axons grew more rapidly than lateral axons (Fig. 2F and G). Dorsal and lateral cortical explants transplanted in the same environment (e.g. at the pallium–subpallium boundary or in the dorsal cortex of organotypic slices) still exhibited the same differences in their axonal outgrowth characteristics, showing that these are intrinsic properties of the cortical neurons. Dorsal and lateral neuritis moreover showed differences in their adhesive properties. Axons of dorsal origin were much more fasciculated than axons of lateral origin. Dissociated cortical cells from dorsal origin formed thick and long bundles that were never observed in cultures of lateral cortical neurons (Fig. 2H1 and H2). This capacity to fasciculate on each other could help dorsal cortical neurons to extend longer axons. 3.2. Differentiation of GABAergic neurons in dorsal cortex explants Culture experiments described above moreover revealed that a small population of cells left the explants of dorsal cortex cultured on laminin. These cells migrated among axons, never far from their explant of origin. In contrast, cells were not or rarely observed around explants of lateral cortex. Therefore, the cells observed around dorsal cortical explants are unlikely to originate from the basal telencephalon. Indeed, if these cells were migrating from the basal telencephalon, the lateral explants that are located on the migratory pathway between the basal telencephalon and the dorsal cortex, would release at least the same number of cells than the dorsal explants. Surprisingly, MGE cells that had already reached the lateral cortex at E12.5 did not leave lateral cortical explants cultured on laminin. MGE cells have been shown to respond to attractive cues expressed in the cortex [30].

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These cues could retain MGE cells within isolated cortical explants. Accordingly, lateral explants grafted in forebrain slices released numerous GABA positive cells that migrated to the host cortex. Culture and grafting experiments show together that a cell population able to migrate along efferent cortical axons both in vitro and in organotypic slices differentiates in the dorsal cortex but not in the lateral cortex. This population differs from the long distance tangentially migrating cells originating in the MGE by its response to attractive cortical cues, and by the amplitude of its migration. After 2 or 3 days in vitro, these cells become strongly GABA immunopositive and express low levels of transcription factors involved in controlling the GABA lineage [8]. These results show that the dorsal and lateral parts of the embryonic cortex differ in their capacity to differentiate a specific GABAergic cell population, both in vitro and in organotypic forebrain slices (Fig. 3). 3.3. Regional variability in the production of tangentially migrating cells in the basal telencephalon The lateral and medial ganglionic eminence (LGE and MGE, respectively) produce a very large number of GABAergic cells. Among them, only a fraction migrate to the cortex and differentiate as cortical interneurons. To know whether cortical interneurons generated at E12.5–E13.5 originate in the entire MGE, we tested small adjacent pieces of GE ventricular zone for their capacity to produce long distance tangentially migrating cells (Fig. 4). Small squares dissected at the ventricular surface of the MGE, LGE and caudal ganglionic eminence (CGE) were either grafted at the pallium–subpallium boundary in homochronic organotypic slices, or deposited on dissociated cortical cells (data not shown). In both experimental designs, cells that migrated away from their explant of origin were identified by the expression of the LacZ transgene (Fig. 4, [60]). In agreement with studies published by several groups [2,28,54], we observed that the MGE produced a considerably higher number of tangentially migrating cells than did any other region in the GE, both at E12.5 and E13.5. More interestingly, we observed that long distance tangentially migrating cells are not produced in equal number in the different parts of the MGE. Indeed, a small area in the center of the MGE produced a very high number of long distance migrating cells, much higher than the number produced by any surrounding area. Regions located ventrally to this “hot spot” produced more tangentially migrating cells than regions located dorsally. Therefore, the differentiation of future cortical interneurons is a regionalized property of the MGE at both E12.5 and E13.5. Recent studies have shown that the CGE produces a subpopulation of cortical interneurons [41,55,56]. Accordingly, we observed that CGE explants released a small number of tangentially migrating cells. A very small number of cells

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Fig. 3. Migration of GABAergic cells from dorsal and lateral cortical explants. (A) GABAergic cells observed around dorsal cortical explants either on laminin or in cortical slices after a few days in culture are likely to originate within the dorsal cortex and express both Dlx and Mash1 (see text). They do not show long distance migration. Cells of lateral cortical explants that show long distance migration in organotypic slices do not migrate from isolated lateral explants. They more likely originate in the basal telencephalon. The percentage of GABA positive cells in this population is close to the percentage of GABA positive cells in tangentially migrating MGE cells [45]. (B1 and B2) GABA immunostaining of dorsal (B1) and lateral (B2) E12.5 cortical explants cultured for 3 days. (C1 and C2) GFP positive cells (green) migrate from GFP expressing cortical explants grafted into a host cortical slice. The GABA is revealed in red. A proportion of cells migrating out of cortical explants are GABA immunopositive (white arrows).

were seen to migrate from LGE and pallial–subpallial boundary explants at E13.5. However, we cannot totally exclude that these migrating cells originate more ventrally in MGE and contaminate regions located on their migratory pathway to the cortex.

