Comprehensive spatiotemporal transcriptomic analyses of the ganglionic eminences demonstrate the uniqueness of its caudal subdivision

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 37 (2008) 845 – 856

Comprehensive spatiotemporal transcriptomic analyses of the ganglionic eminences demonstrate the uniqueness of its caudal subdivision Susan Willi-Monnerat,a Eugenia Migliavacca,b,c,d Didier Surdez,a Mauro Delorenzi,b,c,d Ruth Luthi-Carter,a,1 and Alexey V. Terskikha,e,⁎,1 a

School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland National Center of Competence in Research (NCCR), Epalinges, Switzerland c Swiss Institute for Experimental Cancer Research (ISREC), Epalinges, Switzerland d Swiss Institute of Bioinformatics (SIB), Epalinges, Switzerland e Burnham Institute for Medical Research, La Jolla, California, USA b

Received 15 August 2007; revised 17 December 2007; accepted 7 January 2008 Available online 26 January 2008

The elucidation of mechanisms underlying telencephalic neural development has been limited by the lack of knowledge regarding the molecular and cellular aspects of the ganglionic eminence (GE), an embryonic structure that supplies the brain with diverse sets of GABAergic neurons. Here, we report a comprehensive transcriptomic analysis of this structure including its medial (MGE), lateral (LGE) and caudal (CGE) subdivisions and its temporal dynamics in 12.5 to 16 day-old rat embryos. Surprisingly, comparison across subdivisions showed that CGE gene expression was the most unique providing unbiased genetic evidence for its differentiation from MGE and LGE. The molecular signature of the CGE comprised a large set of genes, including Rwdd3, Cyp26b1, Nr2f2, Egr3, Cpta1, Slit3, and Hod, of which several encode cell signaling and migration molecules such as WNT5A, DOCK9, VSNL1 and PRG1. Temporal analysis of the MGE revealed differential expression of unique sets of cell specification and migration genes, with early expression of Hes1, Lhx2, Ctgf and Mdk, and late enrichment of Olfm3, SerpinE2 and Wdr44. These GE profiles reveal new candidate regulators of spatiotemporally governed GABAergic neuronogenesis. © 2008 Elsevier Inc. All rights reserved. Keywords: Ganglionic eminences; Gene expression profiling; Cerebral cortex; Interneuronogenesis; Cortical interneurons; Rat

⁎ Corresponding author. Burnham Institute for Medical Research, 10901 North Torrey Pines Road, Del E. Webb Center for Neurosciences and Aging, Stem Cells and Regeneration, La Jolla, CA 92037, USA. Fax: +1 858 795 5274. E-mail address: [email protected] (A.V. Terskikh). 1 Equal contributions. Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2008.01.009

Introduction The ganglionic eminence (GE) is a transient embryonic structure composed of three subdivisions, namely the medial (MGE), lateral (LGE), and caudal (CGE) ganglionic eminences. The last is the most recent subdivision to be described as “the region posterior to where the MGE and LGE fuse into a single structure” (Nery et al., 2002). The designation of the CGE as a separate subdivision was primarily motivated by its unique features revealed in fate-mapping and cell migratory studies (Nery et al., 2002). In contrast, molecular distinctions of the CGE that might support its observed functional specialization have not previously been elucidated. An additional level of complexity exists in the distinct temporal birth and rapid turnover of each of the three GE subdivisions (Fig. 1A). As observed in the rat, the MGE is the earliest to emerge at E12, and followed shortly thereafter by the LGE, which arises rostrodorsal to the MGE at E12.5. The separation of these two GEs is visible as a crevice, called the interganglionic sulcus. At approximately E14, the CGE becomes apparent and consequently increases in size, while, in parallel, the MGE begins to regress starting at E15 and essentially disappears by E17 (Jimenez et al., 2002). Upon loss of the MGE, no morphological landmark remains to distinguish between cells of the former CGE and LGE, since the interganglionic sulcus vanishes together with the MGE. Progenitors accommodated in the three GE subdivisions appear to give rise to several distinct differentiated cell populations, most notably including subsets of GABAergic neurons. Indeed, studies of parvalbumin-, somatostatin- and calretinin-expressing cortical interneurons have provided strong support for the notion that distinct subsets of cells would arise from equally unique populations of progenitors housed in spatially restricted GE locations (Xu et al., 2003, 2004; Fogarty et al., 2007).

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Fig. 1. Microarray experiments of the MGE, LGE, CGE and cerebral cortex at E12.5, E14 and E16. (A) Scheme illustrates the development of MGE, LGE and CGE in rat. Red boxes indicate the respective GE of E12.5, E14 and E16 rats used for microarray experiments. (B–G) GEs are located in the embryonic telencephalic hemispheres, and appear as elevations in the lateral ventricle wall. Panels illustrate the stepwise dissection of (B, C) E12.5 MGE and cerebral cortex, (D, E) E14 MGE, LGE, CGE and cerebral cortex, and (F, G) E16 MGE, LGE, CGE and cerebral cortex. (H) Chart visualizes array expression levels of Nkx2.1, Pax6 and Emx2 relative to Gapdh. Error bars indicate standard deviation (n = 3). M: MGE; L: LGE; C: CGE; CTX: cerebral cortex. Scale bars: in panels B–G, 1 mm.

