Contrasting and brain region-specific roles of neurogenin2 and mash1 in GABAergic neuron differentiation in vitro

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Contrasting and brain region-specific roles of neurogenin2 and mash1 in GABAergic neuron differentiation in vitro A-Young Joa,c,d , Chang-Hwan Parkb,c,d , Shinichi Aizawae , Sang-Hun Leea,c,d,⁎ a

Department of Biochemistry and Molecular Biology, Hanyang University, Seoul 133-791, South Korea Department of Microbiology, College of Medicine, Hanyang University, Seoul 133-791, South Korea c Department of Institute of Mental Health, Hanyang University, Seoul 133-791, South Korea d Department of Cell Therapy Research Center, Hanyang University, Seoul 133-791, South Korea e Vertebrate Body Plan Group, RIKEN Center for Developmental Biology (CDB), 2-2-3 Minatojima-minami-cho, Chuou-ku Kobe, Hyogo 650-0047, Japan b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

We have cultivated highly uniform populations of neural precursor cells, which retain

Received 8 May 2007

their region-specific identities, from various rat embryonic brain regions. The roles

Revised version received

of the proneural basic–helix–loop–helix (bHLH) factors neurogenin2 (Ngn2) and Mash1 in

10 August 2007

γ-aminobutyric acid (GABA) neuron differentiation were explored in the region-specific

Accepted 26 August 2007

cultures. Consistent with previous in vivo studies, forced expression of Mash1 promoted

Available online 8 September 2007

GABA neuron formation from the precursors derived from the developing forebrains, whereas Ngn2 displayed an inhibitory role in forebrain GABA neuron differentiation.

Keywords:

Functional analyses of mutant bHLH proteins indicated that the helix–loop–helix domains

γ-aminobutyric acid neuron

of Mash1 and Ngn2, known as the structures for protein–protein interactions, impart the

Basic helix-loop-helix

distinct activities. Intriguingly, the regulatory activities of Mash1 and Ngn2 in GABA

Neurogenin2

neuron differentiation from the hindbrain- and spinal cord-derived precursor cells were

Mash1

completely opposite of those observed in the forebrain-derived cultures: increased GABA neuron yield by Ngn2 and decreased yield by Mash1 were shown in the precursors of those posterior brain regions. No clear difference that depended on dorsal–ventral brain regions was observed in the bHLH-mediated activities. Finally, we demonstrated that Otx2, the expression of which is developmentally confined to the regions anterior to the isthmus, is a factor responsible for the anterior–posterior region-dependent opposite effects of the bHLH proteins. © 2007 Elsevier Inc. All rights reserved.

Introduction A central issue in developmental neurobiology is how brain region-specific neuronal subtypes arise at precise positions in the developing brain along the anterior–posterior and dorsal–

ventral axes. Despite extensive study, these developmental processes have been only partly understood. The major problem comes from the fact that the developmental processes can not be achieved simply by the individual actions of single factors, but must be accomplished in the molecular

⁎ Corresponding author. Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, #17 Haengdangdong, Sungdong-gu, Seoul, 133-791, Korea. Fax: +82 2 2294 6270. E-mail address: [email protected] (S.-H. Lee) 0014-4827/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.08.026

