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The mammalian neocortex contains two main neuron subpopulations, the excitatory glutamatergic projection neurons and the inhibitory GABAergic interneurons. In the rodent brain, projection neurons con-stitute about 80% of all cortical neurons, whereas interneurons constitute about 20%. Although interneurons are less abundant, they have crucial roles in the development and organization of cortical networks that underlie a wide range of cortical and mental functions. Moreover, recent studies in mouse models and humans have indicated that interneuron dysfunction is associated with neurological and psychiatric diseases such as epilepsy, schizophrenia, bipolar disorders and autism1–3.

Studies over the past decade have demonstrated that nearly all neo-cortical interneurons in the mouse brain originate from subcortical tel-encephalic structures: ganglionic eminences and the preoptic area4–6. The mouse ganglionic eminences consist of three subdivisions that have distinct molecular and morphological features: the medial, lateral and caudal ganglionic eminences (MGE, LGE and CGE, respectively)7–11. Although the LGE, MGE and CGE of the human fetal brain have simi-lar morphological features as those of mice12, the origin of neocortical interneurons in humans and nonhuman primates is unclear, as some studies have reported that, in contrast to rodents, large numbers of neo-cortical interneurons in human and nonhuman primates are generated from the cortical ventricular zone and subventricular zone (SVZ)13–17.

Here we show that the expression patterns of several transcription factors that show regional and cell type–specific expression, such as

Nkx2-1, Sox6, Sp8, COUP-TFII (also called Nr2f2), Gsx2 (also called Gsh2), Ascl1 (also called Mash1), Islet-1 (also called Isl1), Pax6 and Tbr2 (also called Eomes), in the developing human and macaque mon-key (nonhuman Old World anthropoid) telencephalon share common features with the expression patterns in rodents, which enabled us to identify the MGE, LGE and CGE. On the basis of the continuity of Sox6, COUP-TFII, Sp8 and GABA expression, we present strong evidence that the majority of primate neocortical interneurons are derived from the MGE, LGE and CGE. Furthermore, these conclusions were strength-ened by ganglionic eminence cell migration assays and cell fate analysis of neocortical progenitors using 5-bromodeoxyuridine (BrdU) labeling in embryonic monkey brain slices and cell cultures. Thus, in conjunc-tion with the conserved patterns of transcription factor expression, the subcortical ganglionic eminence origins and tangential migration of immature GABAergic interneurons to the cortex are also conserved from rodents to humans during the evolution of mammalian brain. This finding provides cogent evidence that these processes are controlled by conserved genetic regulators, an observation that has important impli-cations for understanding the genetics of neuropsychiatric disorders.

RESULTSMultiple progenitor domains of human fetal forebrainOn the basis of molecular features of the mouse subpallium, we first analyzed the expression of the homeodomain transcription factor

1Institutes of Brain Science, Fudan University, Shanghai, China. 2State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, China. 3Department of Human Anatomy, Hebei Medical University, Shijiazhuang, Hebei, China. 4Department of Neonatology, Children’s Hospital of Fudan University, Shanghai, China. 5Division of Developmental Biology, Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. 6Institute for Cell Engineering and Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 7Department of Psychiatry, Nina Ireland Laboratory of Developmental Neurobiology, University of California San Francisco, San Francisco, California, USA. 8These authors contributed equally to this work. Correspondence should be addressed to Z.Y. (yangz@fudan.edu.cn).

Received 21 July; accepted 3 September; published online 6 October 2013; doi:10.1038/nn.3536

Subcortical origins of human and monkey neocortical interneuronsTong Ma1,2,8, Congmin Wang1,2,8, Lei Wang1–3,8, Xing Zhou1,2, Miao Tian1,2, Qiangqiang Zhang1,2, Yue Zhang1,2, Jiwen Li1,2, Zhidong Liu1,2, Yuqun Cai1,2, Fang Liu1,2, Yan You1,2, Chao Chen4, Kenneth Campbell5, Hongjun Song6, Lan Ma1,2, John L Rubenstein7 & Zhengang Yang1,2

Cortical GABAergic inhibitory interneurons have crucial roles in the development and function of the cerebral cortex. In rodents, nearly all neocortical interneurons are generated from the subcortical ganglionic eminences. In humans and nonhuman primates, however, the developmental origin of neocortical GABAergic interneurons remains unclear. Here we show that the expression patterns of several key transcription factors in the developing primate telencephalon are very similar to those in rodents, delineating the three main subcortical progenitor domains (the medial, lateral and caudal ganglionic eminences) and the interneurons tangentially migrating from them. On the basis of the continuity of Sox6, COUP-TFII and Sp8 transcription factor expression and evidence from cell migration and cell fate analyses, we propose that the majority of primate neocortical GABAergic interneurons originate from ganglionic eminences of the ventral telencephalon. Our findings reveal that the mammalian neocortex shares basic rules for interneuron development, substantially reshaping our understanding of the origin and classification of primate neocortical interneurons.

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Nkx2-1 in human fetal brain. Nkx2-1 is a key gene that is required for the formation of the MGE and the production of MGE-derived interneuron subtypes18–21, which account for approximately 60% of mouse neocorti-cal interneurons4,5. We observed a high level of Nkx2-1 expression in the MGE of the human fetal brain at gestation week (GW) 15 (Fig. 1a–e,g). Nkx2-1 expression persisted in subsets of cells in the striatum (including the caudate nucleus, putamen and globus pal-lidus) (Fig. 1f); however, we observed few if any Nkx2-1+ cells in the neocortex at GW15 (Fig. 1h). This pattern is consistent with Nkx2-1 expression in mouse ganglionic eminences and cortex7,18,19,22.