Although the full range of GE cell fates is not yet known, there is strong evidence for the development of several cell types. Progenitor cells in the dorsal periventricular zone of the LGE co-express DLX and ER81 (Stenman et al., 2003) and appear to give rise to GABAergic and dopaminergic neurons comprising the granule and periglomerular cells of the olfactory bulb (Bulfone et al., 1998; Wichterle et al., 2001). In contrast, DLX+/ISL1+ progenitor cells of the ventral LGE are believed to give rise to the GABAergic DARPP32-positive medium spiny striatal projection neurons that compromise approximately 90% of all striatal neurons (Gerfen, 1992). Progenitors of the remaining striatal neurons, including the GABAergic and cholinergic striatal interneurons appear to be localized to the MGE and POa/AEP (Marin et al., 2000). The MGE appears to give rise to a large number of cortical interneurons (Lavdas et al., 1999; Sussel et al., 1999; Wichterle et al., 1999, 2001; Anderson et al., 2001), including parvalbuminand somatostatin-positive subtypes. These fates have been demonstrated using both in vivo transplantation (Wichterle et al., 2001; Valcanis and Tan, 2003; Butt et al., 2005) and Nkx2.1 null-mutant studies (Xu et al., 2004). The MGE additionally gives rise to the globus pallidus, which is also abolished in Nkx2.1 null-mutant mice (Sussel et al., 1999). Other cells known to arise from the MGE are oligodendrocytes (Kessaris et al., 2006), and hippocampal GABAergic interneurons (Pleasure et al., 2000). The fate of the CGE progenitor cells is somewhat less wellcharacterized than those of the other two subdivisions. Nevertheless, in vivo fate-mapping studies suggest that CGE contributes cells to the amygdala and hippocampus (Nery et al., 2002). In vitro cultures (Xu et al., 2004) and in vivo transplantations (Butt et al., 2005) have

also suggested that approximately one-third of calretinin-expressing interneurons originates in the CGE. Although the GE is known to be an important precursor cell source for multiple neural subtypes, the elucidation of the corresponding progenitor cells and study of their distinct developmental pathways has been hindered by the limited understanding of its molecular and cellular heterogeneities. In order to achieve a more comprehensive molecular view of this structure which could facilitate more extensive structural and functional studies, we performed transcriptomic analyses of the rat MGE, LGE and CGE. With particular interest in mechanisms underlying cortical interneuronogenesis, we focused on spatiotemporal aspects reflected by differential gene expression among the GEs as well as common GABAergic functional aspects by opposing the GEs to the primarily glutamatergic cell-providing cerebral cortex. These molecular details provide important groundwork for the study of developmental processes and ultimate fates of GABAergic cells in the forebrain. Results In order to explore the molecular similarities and differences of various GE cell populations, we carried out microarray RNA profiling studies of the MGE, LGE and CGE. Temporal aspects were addressed by sampling at embryonic ages 12.5 (MGE only), 14 and 16 (Figs. 1A–G). The homochronic cerebral cortex was used as reference. In order to identify common regulators of GE-directed GABAergic cell development, we first determined which genes

S. Willi-Monnerat et al. / Mol. Cell. Neurosci. 37 (2008) 845–856

were consistently enriched in all the GE subdivisions relative to the primarily glutamatergic cell-providing cerebral cortex samples of the same embryonic timepoint. Genes meeting criteria of false discovery rate-corrected p b 0.05 and a minimum four-fold GE enrichment are presented in Table 1. Consistent with its important role in generating GABAergic neurons, the GE exhibited enriched expression of the GABA-synthesizing gene Gad1, and the vesicular and plasma membrane GABA transporters Slc32a1 and Slc6a1, respectively (Table 1). Among the distal-less homeobox (Dlx) genes, we detected GE enrichment in Dlx1,

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which together with Dlx2 is essential for late ventral telencephalic neurogenesis as demonstrated by loss of expression studies (Anderson et al., 1997), as well as enrichment of its downstream target Dlx5 (Zerucha et al., 2000). Additional genes implicated in forebrain development, such as the rostrocaudal patterning regulator Six3 (Kobayashi et al., 1998) and the retinol-binding protein Rbp1 (Toresson et al., 1999) also showed GE-enriched expression. The Ascl1/Mash1 gene, required for normal ventral forebrain development as demonstrated by mutant mice showing a severe depletion of ventral forebrain progenitors (Horton et al.,

Table 1 GE-enriched genes as compared to cerebral cortex Probeset ID

1392064_at 1369151_at 1375011_at 1395335_at 1367939_at 1384159_at 1383066_at 1368344_at 1367845_at 1372345_at 1378065_at 1384308_at 1392166_at 1370182_at 1389718_at 1376283_at 1381846_s_at 1387380_at 1368261_at 1398497_at 1387274_at 1376128_at 1368170_at 1395368_at 1379857_at 1370312_at 1375856_at 1389066_at 1376434_at 1377534_at 1380571_at 1377786_at 1378580_at 1382089_at 1369309_a_at 1396852_at 1394577_at 1368044_at

Gene name

Distal-less homeobox 1 (Dlx1) Delta-like 1 homolog (Drosophila) (Dlk1) Transcribed locus Transcribed locus Retinol binding protein 1, cellular (Rbp1) Copine family member IX (Cpne9) /// Non-coding RNA (Evf2) mRNA, complete sequence Unannotated Glutamic acid decarboxylase 1 (Gad1) Neurofilament 3, medium (Nef3) Unannotated Transcribed locus Similar to Homeobox protein Meis1 (Myeloid ecotropic viral integration site 1) Non-coding RNA expressed in the brain, repeat sequence, clone 12 Protein tyrosine phosphatase, receptor-type, N polypeptide 2 (Ptprn2) Transcribed locus Transcribed locus Sine oculis homeobox homolog 3 (Drosophila) (Six3) Solute carrier family 32 (GABA vesicular transporter), member 1 (Slc32a1) Neurexin 3 (Nrxn3) Transcribed locus Distal-less homeobox 5 (Dlx5) Transcribed locus Solute carrier family 6 (neurotransmitter transporter, GABA), member 1 (Slc6a1) Transcribed locus Transcribed locus Spondin 1 (Spon1) Transcribed locus Down syndrome critical region gene 1-like 1 (Dscr1l1) Transcribed locus Serine/threonine kinase 32C (Stk32c) a Purinergic receptor P2X, ligand-gated ion channel 4 (P2rx4) Transcribed locus Transcribed locus Transcribed locus Tachykinin 1 (Tac1) Unannotated Transcribed locus Secretogranin 2 (Scg2)