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context of the developing brain [1]. Specifically, the repertories of region-specific gene expression, established in uncommitted neural precursor cells during early brain development, critically influence the generation of the region-specific types of cells [2,3]. In vivo functional analyses have the benefit that one can appropriately evaluate the normal physiology of the cell fate determination phenomenon resulting from the summation of the interactions between cells, between cells and the surrounding environments, and the existing extrinsic molecules. However, such complicated interactions in in vivo systems often hinder an adequate identification of the contribution by each individual single component, which may be essential for a clear understanding of the developmental processes. In this regard, there is intense interest in studying central nervous system (CNS) stem/precursor cells in culture models, in which each molecular component can be managed, to understand the fundamental cellular, biochemical, and physiological bases of the in situ regulation of cell fate determination and differentiation. Obviously, in vivo cellular characteristics cannot be predicted a priori from culture studies, yet cultures provide a powerful means to test or make hypotheses on the in vivo properties and behavior of cells. In addition, information obtained in the culture system could be applied directly to guide differentiation of neural precursor cells towards specific neuronal subtypes for experimental and transplantation purposes. Neurons secreting γ-aminobutyric acid (GABA) neurotransmitter (GABAergic neurons; GABA neurons), which reside in almost all of the brain regions, are the most common inhibitory neurons in the CNS. The molecular machinery governing GABA subtype specification largely remains to be determined, but recent studies have defined several transcription factors that play essential roles in GABA neuron productions of various brain regions, such as Dlx1, 2 in the forebrain [4,5], Heslike/Megane in the midbrain [6,7], Ptf1a in the cerebellum [8], and Lbx1/Tlx3 in the spinal cord [9]. Proneural basic helix–loop–helix (bHLH) transcription factors are well-acknowledged pan-neurogenic molecules that specify neural precursor cell fates towards neuronal lineages and neuronal differentiation [10,11]. Two families of proneural bHLHs, such as the achaete–scute homolog Mash1 and the atonal-related Neurogenin (Ngn) genes, have been identified in the developing mammalian nervous system [12,13]. A conventional explanation for bHLH's role was that it simply activates generic neuronal differentiation of precursor cells, neurotransmitter subtypes of which had been committed by the actions of regionally-specified proteins. Recent studies, however, have reported that the bHLH proteins are more actively and specifically involved in the processes of neurotransmitter subtype determination [10]. Specifically, in vivo knock-out mouse analyses revealed a critical role for the bHLH factors in forebrain GABA neuron formation [14,15]. In this study, we performed cultures for highly homogenous populations of neural precursor cells from dorsal and ventral parts of rat embryonic brain regions throughout the forebrain to the spinal cord. The precursor cells derived from the different regions of the developing brain shared common properties in their in vitro proliferation and generic differen-

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tiation towards neurons and glia, but retained unique regionspecific neuronal subtype differentiation and gene expression patterns inherited from the regions of the developing brain. The cultures of the region-specific precursors were utilized to obtain further insights into the roles of the proneural bHLH proteins neurogenin 2 (Ngn2) and Mash1 in GABA neuron differentiation. Consistent with previous in vivo findings [14,15], over-expression of Mash1 bHLH factor enhanced GABAergic neuronal yield from precursor cells derived from the developing forebrains [cortex (Ctx) and lateral ganglionic eminence (LGE)], whereas Ngn2 repressed forebrain precursorderived GABA neuron differentiation. Functional analyses of Ngn2 and Mash1 mutants identified the structures of Ngn2 and Mash1 responsible for the activities of GABA subtype differentiation. The GABA differentiation control activities of the bHLH factors, in contrast, were completely opposite to those of the forebrain cultures in the precursors derived from the regions posterior to the isthmus (midbrain–hindbrain junction) such as the hindbrain and spinal cord. We further demonstrate data suggesting that Otx2, expression of which is restricted in the embryonic brain anterior to the isthmus [16– 18], is, at least in part, responsible for the contrasting roles of the bHLH in the anterior and posterior embryonic brain.