Although there was no clear morphological landmark between the LGE and MGE in the GW15 human brain, the pallial-subpallial boundary could be approximated by the ventricular sulcus between the

LGE and CGE and the cortex (Supplementary Fig. 1a). Similar to the embryonic mouse fore-brain, the human cortical ventricular zone and SVZ strongly expressed Pax6, and the LGE and CGE ventricular zone weakly expressed Pax6 (Supplementary Fig. 1a–e). The SVZ of the dorsal LGE (dLGE) and dorsal CGE (dCGE) were characterized by strong expres-sion of Sp8 (Fig. 1 and Supplementary Fig. 1). We also observed weak expression of Sp8 in progenitors of glutamatergic projection

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Sp8 Nkx2-1 Sp8 Nkx2-1Figure 1 Identification of the MGE and the LGE and CGE on the basis of expression of Nkx2-1 and Sp8 in the human fetal brain at GW15. (a–e) GW15 brain coronal sections spanning the rostral-caudal extent of the brain that were double immunostained with Nkx2-1 and Sp8. LV, lateral ventricle; Sep, septum; St, striatum. (f) Higher-magnification image of the boxed area in d showing Nkx2-1+ cells in the striatum. (g–i) Higher-magnification images of the boxed areas in d showing Nkx2-1+ cells in the MGE and LGE but not the neocortex. The MGE was characterized by strong expression of Nkx2-1 in the ventricular zone (VZ) and SVZ, and the dLGE and dCGE was characterized by strong expression of Sp8 in the SVZ. There was also weak expression of Sp8 in the neocortical ventricular zone and SVZ (a–d,i). Scale bars, 1 mm (shown in a, applies to a–e); 50 µm (f and d, inset); 100 µm (shown in g, applies to g–i).

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Figure 2 GABAergic interneurons in GW15 human neocortex appear to derive from the subcortical ganglionic eminences. (a) GABA-immunostained GW15 human brain section. ISVZ, inner SVZ; OSVZ, outer SVZ; IZ, intermediate zone; MZ, marginal zone; SP, subplate; CP, cortical plate; PSB, pallial-subpallial boundary. (b–g) Higher-magnification images of the boxed areas in a showing that neocortical GABA+ cells exhibited morphological features of migrating interneurons; many of them expressed COUP-TFII and/or Sp8. Although robust GABA+ cells were present in the dLGE ventricular zone and SVZ, only a few GABA+ cells were present in the neocortical ventricular zone (b). Scale bars, 200 µm (a); 20 µm (shown in g, applies to b–g).

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neurons in the neocortical ventricular zone and SVZ (Fig. 1 and Supplementary Fig. 1), where Sp8 regulates cortical pattern-ing23–25. Islet-1 was weakly expressed in the ventral LGE (vLGE) SVZ and was strongly expressed in striatal projection neurons (Supplementary Fig. 1f–h).

From the dLGE to the dCGE, COUP-TFII was expressed in increasing rostral-to- caudal gradients, and COUP-TFII+ cells were present mainly in the SVZ (Supplementary Fig. 2a–h). The majority of COUP-TFII+ cells coexpressed Sp8, but not the proliferation marker Ki67, suggesting that they are post-mitotic (Supplementary Fig. 2i). By contrast, in the ventral CGE (vCGE), COUP-TFII was expressed in both the ventricular zone and the SVZ, and the majority of COUP-TFII+ cells expressed Ki67 (Supplementary Fig. 2j)26. We also found that a small population of COUP-TFII+ cells was present in the dorsal and caudal MGE, as they coexpressed Nkx2-1 (Supplementary Fig. 3a–e). Taken together, these transcription factor expression pat-terns clearly defined the LGE, MGE and CGE in the human fetal brain at GW15 (Supplementary Fig. 3f), just as they do in mice.

We then analyzed the expression of these transcription factors in GW18 and GW24 human brains. Several studies have reported that, unlike in rodents, Nkx2-1 is widely expressed in both the human MGE and neocortical (dorsal pallial) ventricular zone and SVZ at midges-tation stages, and Nkx2-1+ cells in the neocortical ventricular zone and SVZ also generate neocortical interneurons15,16. Similar to in the GW15 human fetal brain, we observed that a dense population of proliferating Nkx2-1+ cells was present mainly in the MGE of GW18 and GW24 brains (Supplementary Figs. 4 and 5). We also observed that MGE-derived cells maintained Nkx2-1 expression in the striatum (Supplementary Fig. 5f). Conversely, we found only a small popula-tion of scattered Nkx2-1+ cells in the neocortex. These Nkx2-1+ cells did not express Ki67 (Supplementary Fig. 5i,j), suggesting that they are postmitotic cells but not progenitor cells. Complementing our findings, Nkx2-1 in situ hybridization on fetal human brain sections showed similar observations (Allen Brain Atlas). Thus, our results suggest that when Nkx2-1+ cells tangentially migrate from the MGE into the cortex, expression of Nkx2-1 in the vast majority of these cells is downregulated, as it is in rodents22,27. In the GW24 brain, although we could clearly identify the MGE on the basis of Nkx2-1 expression, the LGE and CGE were not easily distinguished from the cortical ventricular zone and SVZ because there was no obvious sulcus between the LGE and neocortex, and the cortical SVZ and the dLGE and dCGE SVZ were filled with Sp8+ and COUP-TFII+ cells (Supplementary Figs. 4b and 5a).

Origin and migration of human neocortical interneuronsWe next analyzed the migration of human ganglionic eminence–derived cells. At GW15, vast numbers of GABA+ cells appeared to tangentially migrate from ganglionic eminences to the neocortex (Fig. 2). Indeed, we observed that a large stream of GABA+ cells, which exhibited morphological features of migrating interneurons, including a lead-ing process or both leading and tailing processes, left the ganglionic eminence, crossed the pallial-subpallial boundary and entered mainly into the neocortical SVZ (Fig. 2). Within the neocortex, these cells were present in all regions of the neocortex, including the ventricular zone, SVZ, intermediate zone, subplate, cortical plate and marginal zone. Compared to the ventricular zone and SVZ of the ganglionic eminence and other layers in the neocortex (Fig. 2a,c–g), we found fewer GABA+ cells in the neocortical ventricular zone (Fig. 2b), further indicating that they are probably not generated in the neocortex.

Although MGE-derived neocortical interneurons downregulate Nkx2-1 after leaving the MGE22,27, they maintain Sox6 expression. Thus, by identifying Sox6+ cells in ganglionic eminences, striatum and neocortex, we can follow the migration of MGE-derived cells. At GW18, Sox6 was expressed extensively in the MGE (Fig. 3a,d). We also found Sox6+ cells in the striatum and neocortex (Fig. 3a). Because Nkx2-1 is upstream of Sox6 expression in MGE-derived interneurons, and because we did not find proliferating Nkx2-1+ cells in the cortical ventricular zone and SVZ, we propose that Sox6+ interneurons in the neocortex were derived from the MGE. Indeed, a large population of Sox6+ cells appeared to leave the MGE and enter the neocortex in all sections examined; these Sox6+ cells expressed GABA (Fig. 3e–g), suggesting they are interneurons. Consistent with previous reports, we observed that Sox6 was also expressed in the cortical ventricular zone and SVZ and the LGE ventricular zone (Fig. 3a), as Sox6 controls progenitor identity at early developmental

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GW18Figure 3 Sox6+ neocortical interneurons are derived from the human fetal MGE. (a) Sox6 and Pax6 double immunostained GW18 human brain section. (b–d) Higher-magnification images of the boxed areas in a showing Sox6+Pax6+ progenitors of neocortical projection neurons in the neocortical ventricular zone and SVZ (b,c) and Sox6+ Pax6-immunonegative cells in the MGE SVZ (d). (e–g) Sox6+GABA+ interneurons (arrows) did not express Pax6 in the neocortical OSVZ (e,f) or intermediate zone (g). Scale bars, 500 µm (a); 20 µm (shown in g, applies to b–g).