E12.5

E16

GE

CTX

M-value

p-value

GEs

CTX

M-value

p-value

11.90 11.13 11.11 10.60 10.54 10.41

5.36 6.40 7.88 6.80 8.13 6.06

6.54 4.73 3.23 3.80 2.42 4.35

3.90E-07 1.22E-07 1.29E-07 5.72E-06 5.57E-06 3.32E-07

12.05 8.29 9.74 11.29 9.69 11.27

8.12 5.70 7.71 9.20 7.40 7.46

3.94 2.59 2.03 2.09 2.29 3.81

9.29E-08 9.29E-08 3.12E-04 6.52E-03 3.55E-03 6.73E-07

10.34 10.21 10.00 9.88 9.77 9.73

6.81 3.77 7.20 5.93 5.94 6.86

3.53 6.44 2.79 3.95 3.83 2.87

2.02E-06 3.01E-09 6.64E-06 1.53E-05 2.76E-06 8.26E-07

11.03 11.33 10.43 10.85 10.93 9.70

7.95 8.09 8.32 7.70 8.84 7.16

3.08 3.24 2.11 3.14 2.08 2.55

3.43E-04 7.71E-08 2.01E-05 4.64E-04 6.10E-03 9.29E-08

9.64

6.17

3.47

8.73E-06

7.74

5.59

2.15

5.66E-03

9.61

6.97

2.64

5.67E-06

8.88

6.62

2.27

2.37E-08

9.53 9.51 9.22 8.72

4.98 5.13 6.95 4.13

4.56 4.38 2.27 4.59

1.24E-06 3.32E-07 1.15E-04 6.99E-07

6.81 10.59 9.09 9.59

4.46 7.26 4.39 6.23

2.36 3.32 4.71 3.35

4.79E-02 3.31E-09 1.74E-08 1.86E-07

8.71 8.67 8.59 8.56 8.55

5.59 5.81 4.98 5.31 4.40

3.13 2.85 3.61 3.25 4.15

1.36E-04 8.74E-05 8.27E-06 2.06E-06 3.90E-07

10.01 9.17 9.25 7.78 10.20

7.27 6.45 5.61 4.62 7.89

2.74 2.72 3.63 3.16 2.31

1.95E-05 4.88E-05 3.31E-09 1.01E-03 1.53E-06

8.31 8.31 8.29 8.27 7.96 7.91 7.72 7.70 7.66 7.50 7.24 7.14 6.93 6.80 6.35

5.80 5.16 4.26 5.47 5.89 4.54 5.64 4.44 4.30 4.58 4.00 4.94 4.41 3.87 3.87

2.52 3.15 4.03 2.80 2.08 3.37 2.08 3.26 3.36 2.91 3.24 2.20 2.53 2.93 2.49

5.33E-06 1.24E-06 1.32E-07 3.97E-06 1.36E-04 1.32E-07 1.33E-04 2.37E-05 1.44E-07 9.14E-06 1.24E-06 5.06E-06 7.10E-06 3.90E-07 1.23E-05

8.29 9.01 7.12 8.47 8.46 9.86 8.48 9.30 8.63 7.74 8.28 8.58 8.47 8.43 6.82

6.27 6.16 4.26 5.27 5.60 7.41 5.80 5.73 4.97 4.94 5.66 4.43 5.82 4.51 4.19

2.02 2.85 2.86 3.19 2.86 2.46 2.69 3.56 3.65 2.80 2.62 4.15 2.66 3.92 2.62

3.28E-07 2.83E-06 4.36E-05 1.84E-07 1.89E-05 9.71E-07 3.25E-06 1.71E-05 3.31E-09 1.92E-06 4.85E-04 1.79E-04 1.23E-04 7.64E-08 4.91E-03

Listed are genes selected by M-value ≥2 and an adjusted p-value false discovery rate (FDR) cutoff of 5% for GE versus CTX comparisons at both E12.5 and E16. Signal intensities represented in log2 scale are shown. CTX: cerebral cortex; GE: E12.5 MGE; GEs: average of E16 MGE, LGE and CGE. a Predicted genes.