Materials and methods Cultures for neural precursor cells derived from rat embryonic brains Timed-pregnant Sprague Dawley (SD) rats were purchased from Koatech (Seoul; Korea). Embryonic brain tissues were dissected from dorsal and ventral parts of the forebrain, midbrain, hindbrain and spinal cord at embryonic day 13 (E13; day of conception was called day 0). Dissected tissues were mechanically triturated in Ca2+/Mg2+ free Hank's balanced salt solution (HBSS; Invitrogen, Grand Island, NY) and seeded at 19,000 cells/cm2 on 10-cm culture dishes (Corning, Corning, NY) pre-coated with polyornithine/fibronectin [poly-L-ornithine (15 μg/mL; Sigma-Aldrich, St Louis, MO) in a 37 °C incubator overnight followed by incubation with fibronectin (1 μg/mL, Sigma-Aldrich) for at least 2 h]. Neural precursor cells were allowed to proliferate in serum-free N2 media with basic fibroblast growth factor (bFGF; 20 ng/mL; R&D system, Minneapolis, MN) for 4–5 days [19]. Clusters of proliferated cells were then dissociated by trypsin–EDTA (Invitrogen) and plated on freshly coated 12-mm glass coverslips (45,000 cells/ cm2; Carolina Biological Supply Company, Burlington, NC) or 6-cm culture dishes, and grown in N2 + bFGF for an additional 1–5 days. All the experiments were performed in the passaged (P1) cultures. Differentiation of the precursor cells was induced by withdrawal of bFGF from the medium for 3– 6 days. The medium was changed every other day, and bFGF was supplemented daily. Cell cultures were maintained at 37 °C in a 5% CO2 incubator.

Construction of the mutants of Ngn2 and Mash1 The DNA-binding mutants, Ngn2/AQ [20] and Mash1/AQ (modeled after Ngn2AQ), were generated by changing two

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amino acids NR, located in the basic region of the bHLH proteins and crucial for DNA binding, into AQ, by site-directed mutagenesis using the QuickChange System® (Stratagene, La Jolla, CA). Chimeric mutants of Ngn2 and Mash1 were generated by interchanging the basic (B) and helix–loop– helix (HLH) domains in Ngn2 and Mash1. The basic region of Ngn2 was fused to the HLH domains of Mash1, to generate the chimeric proteins NBMHLH. Similarly, the chimera MBNHLH was generated by fusing the basic regions of Mash1 to the Ngn2 HLH domain. Conventional methods for PCR amplification, digestion with restriction enzymes, and ligation reactions were used for the production of the chimeras.

Retroviral infection The PCR-amplified cDNA fragments of the wild-types and mutants of Mash1 (rat), Ngn2 (mouse), and Otx2 (human) were engineered into the retroviral vector pCL [21,22]. For control transduction, a pCL-based LacZ expression vector was constructed. The retroviral vectors were introduced into the retrovirus packaging cell line 293gpg [23] by transient transfection with Lipofectamin™ (Invitrogen). After 72 h, supernatant fractions were harvested and maintained at −70 °C until use. For viral transduction, P1 cultures for neural precursors at 40–60% cell confluency (usually 1 day after cell plating) were incubated with the viral soup containing polybrene (hexadimethrine bromide; 1 μg/mL; Sigma-Aldrich) for 2 h, followed by a medium change with bFGF-supplemented N2. Coexpression studies were carried out by infecting cells with the mixtures of the individual viral constructs (1:1, v/v). Retroviraltransduced precursors were induced to differentiate by withdrawing the bFGF at the day following infection.

Immunocytochemistry Cultured cells were fixed in freshly prepared 4% paraformaldehyde (PFA) or 4% PFA/0.2% glutaraldehyde (for GABA staining) in phosphate-buffered saline except in the case of LeX immunostaining, which has been developed for live cell staining. Conventional methods for immunofluorescent staining were applied. The following primary antibodies were used at the concentrations specified: anti-mouse antibodies for Ki67 (1:200; Novocastra, Newcastle, UK), Mash1 (1:1000; BD Biosciences, Franklin Lakes, NJ), Ngn2 (a kind gift from D.J. Anderson, Howard Hughes Medical Institute, Pasadena), neuron-specific class III β-tubulin (Tuj1; 1:500; Covance, Princeton, NJ), LeX antibody (clone MMA; CD15; 1:200; BD Biosciences), microtubule associated protein 2 (MAP2; 1:200; Sigma-Aldrich), glial fibrillary acidic protein (GFAP; 1:200; MP Biochemicals, Solon, OH), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase; 1:500; SigmaAldrich), serotonin (1:2000; Sigma-Aldrich), anti-rabbit antibodies for Nestin (1:50; Dr. Mackay, NIH, Bethesda, MD), GABA (1:700; Sigma-Aldrich), and tyrosine hydroxylase (TH; 1:250; Pel-Freeze, Roger, AK). Appropriate fluorescencetagged secondary antibodies (Jackson Immuno-Research, West Grove, PA) were used for visualization. Cells were mounted in Vectashield® containing 4′,6 diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA) and analyzed under an epifluorescence microscope (Nikon,

Tokyo, Japan). For LeX immunostaining, live cells were sequentially incubated with the LeX antibody, a Cy3-conjugated secondary antibody, then fixed in 4% PFA and mounted for microscopic examination.