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stages28,29. Extensive analysis of the GW18 brain further revealed that Sox6+ was relatively weakly expressed in cortical ventricular zone and SVZ progenitors, and all of these cells coexpressed Pax6 (Fig. 3b,c) but not GABA (Fig. 3e–g). Thus, progenitors of human cortical glutamatergic projection neurons coexpress Pax6, Sox6 and Sp8, as they do in rodents23–25,28–32.

The human fetal brain contains a robust rostral migratory stream (RMS) with massive immature neuronal progeny migration33–35. We found that in GW15, GW18 and GW24 human forebrains, there were vast numbers of Sp8+ cells in the dLGE and dCGE and the RMS (Fig. 1 and Supplementary Fig. 4). This finding is consistent with previous observations that the dLGE is one of major origins of mouse olfactory bulb interneurons36 and the majority of these interneurons in the RMS express Sp8 (ref. 37). However, very few COUP-TFII+ or COUP-TFII+Sp8+ cells were present in the RMS of GW15 (Supplementary Fig. 2) and GW24 human forebrains (Supplementary Fig. 5a), suggesting that large numbers of COUP-TFII+ and COUP-TFII+Sp8+ cells in the dLGE and dCGE migrate

mainly into the cortex. Because humans have a relatively smaller olfactory bulb compared to the mouse olfactory bulb, we suggest that most of these dLGE- and dCGE-derived cells migrate tangentially into the cortex. However, it is possible that COUP-TFII+Sp8+ cells in the neocortical ventricular zone and SVZ of the GW24 brain also originate from the neocortical ventricular zone and SVZ itself, as previous studies have suggested that this neocortical area is a major source for neocortical interneurons13. If this is the case, given that Gsx2 is upstream of Sp8 in the mouse LGE and CGE37, we predicted that Gsx2, which is required for the specification and maintenance of subcortical progenitors38 and is at the top hierarchy of LGE and CGE identity39, would be also expressed in the cortical ventricular zone and SVZ of the GW24 brain if it generates cortical interneurons. However, we found Gsx2+ cells only in the ventricular zone and SVZ of the LGE, CGE and MGE; we observed no Gsx2+ cells in the corti-cal ventricular zone and SVZ (Supplementary Fig. 6). This result strongly suggests that COUP-TFII+ and Sp8+ cells in the neocortex are derived from the dLGE and CGE. In the neocortex of GW24 brains,

Figure 4 Subcortical origins and migration of human neocortical interneurons. (a,b) Diagrams showing the proposed origin and migration of neocortical interneurons in the human fetal brain at GW15 and GW24. (c–e) COUP-TFII+ and Sp8+ cells in the neocortical ventricular zone and SVZ of GW15, GW24 and full-term (FT) human brains. (f–h) COUP-TFII+, Sp8+ and Sox6+ cells in the neocortex of GW15, GW24 and full-term human brains. In addition to large numbers of Sp8+ and/or COUP-TFII+ cells in the neocortical SVZ, a large number of these cells were also concentrated in the subpial granular layer and marginal zone (f,g). The small tick marks on the right of each image indicate the borders between the zones or areas indicated. iOSVZ, inner OSVZ; oOSVZ, outer OSVZ; L1, layer I; SGL, subpial granular layer; WM, white matter. Scale bar, 50 µm (shown in c, bottom left, applies to c–h).

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we found that the majority of Sp8+ cells expressed COUP-TFII and vice versa (Supplementary Fig. 7). These cells did not express Ki67, Tbr2 or Olig2, which is consistent with a postmitotic status of the interneurons (Supplementary Fig. 7). These findings are in line with the observation that nearly all Sp8+ and/or COUP-TFII+ cells in the neocortex expressed GABA (Fig. 2b–g).

In the neonatal human brain, although many Sp8+ and/or COUP-TFII+ cells already populated the superficial cortical layers (Fig. 4h), large numbers of these cells were still present in the SVZ (Fig. 4e), suggesting that their migration to the cortex continued into the early postnatal stages35. Taken together, our observations suggest that ganglionic eminences are major sources for human neocortical interneurons (Sox6+, CPUP-TFII+ and/or Sp8+) (Fig. 4a), the cortical ventricular zone and SVZ is a major corridor of ganglionic eminence–derived neocortical migrating interneurons (Fig. 4b–e) and many interneurons also migrate through the marginal zone, intermediate zone and subplate (Fig. 4b,f–h).

Classification of adult human neocortical interneuronsDistinct subpopulations of mouse neocortical interneurons have dif-ferent ontogenic origins that are often correlated with their expression

of transcription factors such as Sox6, COUP-TFII and Sp8 (ref. 40). Adult human neocortical interneurons also expressed these tran-scription factors (Fig. 5a–c). We compared the coexpression of these transcription factors with the expression of known markers of human neocortical interneurons, including parvalbumin (PV), calbindin- D-28k (CB, also known as Calb1), calretinin (CR, also known as Calb2), somatostatin (SOM), neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and Reelin (Supplementary Fig. 8). We found that a large majority of PV+, CB+, SOM+, nNOS+ and NPY+ cells expressed Sox6, suggesting they are derived from the MGE (Fig. 5d–g,l,n and Supplementary Fig. 8). In contrast, the majority of CR+, VIP+ and strongly Reelin+ cells expressed COUP-TFII+ and/or Sp8+, suggesting that they are derived from the dLGE and CGE (Fig. 5h–j,m–q and Supplementary Fig. 8). A small population of SOM+ and nNOS+ cells in deep layers expressed COUP-TFII (Supplementary Fig. 8f,h); these cells might be derived from the Nkx2-1+COUP-TFII+ progenitor domain in the MGE (Supplementary Fig. 3a–e)11. Notably, almost all CR+ cells in the neocortex expressed COUP-TFII and/or Sp8 but not Sox6, providing strong evidence that they are derived from the dLGE and CGE but not from the MGE or neocortical ventricular zone and SVZ. Furthermore,