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1999), was found to be slightly upregulated (∼two-fold) in all three GEs relative to cerebral cortex. Novel discoveries of transcripts enriched in all GEs also proved interesting. These included neurexin 3 (Nrxn3), which belongs to a gene family implicated in synaptic maturation (Graf et al., 2004), delta-like 1 homolog (Dlk1), a gene promoting midbrain dopaminergic differentiation (Christophersen et al., 2007), secretogranin 2 (Scg2), encoding a precursor form of secretoneurin which facilitates dopamine release from nigrostriatal neuron terminals (Saria et al., 1993), protein tyrosine phosphatase receptor-type N polypeptide 2 (Ptprn2) (Cui et al., 1996), putative cell migration regulator spondin 1 (Spon1) (Klar et al., 1992), and the ionotropic purinergic receptor P2rx4 shown previously to inhibit neurite outgrowth (Cheung et al., 2005). Importantly, enriched expression of many unannotated sequences was also observed, suggesting roles for other genes in GE cell development. Global molecular relationships of the MGE, LGE and CGE To investigate the molecular heterogeneity among the three GEs, we conducted comparative analyses of the MGE, LGE and CGE at E14 and E16. 1% of all array probesets displayed regional differences at E14, whereas a far larger 4% of probesets displayed differential regulation at E16. Expected patterns of regionally variant expression included enrichment of Nkx2.1 in the MGE (Sussel et al., 1999), and Emx1/2 and Pax6 in the LGE and CGE (Sussel et al., 1999; Muzio and Mallamaci, 2003) (Fig. 1H). Known neuropeptide and calcium-binding protein markers of mature cortical interneurons (Toledo-Rodriguez et al., 2005) including SST, NPY, VIP, SP, CCK, CR, and CB were highly expressed in at least one of the GEs, except for VIP and PV, which are not expressed in the telencephalon before E19 (Alcantara et al., 1996; Graber and Burgunder, 1996). Cck exhibited prominent expression in the CGE, whereas Npy and Sst were detected at high levels in all three GEs. Cr showed temporally variant expression, with dominant expression in the LGE and CGE at E14, and similar expression levels in all three GEs at E16. In order to confirm the array results, a subset of mRNAs exhibiting differential expression across the three GEs was subjected to immunohistochemical or quantitative real-time PCR (Q-PCR) analyses. Consistent with its RNA expression measures from the arrays, DLK1 protein was enriched in the MGE and CGE at E14 (Figs. 2A–E). Both the radial glial marker FABP7 (Feng et al., 1994) and the known muscle cell precursor gene brother of Cdo (Boc) (Kang et al., 2002) showed a distinctive expression pattern across the LGE and CGE (Figs. 2F–K). We also confirmed LGE-enriched expression of the cell growth regulator Gas1 (Del Sal et al., 1992) and MGE-enriched expression of the SOCS-box containing Asb4 (Kile et al., 2000) (Figs. 2L, M). We next explored global relationships between the GE subdivisions using hierarchical clustering. As expected, biological triplicate samples from each subregion formed unambiguous clusters (Figs. 2N, O). Strikingly, differential expression among the GEs was dominated by a set of genes showing enriched expression in the CGE. In pairwise comparisons among the GE subdivisions, the CGE and MGE exhibited the most differences in their transcriptomes at both E14 and E16 (respectively 43% and 59% of all significantly differentially detected probesets) while the most similar gene expression profiles were those of the LGE and CGE (showing differential signals for only 19% and 20% of all differentially detected probesets at E14 and E16, respectively).

Functional heterogeneity of the GEs is observed in proliferation-, migration- and neuronal signaling-associated processes In order to examine functional correlates of differential GE gene expression, we performed molecular pathway analysis of the genes differentially expressed at E16 using GOstat, which examines overrepresentation of functional categories described in the Gene Ontology (GO) database. Not surprisingly, a major contribution was ascribed to genes involved in developmental processes, denoted by the GO terms “multicellular organismal development”, “nervous system development”, “brain development”, and “anatomical structure development” (Table 2). Unexpectedly, and of particular interest to us, was the observed differential regulation of sets of genes represented by the GO terms i) “cell division”, “(regulation of) cell cycle”, “mitosis”, “interphase (of mitotic cell cycle)”, “cytokinesis”, and “chromosome segregation”, ii) “cell migration”, “axon guidance”, “axonogenesis”, and “neurite morphogenesis/ development”, and iii) “synaptic transmission”, “transmission of nerve impulse”, and “neurological process”, which can more succinctly be described as i) proliferation-, ii) migration-, and iii) neuronal signaling processes (Table 2). When specific differences between subregions were considered, we noted that MGE showed enriched expression of genes associated with proliferation (e.g. Birc5, Aurkb, Cdc20, Ccnb1, Cdc25b), while neuron differentiation markers were generally enriched in the CGE and LGE (Table C). LGE- and CGE-enriched mRNAs showed the molecular underpinnings of neuronal signaling activity, including the enriched expression of neurotransmitters (Cck), neurotransmitter-releasing factors (Snap25, Sv2a, Unc13b), ion transporters and channels (Cacng2, Slc12a5 and Scn2a1), and GABA- (Gabra5, Gabra1) and glutamate- (Grik2, Slc17a6) signaling components. Notably, both CGE and LGE also exhibited high expression of genes involved in cell movement. Migration-associated genes showing enriched expression in both the LGE and CGE included those encoding guidance cue molecules such as Cntn2/Tag1 (Denaxa et al., 2001) and Cxcr4 (Stumm et al., 2003), as well as Cspg2, one whose isoforms were previously demonstrated to mediate neurite formation (Wu et al., 2004). CGE also showed enrichment for mRNAs encoding the cell-adhesion molecule L1cam (Kenwrick et al., 2000) and the neurite outgrowth-promoting Gnao (Strittmatter et al., 1994). Molecular heterogeneity of the GEs may correlate with distinct cell populations Immunohistochemical data have suggested the existence of distinct subpopulations of GE cells which may distribute in patterns different from the defined MGE, LGE and CGE anatomical subdivisions (Nery et al., 2002; Wonders and Anderson, 2005; Flames et al., 2007). In order to identify patterns of mRNA expression which might correlate with other such distributions, we plotted absolute expression measures across all GE subdivisions for genes whose expression showed differential regulation using criteria of false discovery rate-corrected p b 0.05 and a minimum two-fold enrichment in one comparison (see also Experimental methods). This approach yielded unique molecular signatures of hypothetically distinguishable GE cell populations, the most prominent of which appeared to comprise CGE-specific and CGE/LGE coresident cells (Fig. 3). Details of differentially expressed genes showing single and complex GE subdivision enrichment are discussed below.