RT–PCR analysis Total cellular RNA was prepared using TRI REAGENT (Molecular Research Center, Inc. Cincinnati, OH) following the manufacturer's recommendations. Messenger RNA was reverse transcribed by Superscript II (Invitrogen), and PCR analysis were performed using standard protocols, as described previously [24]. PCR conditions (sense and antisense primer sequences, cycles, annealing temperatures) applied and product sizes are as follows: GAPDH 5′-GGCATTGCTCTCATTGACAA-3′, 5′-AGGGCCTCTCTCTTGCTCTC-3′, 25 cycles, 60 °C, 165 bp Otx1 5′-GCTGTTCGCAAAGACTCGCTAC-3′, 5′-ATGGCTCTGGCACTGATACGGATG-3′, 30 cycles, 62 °C, 425 bp Otx2 5′-CCATGACCTATATCAGGCTTCAGG-3′, 5′-GAAGCTCCATATCCCTGGGTGGAAAG-3′, 30 cycles, 50 °C, 211 bp Brain factor 1 (BF1) 5′-TCGTAAAACTTGGCAAAGAGGG-3′, 5′-GCTGTTCGCAAAGACTCGCTAC-3′, 32 cycles, 62 °C, 373 bp Emx1 5′-AGCGACGTTCCCCAGGACGGGCTGC-3′, 5′-TGCGTCTCGGAGAGGCTGAGGCTGC-3′, 35 cycles, 68 °C, 182 bp Gbx2 5′-ATGATGATGCAGCGCCCGCTG-3′, 5′-TCAGGGTCGGGCCTGCTCCAG-3′, 33 cycles, 61 °C, 177 bp Engrailed1 (En1) 5′-TCAAGACTGACTACAGCAACCCC-3′, 5′-CTTTGTCCTGAACCGTGGTGGTAG-3′, 35 cycles, 60 °C, 381 bp Pax2 5′-CCAAAGTGGTGGACAAGATTGCC-3′, 5′-GGGATAGGAAGGACGCTCAAAGAC-3′, 35 cycles, 58 °C, 545 bp Bone morphogenetic protein 2 (BMP2) 5′-GAGGCTGCTCACCATGTTTG-3′ 5′-GTGACATCAAAGCTCTCCCACT-3′, 35 cycles, 58 °C, 463 bp Sonic hedgehog (Shh) 5′-GGAAGATCACAAACTCCGAAC-3′, 5′-GGATGCGAGCTTTGGATTCATAG-3′, 33 cycles, 58 °C, 354 bp Nkx2.2 5′-CTCTTCTCCAAAGCGCAGAC-3′, 5′-AACAACCGTGGTAAGGATCG-3′, 35 cycles, 55 °C, 514 bp Nkx6.6 5′-GGATGACGGAGAGTCAGGTCAAGGT-3′, 5′-GCTGCCACCGCTCGATTTGTGCTTT-3′, 30 cycles, 64 °C, 450 bp Nestin 5′-GGAGTGTCGCTTAGAGGT-3′, 5′-TCCAGAAAGCCAAGAGAA-3′, 30 cycles, 60 °C, 327 bp Pax6 5′-CCAAAGTGGTGGACAAGATTGCC-3′, 5′-TAACTCCGCCCATTCACTGACG-3′, 35 cycles, 58 °C, 419 bp