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Figure 5 Classification of human neocortical interneurons on the basis of their ontogenic origins and expression of transcription factors. (a–c) Expression of Sox6, COUP-TFII and Sp8 in the adult human neocortex from the parietal lobe (Brodmann areas 3, 1 and 2). COUP-TFII+ and Sp8+ cells were distributed preferentially in the superficial layers. (d–g) The majority of PV+, CB+, SOM+ and nNOS+ cells expressed Sox6. (h–j) The majority of CR+, VIP+ and strongly Reelin+ cells expressed COUP-TFII and/or Sp8. (k) Pie chart showing the proportion of Sox6+, COUP-TFII+ and Sp8+ cells in the adult human neocortex of the parietal lobe (Brodmann areas 3, 1 and 2). (l–n) The percentage of different subtypes of interneurons that expressed transcription factors. (o–q) The percentage of different transcription factors that expressed interneuron markers in the adult human neocortex (Brodmann areas 3, 1 and 2). ND, no data. Scale bars, 50 µm (shown in a, applies to a–c); 20 µm (shown in d, applies to d–j). Data in the histograms are shown as the mean ± s.e.m. W-Reln, weak expression of Reelin; S-Reln, strong expression of Reelin.

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dLGE- and CGE-derived COUP-TFII+ and/or Sp8+ interneurons preferentially occupied superficial cortical layers (Fig. 5b,c).

We found that the proportions of Sox6+, COUP-TFII+ and Sp8+ interneurons in different parts of the adult human neocortex were different, although MGE-derived Sox6+ interneurons comprised the largest group (Fig. 5k and Supplementary Fig. 9). There was a higher proportion of COUP-TFII+ and/or Sp8+ interneurons in the frontal (Brodmann areas 9) and parietal (Brodmann areas 3, 1 and 2) lobe than in the temporal (Brodmann area 21) and occipital (Brodmann area 17) lobe (Fig. 5k and Supplementary Fig. 9). Accordingly, there was a lower proportion of Sox6+ interneurons in the frontal and parietal lobe than in the temporal and occipital lobe (Fig. 5k and Supplementary

Fig. 9). Consistent with these results the ratio of CR+ cells to PV+ cells was 1:1 in the frontal lobe (Brodmann area 9), whereas in the occipital lobe (Brodmann area 17), this ratio was 1:2.7.

Subcortical origins of monkey neocortical interneuronsThe results described above support the model that subcortical gan-glionic eminences are major sources for human neocortical interneu-rons. We next performed a similar analysis in macaque monkey neocortical interneurons (Fig. 6). In fetal monkey brains at embryonic day (E) 55 and E80, we were able to clearly identify the ventricular zone and SVZ of the cortex, as well as the LGE, MGE and CGE, on the basis of the expression patterns of Nkx2.1, COUP-TFII, Sp8, Gsx2,

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Figure 6 Subcortical origins and classification of monkey neocortical interneurons. (a–d) Images showing the expression patterns of Pax6, Sp8, Gsx2, Nkx2-1, COUP-TFII and Sox6 in E55 monkey coronal brain sections. (e) Many COUP-TFII+ and/or Sp8+ cells appeared to migrate from the dLGE SVZ into the neocortex. (f) In the E55 monkey neocortex, COUP-TFII+ cells were found in the SVZ, subplate and intermediate zone, cortical plate and marginal zone. However, nearly all Sp8+COUP-TFII+ cells migrated into the SVZ; few if any Sp8+ cells were present in the subplate and intermediate zone, cortical plate or marginal zone. By contrast, nearly all COUP-TFII+Sox6+ cells migrated into the subplate and intermediate zone, cortical plate or marginal zone at this time. Mng, meninges. (g,h) COUP-TFII and Sp8 double-immunostained E80 monkey brain sections showing COUP-TFII+Sp8+ cells in the dLGE and neocortex. The RMS contained large numbers of Sp8+ cells and very few COUP-TFII+ cells. (i) In the E80 monkey neocortex, large numbers of COUP-TFII+Sp8+ cells populated the marginal zone. (j,k) COUP-TFII+, Sp8+ and Sox6+ cells in the primary somatosensory cortex (parietal lobe) of neonatal (postnatal day (P) 0) and 17-month-old monkeys. COUP-TFII+ and/or Sp8+ cells were distributed preferentially in the superficial layers. (l) Pie chart showing the proportion of Sox6+, COUP-TFII+ and Sp8+ cells in the 17-month-old monkey primary somatosensory cortex. (m–o) Quantification data from a 17-month-old monkey brain showing that the majority of PV+, CB+, SOM+, nNOS+, NPY+ and weakly Reelin+ cells expressed Sox6, whereas the majority of CR+, VIP+ and strongly Reelin+ cells expressed COUP-TFII and/or Sp8 in the primary somatosensory cortex. Scale bars, 1 mm (a–d,g); 50 µm (e,f,i–k); 100 µm (h). Data in the histograms are shown as the mean ± s.e.m.

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Pax6 and Sox6 (Fig. 6a–i and Supplementary Figs. 10 and 11). As with the human fetal brain, multiple lines of evidence indicated that monkey neocortical Sox6+, COUP-TFII+ and Sp8+ interneurons are derived from the subcortical ganglionic eminences: (i) few if any Nkx2-1+ or Gsx2+ cells were present in the embryonic neocortical ventricular zone and SVZ (Supplementary Figs. 10 and 11); (ii) Sp8+ and Sox6+ cells in the neocortical ventricular zone expressed Pax6, and thus they were progenitors of excitatory projection neurons (Fig. 6a,d and Supplementary Fig. 11s); (iii) Sox6+, COUP-TFII+ and/or Sp8+ neocortical interneurons appeared to originate from ganglionic eminences (Fig. 6a–i and Supplementary Fig. 10); and (iv) the vast majority of Sox6+ and nearly all COUP-TFII+ and/or Sp8+ cells in the cortex expressed interneuron markers.