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Fig. 2. Molecular similarities and differences of E14 and E16 GEs. (A–M) Expression analysis of Dlk1, Fabp7, Boc, Gas1 and Asb4 in E14 and E16 rats. Immunohistochemical studies show expression of (A–D) DLK1 predominantly in the MGE and CGE, while (F–I) FABP7 is mostly expressed in the LGE and CGE. Array log2-fold changes (FC) are shown for (E) Dlk1 (Affymetrix ID: 1369151_at) and (J) Fabp7 (Affymetrix ID: 1370024_at). Histograms exhibit the expression of (K) Boc, (L) Gas1 and (M) Asb4 detected by microarray and quantitative real-time PCR (Q-PCR). Expression levels were normalized to Gapdh. Error bars indicate standard deviation for microarray (n = 3) and Q-PCR (n = 2) experiments. (N, O) All probesets showing differential expression across the (N) E14 and (O) E16 GEs were hierarchically clustered using Cluster and Treeview. Each column corresponds to a single array experiment and each row to a probeset. Each GE sample was performed in triplicate. Colors indicate up- (red) and down- (green) regulation of probesets. The list of genes is supplied as supplemental data (Tables A and B). M: MGE; L: LGE; C: CGE; CTX: cerebral cortex; MGE vs LGE: MGE versus LGE; MGE vs CGE: MGE versus CGE; LGE vs CGE: LGE versus CGE. Scale bar: in panel A: 200 µm, for panels A–D, F–I.

CGE-enriched genes (C NM,L) A substantial number of differentially expressed genes (138) mapped to the CNM,L compartment representing selective enrichment in the CGE, further supporting the idea that the CGE is molecularly distinct from the MGE and LGE. These included Nr2f2/CoupTfII, which was recently demonstrated to influence GE-directed neuronal migration (Tripodi et al., 2004). Genes encoding the fatty acid-metabolizing enzymes CYP26B1 (White et al., 2000) and CPTA1 (Britton et al., 1997) also displayed a CNM,L expression

pattern. Additional CGE-enriched genes included the axon morphology regulator Slit3 (Ma and Tessier-Lavigne, 2007), the transcriptional regulators Hod (Chen et al., 2002) and Egr3 (Patwardhan et al., 1991), and the RWD domain-containing Rwdd3 (Bonaldo et al., 1996), whose function is yet to be determined. CGE/LGE-enriched genes (C,LNM) Interestingly, the largest number of genes (155) distributed to the compartment representing enriched expression in both the CGE and

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Table 2 Functional differences between MGE, LGE and CGE at E16

Differentially expressed genes meeting criteria of |M| ≥ 1 and false discovery rate (FDR) cutoff of 5% were analyzed using GOstat. List of differentially expressed genes attributed to overrepresented GOstat functions is supplied as supplemental data (Table C). GO ID: Gene Ontology identifier. Proliferation-associated process. Neuronal signaling-associated process. Migration-associated process.

LGE. These GE subdivisions are adjacent to the cerebral cortex and accordingly revealed enriched expression of the dorsalization factor Pax6 (Sussel et al., 1999). Other coordinately CGE and LGEenriched genes included several GE cell-associated guidance mole-

cules, such as Cntn2/Tag1 (Denaxa et al., 2001), reelin (Lavdas et al., 1999), Cxcr4 (Stumm et al., 2003) and Ntrk2/TrkB (Polleux et al., 2002), with loss of function of Cxcr4 and Ntrk2 having been shown to result in severe impairments of tangential migration (Polleux et al.,

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2002; Stumm et al., 2003). The CGE/LGE-enriched set of genes also included the known GE cell specification factor Ebf1 (Nery et al., 2002), the known or predicted transcription factors Ebf3 (Wang et al., 1997), Neurod1 (Lee et al., 1995), Ldb2 (Bach et al., 1997), Foxp4 (Teufel et al., 2003), and Tcfap2c (Werling and Schorle, 2002). Other genes displaying enriched expression in both the LGE and CGE included the neurite outgrowth-promoting gene neuritin (Naeve et al., 1997), and the growth arrest and cell differentiation gene Ndrg1 (Kalaydjieva et al., 2000). MGE-enriched genes (M NL,C) Despite the particularly important function of the MGE in cortical interneuronogenesis, very few genes (32) were found to exhibit

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enriched expression in the MGE. Prominent members of this group encode proteases, structural and scaffolding proteins, and growth factor-related proteins, including the previously MGE-associated growth factor receptor Pdgfra (Nery et al., 2001) and homeoboxcontaining Gbx2 (Waters et al., 2003). Figuring among newlyidentified MGE-enriched genes were those encoding ADAMTS5, a protease that remodels extracellular matrix constituents (Glasson et al., 2005; Stanton et al., 2005), the ion channel ACCN4/ASIC4 (Grunder et al., 2001), the retinol-binding RBP4 (Rask et al., 1987), and the MAP3K12-inhibiting protein MBIP (Fukuyama et al., 2000). Note that Titf1/Nkx2.1, which is essential for proper MGE development as shown by its transformation into a LGE-like structure in mutants (Sussel et al., 1999), belonged to the MNL,C cluster at E14 (deducible from Table A), but appeared to expand its

Fig. 3. Spatial expression patterns for heterogeneously expressed genes. Genes differentially expressed across the MGE, LGE and CGE at E16 were binned according to their relative expression patterns. Expression measures are represented by lines for each gene, and the total number of genes contained in each bin is indicated in its respective panel. The table below shows representative RNAs in each bin, with array signal intensities represented in a log2 scale. Predicted genes are marked with an asterisk. The full list of genes for each bin is supplied as supplemental data (Table D). M: MGE; L: LGE; C: CGE.