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Pax7 5′-CATGGTTGTGTCTCCAAGATTCTG-3′, 5′-ATTCCACACCTGACCCCTCATC-3′, 35 cycles, 58 °C, 376 bp GAD67 5′-AATTGCACCCGTGTTTGTTCTTAT-3′, 5′-AGCGCAGCCCCAGCCTTCTTT-3′, 30 cycles, 56 °C, 338 bp Ngn2 5′-CCAGCGCGGACGAGGAGGAG-3′, 5′-TCGGTGAGCGCCCAGATGTG-3′, 35 cycles, 62 °C, 394 bp Mash1 5′-GGCTCAACTTCAGTGGCTTC-3′ 5′-TGGAGTAGTTGGGGGAGATG-3′, 28 cycles, 55 °C, 291 bp RT–PCR products were analyzed by 2% agarose gel electrophoresis.

Western blot analysis Proteins were extracted from cultures, electrophoresed by SDS–PAGE gel and transferred to a nitrocellulose membrane, as reported previously [24]. Membrane-transferred proteins were incubated in 5% bovine serum albumin or in 5% non-fat milk to block non-specific binding. The blot was probed with anti-mouse TuJ1 (1:2000; Covance), Mash1 (1:1000; BD Biosciences), Ngn2 (1:1000; a kind gift from D. J. Anderson, Howard Hughes Medical Institute, Pasadena, CA), β-actin (1:5000; Abcam, Cambridge, UK), anti-rabbit glutaraldehyde decarboxylase (GAD67; 1:500; Chemicon, Temecula, CA) followed by peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (1:2000; Cell Signaling Technology, Beverly, MA, USA, for both antibodies). Bands were visualized by enhanced chemiluminescence (ECL detection kit; Amersham-Pharmacia Co., Buckinghamshire, UK).

Cell counting and statistical analysis Cell counting was performed in 10–40 randomly chosen microscopic fields for each coverslip using an eye piece grid at a final magnification of 200 or 400. One to three cultured coverslips were analyzed in each independent experiment. Data are expressed as mean ± SEM of 3–4 independent experiments. Statistical analyses were made by one-way ANOVA (SPSS12.0; SPSS Inc., Chicago, IL).

Results General proliferative and developmental properties of cultured neural precursor cells derived from different brain regions of rat E13 embryos We isolated neural precursor cells from dorsal (d) and ventral (v) parts of the forebrain [dorsal forebrain (dF, Ctx) and ventral forebrain (vF, LGE)], midbrain (dM and vM), hindbrain (dH and vH), and spinal cord (dS and vS) of rat embryos at embryonic day 13 (E13) and cultured them in the presence of bFGF, which is known to be a mitogen for neural precursor cells. All the cells from the different brain regions were proliferated in response to bFGF, and formed cell clusters after several days of in vitro culture. Although major