To directly assess tangential migration of ganglionic eminence cells in the embryonic monkey brain, we microinjected a GFP-expressing adenovirus (adenoGFP) into the MGE, LGE or CGE in a total of five brain slices prepared from a macaque monkey at E55. Twenty-four hours after adenoGFP injection, we found many GFP-labeled cells in the ventricular zone and SVZ of the MGE, LGE and CGE of the cultured slices (Supplementary Movies 1–4). At this stage, a very small number of GFP+ cells were also present in the neocortex (Supplementary Movies 1–4). We therefore started long-term (5 d total) time-lapse imaging to monitor ganglionic eminence cell migra-tion in one cultured slice that contained both the MGE and LGE. We observed 11 ganglionic eminence cells that migrated tangentially from the MGE toward the LGE (Supplementary Movies 1 and 2) or from the LGE toward the neocortex (Supplementary Movies 3 and 4), providing direct evidence that tangential migration takes place in nonhuman primates. Although we observed tangential migration of ganglionic eminence cells toward the cortex, we were not able to observe cells tangentially migrating from the ganglionic eminence into the neocortex. We are uncertain why this was the case, but it could be because of inefficiencies of tangential migration in the slice culture method used with the monkey telencephalon and/or technical limitations (for example, in 300-µm-thick cultured slices, we can only monitor cells in a 100-µm-thick sample). We were also surprised to find that 5 out of 11 migrating cells maintained their ability to concur-rently undergo proliferation (Supplementary Movies 1–4).

The E55 monkey neocortex does not produce interneuronsWe next tested whether the E55 monkey neocortex generates GABAergic interneurons. We cultured slices of neocortical tissue and

MGE tissue for 6 d and then immunostained for GABA (Fig. 7a). In the cultured MGE slices, we observed massive numbers of GABA+ cells, whereas in cultured neocortical slices, we found only a few scat-tered GABA+ cells (Fig. 7a). We also cultured nine neocortical slices with BrdU for 6 d and then double immunostained them for BrdU and GABA. We found approximately 240 GABA+ cells in these slices; however, none of these cells was BrdU+ (Fig. 7b).

We then prepared single-cell suspensions from E55 monkey neocortex, MGE and CGE and cultured these dissociated cells in a 24-well plate containing BrdU for 12 d (Fig. 7c). Triple immuno-staining with BrdU, GABA and Tuj1 revealed that the total numbers of BrdU+GABA+ cells as compared to GABA+ cells per well were 138 compared to 336 (MGE cultures), 59 compared to 185 (CGE cultures) and 1.5 compared to 9 (neocortical cultures). Thus, mon-key cortical cultures generated very few BrdU+GABA+ cells (65-fold fewer BrdU+GABA+ cells as compared to in ganglionic eminence cell cultures after a 12-d culture period; Fig. 7c), which is similar to what we found in mouse cortical cell cultures41. Cortical GABA neuro-genesis may correspond to the generation of olfactory bulb GABA+ interneurons41,42.

In conclusion, we measured very little production of GABA+ cells in the neocortex of E55 monkeys using slice and dissociated culture assays. Combined with our evidence of tangential migration from the ganglionic eminences toward the cortex and the robust expression of transcription factors that are known to mark subpallially derived cortical interneurons in streams of cells migrating to the cortex, we conclude that the subpallium is the source for most monkey cortical interneurons, at least before E55.

Classification of monkey neocortical interneuronsIn the monkey neocortex (from 6 months to 10 years of age), a large majority of PV+, CB+, SOM+, nNOS+, NPY+ and weakly Reelin+ cells expressed Sox6, suggesting that they derive from the MGE (Figs. 6m and 8 and Supplementary Fig. 12). By contrast, the majority of CR+, VIP+ and strongly Reelin+ cells expressed COUP-TFII+ and/or Sp8+, suggesting that they derive from the dLGE and CGE (Figs. 6n,o and 8 and Supplementary Fig. 12). Unlike CR+ interneurons in adult humans, a small number of CR+ interneurons in deep layers of the monkey neocortex were also derived from the MGE, as they expressed Sox6, SOM and nNOS (Fig. 8i,q).

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Figure 7 Lack of GABA+ cell production within the embryonic monkey neocortex. (a) Slices of E55 monkey neocortical or MGE tissue that were cultured for 6 d. Whereas massive numbers of GABA+ cells were present in the MGE, only a few of these cells were present in the neocortex. (b) E55 monkey neocortical slices that were cultured with BrdU for 6 d. The image on the right is a higher-magnification image of the boxed area on the left. No BrdU+GABA+ cells were present in cultured cortical slices. (c) Neocortical, MGE and CGE cells dissociated and cultured with BrdU for 12 d that were immunostained with BrdU, GABA and Tuj1. Quantitative data (right) revealed very few GABA+ and BrdU+GABA+ cells in cultured cortical cells. Scale bars, 50 µm (a–c). Data in the histograms are shown as the mean ± s.e.m.

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Once again, MGE-derived Sox6+ interneurons were the larg-est group of monkey neocortical interneurons (Fig. 6l and Supplementary Fig. 12), and LGE- and CGE-derived interneurons

preferentially occupied the superficial cortical layers (Figs. 6j,k and 8b). In general, there was a higher proportion of COUP-TFII+ and/or Sp8+ interneurons in the frontal (prefrontal cortex) and parietal

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Figure 8 The subtypes of monkey neocortical interneurons that express the transcription factors Sox6, COUP-TFII and Sp8. (a–n) Brain sections of the primary somatosensory cortex from a 17-month-old monkey immunostained with interneuron markers and transcription factors. The majority of PV+ (a), CB+ (c), SOM+ (e), nNOS+ (g) and weakly Reelin+ (j) cells expressed Sox6. Few if any PV+ (b), CB+ (d) and NPY+ (i) cells expressed COUP-TFII or Sp8. A small number of SOM+ (f) and nNOS+ (h) cells in deeper layers expressed COUP-TFII. The majority of CR+ (m), VIP+ (n) and strongly Reelin+ (k) cells expressed COUP-TFII and/or Sp8. A small number of CR+ cells in deeper layers also expressed Sox6 (l). (o–t) Some interneuron markers were coexpressed in a subpopulation of neocortical interneurons. There was a wide range overlap of SOM+, nNOS+ and CB+ cells (o). The majority of NPY+ cells expressed SOM and nNOS (p). A small number of CR+ cells expressed SOM and nNOS (q). The majority of weakly Reelin+ cells expressed nNOS (r). Some strong Reelin+ cells in superficial layers and some weakly Reelin+ cells in deeper layers expressed CR (s). Nearly all VIP+ cells expressed CR (t). The insets show higher-magnification images of the boxed areas above. Scale bars, 100 µm (shown in a, applies to a–t); 50 µm (shown in the inset in a, applies to the insets in a–t).