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Table 3 Functional differences between E12.5 and E16 MGE

Differentially expressed genes meeting criteria of |M| ≥ 1 and false discovery rate (FDR) cutoff of 5% were analyzed using GOstat. List of differentially expressed genes attributed to overrepresented GOstat functions is supplied as supplemental data (Table F). GO ID: Gene Ontology identifier. Cell specification-associated process. Neuronal signaling-associated process. Migration-associated process.

expression domain into the CGE (MNCNL) at E16 consistent with previous expression studies (Nery et al., 2002).

finding suggests that the RNAs, exhibiting most differential expression across subdivisions identify local cells or states that are not temporally variant rather than reflecting cell migration events.

LGE-enriched genes (L NM,C) Temporal dynamics of the MGE The molecular signature of the LGE appeared to be intermediate between those of the other two anatomical subdivisions, but generally showed a higher transcriptomic overlap with the CGE. Consistent with this finding, we observed a low number of genes (21) to be selectively expressed in LGE. Nonetheless, those genes showing selective expression had obvious relevance, including the striatal medium spiny neuron markers Ppp1r1b/Darpp32 (Anderson and Reiner, 1991), the dopamine receptor Drd1a (Gerfen et al., 1990), and tachykinin-1, which encodes the precursor of substance P. Novel LGE-enriched genes included Edg2, which has been reported to be expressed in neurogenic regions of the brain (Hecht et al., 1996), and an unannotated RNA measured by probeset 1383469_at. Stability of differential mRNA distribution Many of the genes showing unique expression patterns at E16 showed a similar distribution at E14 (deducible from Table A). This

To investigate temporal dynamics of the GEs which might be reflected by changes in gene expression, we conducted a longitudinal analysis of the MGE. Examining the most extreme stages of the MGE developmental spectrum, we analyzed differential gene expression at E12.5 and E16 timepoints, which fall within the first and last days of its lifetime. A sizeable 1027 genes were differentially regulated (Table E), with the majority of these (68%) showing enriched expression at E16. Consistent with previous findings, early MGE-enriched genes included Punc, a putative neuronal cell-adhesion molecule (Salbaum, 1998), and Cxcl12, encoding a chemoattractant for Cajal–Retzius cells (Takiguchi-Hayashi et al., 2004; Borrell and Marin, 2006). Early MGE-enriched genes also included several extracellular matrix (ECM) constituents (collagens Col1a1, Col1a2, Col3a1 and glypican Gpc3) and ECM-interacting proteins (Lum, Filip1). These findings are consistent with the early embryonic establishment of a specialized

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extracellular environment favoring differentiation and migration (Fleming et al., 1997). In contrast, late MGE exhibited enriched expression of genes encoding plasma membrane proteins that play a role in neuronal signal transduction (Table 3), consistent with the late development of neuronal activity in the MGE. These included expression of transcripts encoding GABA receptors (Gabra5, Gabra1, Gabbr1, Gabbr2, Gabrg2), GABA transporters (Slc6a1, Slc32a1), and glutamate signaling components (Grik2, Grik5, Gria2, Slc1a1) (Table 3 and Table E). GOstat analyses of the differentially expressed RNAs indicated that differences between the early and late MGE represent a broad variety of development-linked processes, notably including neurogenesis (Table 3). We also observed an enrichment of genes implicated in cell specification- and migration-associated processes. Specification genes included E12.5-enriched Hes1, Lhx2 and E16-enriched Olfm3, Prg1 whereas migration genes included E12.5-enriched Ctgf, Mdk and E16-enriched SerpinE2, Wdr44 (Table F). Two non-exclusive possibilities are that these moleculartemporal heterogeneities may reflect differences in targeting (such that early- and late-born neurons follow distinct migratory pathways terminating in deep and outside layers of the cerebral cortex, respectively, as suggested by Valcanis and Tan (2003) and Butt et al. (2005)) or cell type specification (which generates distinct cortical interneuron and/or other GABAergic neuron subtypes in E12.5- and E16-derived MGE neurons). Discussion Apart from the effects of a few selected genes (Toresson et al., 1999; Faedo et al., 2004; Holm et al., 2007), crucial molecular regulators of ganglionic eminence function have remained largely unknown. We present here an extensive transcriptomic study which identifies numerous additional genes whose functions may be important in GE-derived cell development. We first explored universal molecular correlates of GABAergic neuron development by comparing all GE subdivisions to the homochronic cerebral cortex (which primarily gives rise to glutamatergic cells). Beyond detection of the expected genes encoding GABA-related and DLX proteins, we also identified additional GE-enriched genes including Nrxn3, Scg2, Ptprn2, Dlk1, Spon1 and P2rx4. It is likely that these or other novel molecules encoded by the newly-identified GE-enriched transcripts have crucial influences on GABAergic cell generation. Cortical interneurons comprise a diverse array of GABAergic cells, a substantial number of which derive from the MGE. In our temporal analysis of the MGE we detected discrete sets of genes enriched at early (E12.5) and late (E16) developmental stages. Molecular pathway analysis showed differential regulation of genes such as Ctgf, Wdr44, Olfm3 and Prg1 which are involved in cell specification and migration processes. These results suggest that temporally restricted events may also govern cortical interneuron and/or other GABAergic cell fate and development. Various GE subcompartments showed enrichment of particular sets of mRNAs, consistent with the model fact that spatially segregated precursors supply the brain with different types of cells. The updated view of molecules and compartments our study provides will likely contribute to the further discrimination of GE cell populations. Surprisingly, transcriptome-wide comparison of the three GE subdivisions showed that the least well-characterized CGE was the most unique at molecular level. Although MGE appeared as dis-