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populations of cell clusters were positive for neural precursor cell markers, up to 30% of the cells were postmitotic neurons positive for the neuronal marker TuJ1 after cell proliferation in the bFGF-supplemented cultures (unpassaged, P0; data not shown) [19]. In order to obtain a homogeneous population of neural precursor cells, the clusters of cells were dissociated into single cells, plated on fleshly prepared culture dishes at 45,000 cells/cm2, and induced by additional bFGF proliferation of the precursor cells (see Materials and methods for the detailed procedures). This procedure allows elimination of differentiated cells by mechanical passage as well as further selective expansion of neural precursors, resulting in a uniform population of neural precursor cells in the passaged (P1) cultures (Figs. 1F–I) [24]. Total cell numbers increased steadily during 5 days of P1 proliferation. Forebrain precursors (Ctx and LGE) grew faster than the cells from the other brain regions, and reached similar cell numbers 1– 2 days earlier than the precursors from the other regions (Fig. 1A). The percentages of the cells positive for the markers specific to neural precursor cells nestin [25], Lex [26], and proliferating cell marker Ki67 reached their maximum levels after 2–3 days of P1 proliferation. Specifically, % nestin+ cells out of total (DAPI+) cells were 99.1 ± 0.2% (Ctx; dF), 98.3 ± 0.5% (LGE; vF), 98.6 ± 0.3% (dM), 98.5 ± 0.1% (vM), 99.1 ± 0.2% (dH), 99.6 ± 0.1% (vH), 99.7 ± 0.1%(dS), and 99.6 ± 0.1%(vS). % Lex+ cells were 95.8 ± 1.0% (dF), 91.5 ± 1.9% (vF), 93.4 ± 1.5% (dM), 91.5 ± 1.7% (vM), 88.7 ± 2.2% (dH), 88.8 ± 2.8% (vH), 84.5 ± 3.8% (dS), and 88.3 ± 2.2% (vS). Percentages of Ki67+ cells out of total cells were 99.2 ± 0.2% (dF), 99.4 ± 0.1% (vF), 92.8 ± 1.1% (dM), 93.9 ± 0.6% (vM), 91.6 ± 1.1% (dH), 98.2 ± 0.5% (vH), 91.0 ± 0.9 % (dS), and 94.3 ± 1.2% (vS). Cell apoptosis, estimated by the % of cells with apoptotic nuclei (Fig. 1C) and cleaved caspase-3 (Fig. 1D), was greatly decreased by 2 or 3 days of proliferation. Further cell expansion for longer periods (up to 5 days) had a rather detrimental influence on cell survival, maybe because of ‘cell contact inhibition’ by the cells overgrown in the clusters, resulting in increased cell apoptosis (Figs. 1C and D). Therefore, all the tests in this study have been performed after 2 (Ctx, LGE) or 3 days (midbrain, hindbrain, spinal cords) of P1 proliferation at a cell density of 2.2–3.8 × 105 cells/cm2 (30–60% cell confluency), unless otherwise noted. Only < 1%, 0%, and 0% of cells were positive for the differentiated cell markers TuJ1 (neuron), GFAP (astrocyte), and CNPase (oligodendrocyte), respectively. Withdrawal of bFGF readily induced differentiation of the precursors towards their differentiated neuronal and glial progenies (Figs. 2A and B): for instance, 30.9 ± 2.7%, 23.5 ± 4.7%, and 8.7 ± 0.5% of total cells were positive for TuJ1, GFAP, and CNPase in the differentiated cortical cultures. These findings together indicate cultivation of homogenous populations of neural precursor cells with self-renewal and multi-developmental capacities, irrespective of the embryonic brain regions. We further explored precursor differentiation into several neurotransmitter subtypes of neurons. Cells positive for TH (dopamine neuron marker) and serotonin (serotonin neuron marker) were exclusively detected in the differentiated cultures of the precursors derived from ventral midbrain and ventral hindbrain (Figs. 2E and F). This finding is consistent with the idea that major populations of dopamine

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Fig. 1 – Uniform populations of neural precursor cells in the cultures derived from various regions of rat embryonic brains along dorsal–ventral and anterior–posterior axes. Precursor cells were isolated from dorsal and ventral regions of the forebrain, midbrain, hindbrain and spinal cord of rat embryonic brains at embryonic day 13, and proliferated in vitro with bFGF. The bFGF-proliferated precursors were subcultured and further expanded for 5 days. (A–E) Cell growth, proliferation, and apoptosis over the expansion period were analyzed by total cell number (A), % Ki67+ cells (B), % cells with fragmented or condensed apoptotic nuclei (C), cleaved caspase3+ cells (D) and nestin+ cells (E). (F–I) cells positive for neural precursor-specific markers. Shown are representative images of Ki67+/nestin+ cells (F) and Lex+ cells (H) at in vitro proliferation day 2 (forebrain) and 3 (midbrain, hindbrain, spinal cord). Note that percentages of neural precursor cells are high and indistinguishable among the region-specific cultures with minimal cell apoptosis (C, D) under the conditions. Scale bars: 20 μm. Panels G and I are the graphs depicting the % Ki67+, Nestin+ cells and the % Lex+ cells, respectively.