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(primary somatosensory cortex) lobe, whereas there was a higher proportion of Sox6+ interneurons in the temporal (lateral tempo-ral cortex) and occipital (primary visual cortex) lobe (Fig. 6l and Supplementary Fig. 12). For example, in the frontal lobe (prefrontal cortex), the ratio of CR+ cells to PV+ cells was 1:0.9, whereas in the occipital lobe (primary visual cortex), this ratio was 1:3.1.

DISCUSSIONThe basic organization of cortical microcircuits is conserved across mammals but also shows species differences43–46. Here we provide strong evidence that the majority of primate neocortical interneurons that express Sox6, Sp8 or COUP-TFII are derived from the subcortical ganglionic eminences, as they are in rodents. Although our results cannot eliminate the possibility that a small population of neocortical interneurons is generated from the human neocortical ventricular zone and SVZ, we conclude that it is the subpallium, not the pallium, that is the major source of neocortical GABAergic interneurons in humans and nonhuman primates (Supplementary Fig. 12k). Thus, genetic analyses of rodent cortical interneuron development and function should provide direct insights into the genetic and molecu-lar mechanisms that can underlie human neuropsychiatric disorders involving interneuron dysfunction.

Using DiI to label ganglionic eminence cells in human fetal brain slices, previous studies have shown that a subpopulation of cells migrate into the neocortex from the ganglionic eminence13. In the present study, we also found that some adenoGFP-labeled cells migrated tangentially from ganglionic eminences toward the neocor-tex in E55 monkey brain slice cultures. Thus, tangential migration of interneurons from the subpallium to the pallium in humans, and per-haps all vertebrates, appears to be a shared trait47. In rodents, nearly all cortical interneurons originate from the MGE, the dLGE and CGE and the preoptic area4–6. However, in the human and monkey neocortex, the proportion of subcortical-derived interneurons is not clear. Indeed, it has been reported that Ascl1-expressing progenitors in the human neocortical proliferative zone give rise to two-thirds of neocortical GABAergic interneurons13; however, it is not known into which subpopulation of interneurons they differentiate. By contrast, our study shows that Sox6+, Sp8+ and COUP-TFII+ interneurons that comprise the majority of primate neocortical interneurons are generated from ganglionic eminences. Previous studies have clearly identified that Ascl1 is expressed by progenitors for human neocor-tical glutamatergic projection neurons at GW15 (ref. 31); similar findings have been obtained in mice48,49. Thus, Ascl1 is not a spe-cific marker of GABAergic lineages. Likewise, in our current study we found strong expression of Ascl1 in the ganglionic eminences; the majority of Ascl1+ cells in the cortical ventricular zone and SVZ expressed Pax6 and/or Tbr2 in E80 monkey brains and GW18 and GW24 human fetal brains (Supplementary Figs. 13–15). Moreover, none of these Ascl1+ cells in the neocortical ventricular zone and SVZ expressed GABA, GAD65 or GAD67 (Supplementary Fig. 14f,g). Thus, in rodents and primates, Ascl1 is a marker for progenitors of projection neurons in the pallium only. In the subpallium, Ascl1 is a marker for the progenitors of interneurons.

It is worth noting that cultured neocortical ventricular zone and SVZ progenitors from GW15 human fetal brains did not produce GABA+ interneurons31. In our current study, we cultured E55 mon-key neocortical slices and dissociated cells with BrdU and found that cortical progenitors produced very few BrdU+GABA+ cells. Thus, our cell fate analyses in brain slices and cell cultures further suggest that embryonic primate neocortical progenitors do not generate a large proportion of neocortical GABAergic interneurons.

In the early development of the mouse telencephalon, both Sox6 and Sp8 are expressed by the progenitors of glutamatergic projection neurons in the pallial ventricular zone and SVZ, in which they control pallial progenitor identity and regulate cortical patterning23–25,28. In the subpallium, Sox6 is expressed mainly in the SVZ but not the ven-tricular zone of the MGE, although a small population of Sox6+ cells in the MGE SVZ are mitotic29. In contrast, Sp8 is expressed mainly in the SVZ of the dLGE and CGE but not the MGE; many Sp8+ cells in the dLGE and CGE are capable of proliferation37. Accordingly, Sox6 and Sp8 are expressed by nearly all MGE-derived and a large number of dLGE- and CGE-derived mouse cortical interneurons, respectively, from migrating to mature stages11,29,40. The transcrip-tion factor COUP-TFII is expressed mainly in the mouse CGE9–11, but a small population of COUP-TFII+ cells is also observed in the MGE and LGE11. In the adult mouse neocortex, the majority of CGE- and dLGE-derived and a subset of MGE-derived interneurons continuously express COUP-TFII11,29,40. We also note that Sox6+, COUP-TFII+ and Sp8+ cells account for more than 90% of the neo-cortical interneurons in adult mouse brains40. In our current study, we observed that the expression patterns of Sox6, COUP-TFII and Sp8 in developing and adult human and macaque monkey brains were very similar to those in mice. Furthermore, we observed expression of Gsx2 and Nkx2-1, fundamental features of mouse subpallial pro-genitors, only in the subpallium of developing human and macaque monkey brains. We therefore propose that the majority of neocortical interneurons in monkey and human brains originate from subcortical ganglionic eminences of the ventral telencephalon. It was recently found that the numbers of SOM+, NPY+ and nNOS+ neocortical interneurons are substantially reduced in fetal and infant cases of human holoprosencephaly with severe ventral forebrain hypoplasia50, further indicating their subcortical developmental origins.

In the adult monkey and human neocortex, MGE-derived interneu-rons (Sox6+) account for about 60% of all cortical interneurons, although the proportions in different cortical areas are different. Sp8 is expressed extensively from the rostral to caudal extent of the telencephalon, including primarily the olfactory bulb, RMS, dLGE and CGE. However, we observed few Sp8+COUP-TFII+ cells in the monkey and human RMS and olfactory bulb. This suggests that Sp8+COUP-TFII+ cells in the primate dLGE and CGE migrate mainly into the cortex. On the basis of the expression pattern of COUP-TFII in the LGE and CGE (COUP-TFII is expressed in increasing rostral-to-caudal gradients in the subpallium) and the presence of COUP-TFII+ interneurons in the adult neocortex, we estimated that the number of CGE-derived interneurons appeared to be more promi-nent than those derived from the LGE. Thus, as in rodents, although it is difficult to identify the boundary between the LGE and CGE, human and monkey LGE-derived cortical interneurons may contrib-ute less substantially to the populations of cortical interneurons than MGE- and CGE-derived populations.