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tinct as CGE in regard of all GE transcriptomic profiles, it was particularly the CGE that comprised a large set of upregulated genes, including Hod, Rwdd3, Nr2f2, Egr3, Cpta1, Cyp26b1, and Slit3. This unique transcriptomic signature thus provides unbiased genetic evidence for its differentiation from MGE and LGE. This pool of newly-identified CGE-enriched genes will facilitate prospective cell-tracking approaches to address additional aspects of CGE-derived cell fate. The uniqueness of CGE notwithstanding, the considerable number of genes whose expression is robust in both the CGE and LGE suggests that the CGE shares some molecular and cellular aspects with the LGE. One explanation for this pattern is that the CGE might be composed of more than one cell population, with certain subpopulation(s) confined to the CGE and others extending into the territory of the LGE. In fact, the latter distribution is seen for Boc mRNA and protein (Figs. 2K and S. Willi-Monnerat, unpublished), as well as for CRBP1 (Nery et al., 2002). Recent studies of transcription factor expression also observed an extension of MGE- and LGE cell population domains into the CGE (Flames et al., 2007). Together with other immunohistochemical studies (Figs. 2F–J), we thus strongly support the idea that some cell subpopulations may not fall within strict MGE–LGE–CGE boundaries. Collectively, the full transcriptomic analysis of the MGE, LGE and CGE lays important groundwork for the elucidation of molecular mechanisms underlying GABAergic cell generation. This is particularly important with respect to the genesis of cerebral cortical interneurons, but is also relevant to the development of important populations of striatal, amygdalar, pallidal, hippocampal, and olfactory bulb neurons. Understanding the processes through which these cells arise will contribute significantly our overall understanding of how the brain develops. Furthermore, such knowledge will facilitate the identification of molecular causes of neurological and psychiatric disorders such as epilepsy and schizophrenia that may result from abnormalities in the developing ganglionic eminence. Experimental methods Animals Wild-type Sprague–Dawley (OFA) rats were obtained from Iffa Credo (France). Mating of animals was carried out during 4 h, and plug detection was designated embryonic day 0 (E0). The experiments were carried out in accordance with the European Community Council Directive (86/609/EEC) for the care and use of laboratory animals. Ganglionic eminence and cerebral cortex tissue sampling Timed-pregnant rats were sacrificed by decapitation, and embryos were collected in a Petri dish placed on ice. Dissections of the MGE, LGE, and CGE (including ventricular, subventricular and mantle zones) and cerebral cortex, while both LGE and CGE were separated from cerebral cortex using the morphological boundary visible as change in thickness of the telencephalic wall, were performed in ice-cold Ca2+- and Mg2+-free phosphate-buffered saline (PBS) supplemented with 0.6% D-Glucose, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen). Microarray sample preparation and hybridization Total RNA was isolated from rat MGE, LGE, CGE and cerebral cortex samples at E12.5 (MGE only), E14 and E16 using the RNAqueous kit (Ambion). The quality of ethanol-precipitated RNA was examined with a Bioanalyzer 2100 (Agilent). Double-stranded cDNA was synthesized from

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2 µg (E12.5 and E16) or 5 µg (E14) total RNA using the CodeLink Expression Assay Reagent kit (Amersham). Quality of cRNA was examined on the Bioanalyzer before and after fragmentation. 11 µg fragmented cRNA was hybridized to a GeneChip Rat Expression Array 230A (E14) or 230 2.0 (E12.5 and E16) (Affymetrix). Hybridization, washing and scanning (GeneChip Scanner 3000 High-Resolution) were performed according to the Affymetrix manual. Scanned chips were controlled for standard quality parameters. Grid alignment, background noise, control targets (B2 oligo, bioB, bioC, bioD, cre, Gapdh), and percent presence calls confirmed good quality of raw array data. Additionally, the affyPLM package showed density plots of uniform probe intensity distribution. The raw microarray datasets were deposited in the European Bioinformatics Institute ArrayExpress data repository (http://www.ebi.ac.uk/arrayexpress/) according to MIAME guidelines (Brazma et al., 2003) under the accession number E-MEXP-570. Microarray expression analysis Gene expression values were obtained from the GeneChip array scans with R software packages in BioConductor (http://www.bioconductor.org/). Normalization and quantification were performed with the Robust Multichip Average (RMA) method. Moderated F-statistics (Smyth, 2004) was used to assess differential expression. Genes that met both the adjusted p-value false discovery rate (FDR) cutoff of 5% and |M| ≥ 1 were considered to be differentially expressed. Hierarchical clustering was carried out using Cluster and Treeview (Eisen et al., 1998) by employing the Pearson correlation as a measure of similarity along with an average linkage algorithm. Genes identified as differentially expressed across the MGE, LGE and CGE subdivisions were binned according to specific expression patterns, which were described with the use of the “N” sign (e.g. LNC) representing |M| ≥ 1 and a comma (e.g. L,C) for |M| b 1. GOstat (Beissbarth and Speed, 2004) was used to assess overrepresented biological processes with a significant p-value b0.01, corrected for multiple testing using the method of Benjamini and Hochberg (1995). Quantitative real-time polymerase chain reaction (Q-PCR) Total RNA samples from the MGE, LGE, CGE and cerebral cortex used for microarray analysis was treated with 40 U/ml DNase I (Ambion). Triplicate samples of each ganglionic eminence and cerebral cortex were processed for one-step real-time Q-PCR with a LightCycler instrument (Roche) in a total volume of 12 µl containing 200 ng of total RNA, 3.25 mM Mn(OAc)2, primers at concentrations of 0.3 µM (Boc, Gas1, Asb4) or 0.6 µM (Gapdh), and 1× LightCycler RNA Master SYBR Green I (Roche) containing the Tth DNA polymerase. Reverse transcription was performed at 61 °C for 20 min. Denaturation and amplification conditions were set to 95 °C for 30 s, followed by 45 cycles of i) denaturation at 95 °C for 1 s, ii) primer annealing at 60 °C for 5 s (Asb4), 62 °C for 2 s (Gapdh), 62 °C for 5 s (Gas1) or 64 °C for 2 s (Boc), and iii) elongation at 72 °C for 12 s (Gapdh, Boc), 13 s (Gas1) or 14 s (Asb4). After amplification, a melting curve was obtained with fluorescence data collection at 0.1 °C/s intervals. The following primers were used for Q-PCR: Boc fwd 5′-GTCGAGTTGGCGGAAG-3′ and rev 5′-TGCCACCGATTATTGGTT-3′; Gas1 fwd 5′-ATTCAGGACACCTTGCC-3′ and rev 5′-GTTTCTCAGTGGTCACG-3′; Asb4 fwd 5′-GGTCAATGCCTACGAGC-3′ and rev 5′-GCAAACTGCGTCTACCC-3′; Gapdh fwd 5′-GGCTGCCTTCTCTTGT-3′ and rev 5′TCTCGCTCCTGGAAGAT-3′. Quantitative analysis was performed employing LightCycler analysis software. Immunohistochemistry Timed-pregnant rats were administered 40 mg/kg sodium pentobarbital intraperitoneally and perfused transcardially with ice-cold PBS containing 4% paraformaldehyde (PFA). Embryos were removed, postfixed in 4% PFA/ PBS for 4 h at 4 °C, and cryoprotected in 25% sucrose/PBS for 2 days at 4 °C. Heads were embedded in Tissue-Tek O.C.T (Sakura) and sectioned coronally at a thickness of 14 µm. Immunohistochemistry was performed using a Discovery automated staining instrument manufactured by Ventana Medical