and serotonin neurons arise in the ventral parts of the embryonic midbrain and hindbrain by the combined actions of sonic hedgehog (Shh) and FGF8, secreted from the floor plate and isthmus, respectively [27]. Although GABA neurons are ubiquitously detected in the differentiated cultures of all the brain regions, significant variation in GABA neuron yields from the precursor cells was observed in the cultures of different brain regions (Figs. 2C and D). The cultures derived from the ventral forebrain (LGE) exhibited the highest GABA neuron yield. In general, GABA neuron yields were greater in the cultures derived from the ventral

domains of the forebrain, midbrain and hindbrain than from the respective dorsal domains. These findings are consistent with the previous in vivo studies demonstrating GABA neuron generation in the ventral parts of the forebrain [11,28] and midbrain [6,7]. The GABAergic neuron yield of the precursors from spinal cords was lowest among the precursors tested, and no significant difference between ventral and dorsal domains was shown. These findings collectively suggest that brain region-specific neuronal subtype differentiation is retained in the cultures of neural precursor cells.

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Fig. 2 – Differentiation phenotypes of the precursor cells derived from various regions of rat embryos. Neural precursor cells were cultured from the dorso-ventral regions of the developing rat forebrain, midbrain, hindbrain, and spinal cord at E13. Differentiation phenotypes were determined by immunocytochemical analysis 6 days after in vitro differentiation. (A,B) General neuronal and glial differentiation estimated by % cells positive for TuJ1 (neuron), GFAP (astrocyte), and CNPase (oligodendrocyte). (C–F) Neuronal subtype differentiation. % GABAergic (GABA+, C and D), dopaminergic (TH+, green of panels E and F), and serotonergic (serotonin+, red of panels E and F) neurons were determined in the differentiated cultures for the region-specific precursor cells. Scale bars: 20 μm.

Cultured neural precursor cells retain expression patterns of the genes specific for embryonic brain regions We next examined the expression of various region-specific markers in the cultures of the precursors derived from the different brain regions using semi-quantitative RT–PCR analyses (Fig. 3). All the cultures and tissues of the brain regions at E13 expressed the general CNS precursor cell markers nestin and Pax6, and the levels of expression of those markers were not significantly different among the cultures or tissues. Otx1 and 2 are expressed in the brain

regions anterior to the midbrain–hindbrain junction (isthmus) during early brain development, whereas Gbx2 expression is detected in the regions posterior to the isthmus [29]. Similar to the expressions of E13 tissues, Otx1 expression was exclusively observed in the cultures of the precursors isolated from the brain regions anterior to the isthmus, such as the forebrain and midbrain. It has been demonstrated that the expression of Otx2 disappears from the forebrain after mid-gestation of mouse embryos [29,30], although its expression is exactly overlapped in the Otx1-expressing domains in the early embryonic brain.

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Fig. 3 – Inherited expression of region-specific genes in cultures of neuronal precursor cells. RNA was isolated from primary tissues (8 lanes in left) of the dorsal and ventral regions of the developing forebrain (dF, vF), midbrain (dM, vM), hindbrain (dH, vH), and spinal cord (dS, vS) or cultures for the precursor cells (8 lanes in right) derived from those brain regions. The mRNA expression of candidate genes was analyzed by semi-quantitative PCR, followed by visualization with EtBr staining.

Consistently, Otx2 expression was either not or slightly detected in E13 forebrain cultures and tissues. In contrast, Gbx2 expression was obvious in the cultures derived from the posterior brain regions such as the hindbrain and spinal cord, but not in the forebrain-derived cultures. Slight levels of Gbx2 expression in the midbrain cultures and tissues (Fig. 3) are probably attributable to imperfect dissection of the midbrain from the neighboring hindbrain. These findings showed that the expression patterns of these anterior– posterior regionalization genes were preserved in the neural precursor cells after in vitro culturing. Emx1 and Bf1 are organizing molecules of forebrain development [31–33]. Similar to the Emx1 and Bf1 expression patterns in E13 tissues, their expression was observed exclusively in the cultures from dorsal (Ctx, dF) and ventral forebrains (LGE, vF). En1 and Pax2 are expressed in embryonic midbrain and hindbrain, and function as midbrain–hindbrain organizers [34–36]. In addition, they are also expressed in the developing spinal cord, and play roles in interneuron cell type specification [36]. Consistently, expression of En1 and Pax2 was detected in the cultures from the E13 midbrain, hindbrain, and spinal cord. However, in contrast to the absence of En1 expression in E13 forebrain tissues, En1 expression was detected in the cultures of the forebrain, especially ventral forebrain precursor cells. How En1 expression is acquired in the forebrain precursors during in vitro culture remains to be elucidated. Previous studies have demonstrated the regulatory roles of bFGF in dorso-ventral