Our classification of cortical interneurons in primates in the present study was based largely on the continuity of Sox6, COUP-TFII and Sp8 transcription factor expression. Our analysis revealed that, similarly to mouse mature neocortical interneurons, PV+ and SOM+ cortical interneurons in primates were derived from the MGE, whereas CR+ cortical interneurons were derived mainly from the CGE (nearly all CR+ interneurons expressed COUP-TFII and/or Sp8) (Supplementary Fig. 12k). Moreover, the laminar distribution of inhibitory interneurons in the mouse, monkey and human neo-cortex also share common features. For example, LGE- and CGE-derived COUP-TFII+ and/or Sp8+ interneurons preferentially occupy superficial cortical layers. This further suggests conserved molecular

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mechanisms that regulate the development of cortical interneurons in the mammalian brain.

METHODSMethods and any associated references are available in the online version of the paper.

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

AcknowledgMentSThis work was supported by the National Basic Research Program of China (2011CB504400 and 2010CB945500) and the National Natural Science Foundation of China (30990261, 31028009, 31121061 and 91232723). We thank the staff at the Chinese Brain Bank Center, Wuhan, China and the Red Cross Society of China, Shanghai Branch at Fudan University for providing access to donated adult human brains.

AUtHoR contRIBUtIonST.M. and Z.Y. designed the study, acquired and interpreted experimental data and prepared the manuscript. C.W. and L.W. acquired and interpreted experimental data and prepared the manuscript. X.Z., M.T., Q.Z., Y.Z., J.L., Z.L., Y.C., F.L., Y.Y. and C.C. assisted with experiments and data collection. J.L. and Y.C. carried out slice culture and time-lapse imaging experiments. K.C., H.S., L.M. and J.L.R. designed some experiments and assisted with manuscript preparation. T.M., C.W., L.W., Z.Y. and C.C. collected specimens. C.C. assisted with neuropathological review. Z.Y. wrote the paper.

coMPetIng FInAncIAl InteReStSThe authors declare no competing financial interests.

Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

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ONLINE METHODSAnimal tissue preparation. All animal care and experiments were conducted in accordance with the Fudan University Shanghai Medical College guidelines. E13.5 mouse brains were dissected and fixed overnight in 4% paraformaldehyde (PFA) at 4 °C. Brains were cryoprotected for at least 24 h in 30% sucrose in 0.1 M phosphate buffer and then frozen in embedding medium (optimal cutting temperature compound (OCT); Sakura Finetek) on a dry ice and ethanol slush.

Embryonic, neonatal, juvenile and adult macaque monkeys, both rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) of both sexes at vari-ous ages (Supplementary table 1), were obtained from the Kunming Primate Research Center of Chinese Academy of Sciences at Kunming, China and Suzhou Xishan Zhongke Laboratory Animal Co., Ltd., Suzhou, China. For immunohis-tochemical staining, monkeys were deeply anesthetized and then perfused with PBS followed by 4% PFA. The brains were removed and postfixed with 4% PFA for 12–48 h. Postnatal brains were then cut coronally into approximately 1.0- to 2.0-cm slabs and cryoprotected in 30% sucrose in 0.1 M phosphate buffer at 4 °C for 72 h. The brain tissue samples were frozen in embedding medium (OCT; Sakura Finetek) on a dry ice and ethanol slush.

Human tissue collection. All human tissues (Supplementary table 2) were collected with informed consent and in accordance with the Fudan University Shanghai Medical College guidelines, and the study design was approved by the institutional review board (ethics committee) of the Fudan University Shanghai Medical College (20110307-085 and 20120302-099).

Human fetal brains were obtained at autopsy within 3 h of spontaneous abor-tion. Brains were examined only with the informed consent of the patients. Brain tissue was fixed in 4% PFA at 4 °C for 1 week and then cryoprotected in 30% sucrose in 0.1 M phosphate buffer for 72 h at 4 °C. The brain tissue samples were frozen in OCT on a dry ice and ethanol slush.

Neonatal and adult human brains were obtained at autopsy from subjects of different ages (Supplementary table 2) with no history of neuropathology. The average postmortem delay was less than 10 h. Some specimens were obtained as early as 3 h postmortem33. Brains were either removed at autopsy and suspended in 4% PFA for 24 h or fixed by bilateral perfusion with 4% PFA for 1–2 h through the internal carotid arteries. The brains were then cut into 1.0- to 2.0-cm-thick slabs. The slabs were postfixed in 4% PFA for 1 week and then cryoprotected in 30% sucrose for 72 h at 4 °C. The brain tissue samples were frozen in OCT on a dry ice and ethanol slush.

Immunohistochemistry. Immunohistochemical staining was performed on 30- to 60-µm (embryonic and neonatal brains) or 30- to 40-µm (adult brains) free-floating serial coronal sections in 12- or 24-well cell culture plates. Some sections were subjected to an antigen retrieval protocol. Briefly, cryosections were mounted on glass slides and boiled in a microwave oven in 10 mM sodium citrate, pH 6.0. Sections were blocked for 1–3 h in Tris-buffered saline with 1% Triton X-100 and 10% normal donkey serum. The primary antibodies used in this study are listed in Supplementary table 3. All primary antibodies were incubated for 48 h at 4 °C.

Secondary antibodies against the appropriate species were incubated for 4 h at room temperature (all from Jackson, 1:200). Fluorescently stained sections were then washed, counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma, 200 ng ml−1) for 2–5 min and coverslipped with Gel/Mount (Biomeda, Foster City, CA). When needed, Autofluorescence Eliminator Reagent (Millipore, 2160) was used to reduce lipofuscin autofluorescence in aged tissue. Streptavidin and diaminobenzidine (DAB) were used to visualize the reaction product for bright-field staining sections. Omission of primary antibodies eliminated the staining.