Systems (Tucson, AZ). Standard detection without pre-treatment was accomplished using the iView 3,3′-diaminobenzidine detection kit (Ventana). Primary and secondary antibodies were incubated at 37 °C for 32 min and 16 min, respectively. All slides were counterstained with hematoxylin and bluing agent (Ventana). Primary antibodies included the polyclonal rabbit anti-DLK1 antibody (1:1000; kindly provided by Prof. M. Meyer, Odense, Denmark) and the polyclonal rabbit anti-FABP7 antibody (1:100; kindly provided by Prof. C. Birchmeier, Berlin, Germany). DLK1 immunoreactivity was detected using polyclonal biotin-conjugated goat anti-rabbit IgG antibody (1:100; Vector) diluted in Reaction buffer (Ventana), and FABP7 immunoreactivity was visualized with polyclonal biotin-conjugated donkey anti-rabbit IgG antibody (1:100; Jackson ImmunoResearch) diluted in Antibody diluent (Ventana).

Acknowledgments We would like to thank Prof. C. Birchmeier and Prof. M. Meyer for providing antisera for FABP7 and DLK1, respectively. Special thanks are expressed to the members of the DNA Array Facility Lausanne (DAFL) including Dr. K. Harshman, Dr. O. Hagenbüchle, Dr. J. Weber, J. Wyniger, S. Wicker, M. Bueno and J. Thomas for their excellent advice and technical assistance, and to Dr. B. Sick for the supervision of preliminary microarray analyses. Thanks also to Dr. E. Regulier and Dr. D. Zala for their technical supervision, and to Prof. J.-P. Hornung, Prof. H. Markram, Prof. D. Kirik and Prof. M. Matz for sharing their insights regarding the complexity of GE development. This work was supported by the Swiss National Science Foundation and the EPFL. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mcn.2008.01.009. References Alcantara, S., de Lecea, L., Del Rio, J.A., Ferrer, I., Soriano, E., 1996. Transient colocalization of parvalbumin and calbindin D28k in the postnatal cerebral cortex: evidence for a phenotypic shift in developing nonpyramidal neurons. Eur. J. Neurosci. 8, 1329–1339. Anderson, K.D., Reiner, A., 1991. Immunohistochemical localization of DARPP-32 in striatal projection neurons and striatal interneurons: implications for the localization of D1-like dopamine receptors on different types of striatal neurons. Brain Res. 568, 235–243. Anderson, S.A., Qiu, M., Bulfone, A., Eisenstat, D.D., Meneses, J., Pedersen, R., Rubenstein, J.L., 1997. Mutations of the homeobox genes Dlx-1 and Dlx-2 disrupt the striatal subventricular zone and differentiation of late born striatal neurons. Neuron 19, 27–37. Anderson, S.A., Marin, O., Horn, C., Jennings, K., Rubenstein, J.L., 2001. Distinct cortical migrations from the medial and lateral ganglionic eminences. Development 128, 353–363. Bach, I., Carriere, C., Ostendorff, H.P., Andersen, B., Rosenfeld, M.G., 1997. A family of LIM domain-associated cofactors confer transcriptional synergism between LIM and Otx homeodomain proteins. Genes Dev. 11, 1370–1380. Beissbarth, T., Speed, T.P., 2004. GOstat: find statistically overrepresented Gene Ontologies within a group of genes. Bioinformatics 20, 1464–1465. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. JRSSB 57, 289–300. Bonaldo, M.F., Lennon, G., Soares, M.B., 1996. Normalization and subtraction: two approaches to facilitate gene discovery. Genome Res. 6, 791–806. Borrell, V., Marin, O., 2006. Meninges control tangential migration of hemderived Cajal-Retzius cells via CXCL12/CXCR4 signaling. Nat. Neurosci. 9, 1284–1293.

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