4. Discussion The parcellation of the cerebral cortex in functionally and morphologically specialized areas is a phenomenon that progressively occurs during embryonic development and that is fully established after birth. Our culture and grafting experiments nevertheless reveal that a regional specification of cortical neurons occurs from the first stages of the corticogen-

esis. Firstly, early projection neurons in the lateral and dorsal embryonic cortex differ in their axonal outgrowth properties. Secondly, the lateral and dorsal cortex differ in their capacity to differentiate tangentially migrating and GABAergic cells. Finally, we show that the differentiation of long distance migrating cortical interneurons is mostly restricted to a small area in the MGE. Signaling proteins that determine dorsal and ventral identities throughout the neuraxis control the dorso-ventral identity of telencephalic progenitors ([42,47] for reviews). In forebrain, BMPs and WNTs are released from dorsal signaling centers and SHH from bilateral ventral signaling centers [11,26]. In the MGE, the differentiation of cortical interneuron depends on SHH signaling [26,27]. The differentiation in the rostral MGE of a small area producing a much higher

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Fig. 4. Production of long distance tangentially migrating cells in the basal telencephalon. (A) Schemes on the left show the ventricular surface of right telencephalic vesicles (olfactory bulb and hippocampus have been removed). Outlined areas were used to prepare explants comprising the VZ and the SVZ. They were dissected in transgenic embryos ubiquitously expressing LacZ (Rosa26 line), and grafted in the pallial–subpallial boundary of homochronic organotypic forebrain slices. Explants and cells that migrated in the host slice were colored in blue by the X-Gal reaction. (B) Illustrates the migration observed in slices grafted with explants dissected in areas 2, 4, 6, 9 of the top left hand scheme in (A). The number of slices grafted with each type of explant is shown in table. Results are summarized by a color code on the top scheme in (A) (darker regions produced the more migrating cells). (C) illustrates the migration observed in slices grafted with explants numbered from 1 to 9 on the bottom left hand scheme in (A). Four slices were grafted with each kind of explant and illustrations show representative examples. The ventro-medial part of the GE (MGE + CGE) produced much more tangentially migrating cells than did dorso-lateral regions (LGE and rostral furrow between the MGE and LGE). Within the ventro-medial region, a limited sector in the rostral MGE produce a particularly high number of tangentially migrating cells.

number of cortical interneurons than the surrounding regions could be related to the dose-dependent effect of SHH [11]. Additional regulatory processes might also be involved in the differentiation of this area. Recent studies have shown that SHH can induce the differentiation of interneurons in the dorsal telencephalon. The dorsally secreted morphogens BMPs seem also involved in the specification of cortical interneurons, but their role is debated [21,59]. Therefore, the lack of interneuron differentiation in the lateral cortex could either result from the selective expression in the dorsal cortex of interneuron inducer(s), comprising SHH [52], or result from the expression in the lateral cortex of factor(s) able to antagonize interneuron inducer(s). Indeed, it has been recently shown that a thin intermediate domain that differentiates between the dorsal Emx1 positive and the ventral Dlx2 positive domains at the pallium–subpallium boundary [16] could play the role of a signaling center to pattern the lateral cortex [5]. This intermediate domain contributes to the amygdala and to the piriform cortex [19]. Therefore, the lateral cortical domain that we identified in E12.5–E13.5 embryos could participate either in the piriform cortex or in the lateral part of the neocortex. The first possibility is unlikely because the lateral cortical domain extends dorsally to the lateral angle of

the ventricle, at both E12.5 and E13.5. The lateral embryonic cortex might then give rise to the lateral or limbic neocortex, two cortical areas that are already committed to express specific molecular markers by E12.5 in the rat [3,4,6,24] and E11.5 in the mouse [14]. At E11.5 and E12.5, when early projecting cortical neurons were born, the lateral and dorsal cortical VZ transiently exhibit differences in Pax6 and Emx2 expression [20,22,40,48,53,57]. This suggests that subtle differences in gene expression might be sufficient to induce the differentiation of functionally distinct neurons. Early cortical neurons generated in the lateral and dorsal parts of the embryonic cortex differ in functional properties that influence the development of the pioneer efferent cortical projection. Differences in adhesion properties might moreover contribute to establish specific interactions between the projections of each cortical sector and cues on their pathway, or between efferent and afferent projections [36]. These precocious neurons might then control the development of later structures. Whatever the later fate of the lateral embryonic cortex (lateral neocortex, limbic neocortex or piriform cortex, [37]), the early regionalisation of the cortical plate that we have shown in vitro might be the first step in the establishment of a major functional subdivision of the mature cortex.

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Acknowledgements This work was supported by grant No. 5934 from Association pour la Recherche contre le Cancer (ARC) to C.M., and fellowships from ARC and Association des Amis de la Science to A.B. We thank Patricia Gaspar for helpful comments on the manuscript.

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