For COUP-TFII (mouse anti–COUP-TFII), Reelin (mouse anti-Reelin) and Sp8 (goat anti-Sp8) triple immunostaining, we first double immunostained for Sp8 and COUP-TFII in brain sections, as they are expressed in the nucleus. After incubation of second antibodies against mouse and goat, Reelin immunostaining was then performed using the standard protocol as described above. For Sox6 and NPY (rabbit anti-Sox6 and rabbit anti-NPY) double immunostaining, we first immunostained for Sox6 in brain sections. After incubation of second antibodies against rabbit, NPY immunostaining was then performed.

Microscopy. Fluorescently immunostained sections were analyzed on an Olympus FV1000 confocal laser scanning microscope. Confocal Z sectioning

was performed using a 20× (numerical aperture (NA) 0.75), 40× (NA 1.00) or 60× (NA 1.42) objective for single, double and triple immunostaining. The Kalman filter mode was used during scanning. Images were acquired, and a Z-stack was reconstructed using the Olympus FV10-ASW software and was cropped, adjusted and optimized in Photoshop CS2. Images of enzyme histochemistry-labeled sec-tions and some fluorescently immunolabeled sections were acquired using an Olympus BX 51 microscope.

cell counting. Brains from a 6-month-old monkey, a 17-month-old monkey and a 39-year-old human were selected for quantification of neocortical interneu-rons (Supplementary tables 1 and 2). The neocortex from the frontal, parietal, temporal and occipital lobe were cut into 30-µm sections. After fluorescently immunostaining, sections were analyzed on an Olympus FV1000 confocal laser scanning microscope. Images covering all layers of the neocortex were first taken by confocal Z sectioning using a 20× (NA 0.75) objective and then photomerged using Photoshop CS2. We then counted neocortical interneurons that expressed transcription factors in all layers of the neocortex. Cortical layers were delineated by NeuN and DAPI staining. The data are presented as the mean ± s.e.m. (n = 3 sections from each part of the brain).

For quantification of COUP-TFII+, Sp8+, CR+, Ascl1+ and Ascl1+Pax6+Tbr2+ cells in E55 and E80 monkey brains and human fetal brains at GW15, GW18 or GW24, three coronal sections through the intermediate brain were selected. Cells were counted in the confocal images. The data are presented as the mean ± s.e.m. (n = 3 sections from each brain).

embryonic monkey brain slice cultures and time-lapse imaging. An E55 mon-key brain was dissected out into ice-cold Hanks’ balanced salt solution (HBSS; Invitrogen, 14175-095). The brain was embedded in 4% low–melting temperature agarose in artificial cerebrospinal fluid containing (in mM) 125 NaCl, 5 KCl, 1.25 NaH2PO4, 1 MgSO4, 2 CaCl2, 25 NaHCO3 and 20 glucose, pH 7.4, 310 mOsm l−1, and sectioned at 300 µm using a vibratome (Leica Microsystems). Brain slices then were transferred onto a slice culture insert (Millicell) in a glass-bottom Petri dish (MatTek) with culture medium containing (by volume) 66% Basal Medium Eagle (BME), 25% HBSS, 5% fetal bovine serum, 1% N-2, 1% penicillin, streptomycin and glutamine (all Invitrogen) and 0.66% d-(1)-glucose (Sigma). Cultures were maintained in a humidified incubator at 37 °C with a constant 5% CO2 supply. For ganglionic eminence cell migration analysis experi-ments (Supplementary Movies 1–4), 50 nl adenoGFP (1.5 × 1011 colony forming units ml−1; GeneChem, CO Ltd, Shanghai, China) was manually microinjected into the MGE or CGE of the cultured brain slice using a calibrated glass micro-pipette (Drummond Scientific). The time-lapse images were captured by the PerkinElmer UltraView live cell imaging system. The recording was started at around 24 h after virus injection. Fluorescence images were captured at 10× magnification, and all images were stitched automatically. The recording time lasted approximately 120 h (5 d). The recording interval was 25 min.

E55 monkey neocortical slices were also cultured with the culture medium described above plus BrdU (Sigma; 15 µg ml−1) for 6 d. The slices were then fixed and immunostained with GABA and/or BrdU. For GABA immunostaining, some cultured slices were resectioned (into sections 40 µm thick). For BrdU detection, slices were pretreated in 2 N HCl for 1 h at room temperature to denature DNA. The total numbers of GABA+ and BrdU+GABA+ cells in the neocortex were counted manually under an Olympus BX 51 microscope.

dissociated cortical and ganglionic eminence cell cultures and immuno-cytochemistry. Neocortical, MGE and CGE tissue fragments from an E55 monkey brain were pooled into tissue tubes and dissociated using medium that contained Dulbecco’s modified Eagle’s medium (DMEM) and F12 (1:1) (Invitrogen, 10565-018), DNase I (Invitrogen, 18047-019) and 10 mg ml−1 Dispase II (Roche, 04942078001). The tissue tubes were placed on a rotator for 30 min at a low speed (12 r.p.m.) at 37 °C. After dissociation, the tissues were rinsed in the resuspending medium containing DMEM and F12 and 1 mM N-acetylcysteine (NAC; Invitrogen, A9165-5G), pelleted by centrifugation at 200g for 5 min, resuspended and triturated 10–20 times using a 1-ml pipette. Cells were centrifuged at 300g for 5 min and ultimately resuspended in 1 ml culture medium containing DMEM and F12, B27 (Invitrogen, 17504-044), N2 (Invitrogen, 17502-048), 1 mM NAC, 10 ng ml−1 fibroblast growth factor 2 (FGF2) (Invitrogen, PMG0034) and 1% penicillin-streptomycin (Invitrogen,

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15070-063). The cell density was calculated using the counting chamber. Cells (1.0 × 105 cells per well) were plated on 24-well plates (Costar, 3524) coated with poly-l-lysine (Sigma) plus BrdU (Sigma, 15 µg ml−1) and cultured at 37 °C, 5% CO2 with 100% humidity for 12 d. Half of the culture medium (plus BrdU) was changed every 3 d. Cells were fixed in 4% PFA for 20 min, followed by triple immunostaining with BrdU, GABA and Tuj1. For BrdU detection, cells were

pretreated in 2 N HCl for 20 min at room temperature to denature DNA. The total numbers of GABA+ and BrdU+GABA+ cells were counted manually per well, and at least two wells per group were analyzed.

Additional information. An additional 43 supporting figures are available at http://iobs.fudan.edu.cn/En/team_view.asp?ID=13.

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