brain patterning factors. Growth in bFGF promotes expression of the ventral markers such as SHH and Nkx2.2 with a gradual loss of the dorsal marker Pax7 expression in cultured neural precursor cells, suggesting bFGF-mediated ventralization of neural precursor cells [37]. In contrast, an opposite effect, such as inhibition of SHH signaling by bFGF in neural precursors, has recently been reported [38]. Higher expression of the ventralization signal molecules SHH, Nkx2.2, and Nkx6.6 was observed in the cultures derived from the ventral parts of the brains than those of the respective dorsal brain regions (Fig. 3). Reverse patterns of BMP2 and Pax7, dorsal markers of embryonic brain, were observed. Thus, although the reported regionalizing effect of bFGF may be present, it does not seem to make a large contribution in our cultures, considering the dorso-ventral marker expression patterns were maintained in the cultures derived from the dorsal and ventral brain regions. These findings together suggest that the expression of the region-specific genes can be inherited in neural precursor cells through multiple divisions outside of the embryonic environment. Considering the role of region-specific factors in the neurotransmitter subtype specification of neurons [39–43], retained expression of the region-specific genes in the cultures, combined with the uniform populations of neural precursor cells with self-renewal and multi-developmental properties, provide a rationale for the precursor cell cultures to be used as an experimental system to investigate neuronal subtype specification.

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Brain region-dependent difference in the roles of Ngn2 and Mash1 in GABAergic neuronal differentiation Recent studies have revealed roles for proneural bHLH factors in the neurotransmitter subtype specification of neurons, such as GABA-glutamertergic neurons in the forebrain [14,15], DA neurons in the midbrain [21,22,43,44], and motor [45,46] and serotonin neurons in the hindbrain [46]. Based on the fact that the GABA neuron is the most common inhibitory neuronal subtype ubiquitously resident in most brain regions and that the expressions of Ngns and Mash1 bHLHs are also detected in various regions of the developing brains, we wondered whether GABA neuron formation in the other brain regions, besides the forebrain, may also be regulated by the bHLH factors. This study, therefore, exploited cultures for neural precursors from various regions of E13 rat brains to gain insight into the roles of Ngn2 and Mash1. Basal expressions of Ngn2 and Mash1 were marginal in untransduced precursor cells when analyzed by both immu-

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nocytochemistry and RT–PCR. Proneural bHLH gene expression is not detected in early neuroepithelial or neural precursor cells, but it is transiently expressed in late or committed precursor cells during the neurogenic period of the developing brain [47]. Therefore the marginal Ngn2 and Mash1 expression, combined with the immnocytochemical phenotypes of the cultures (Fig. 4A), indicate that our culture system allowed a selective cultivation of relatively early and uncommitted types of neural precursor cells which are adequate for studying cell fate determination. Cells were retrovirally transduced with the constructs of Ngn2, Mash1, and LacZ (control), and precursor differentiation was initiated 1 day after the infections. Ngn2 transduction resulted in repression of basal endogenous Mash1 expression in all the cultures tested (Figs. 4B and C). However, Ngn2 expression was not significantly altered by forced Mash1 expression. These findings are consistent with the previous in vivo data showing that the complementary patterns of Ngn1/2 and Mash1 expression in the developing forebrain and spinal cord are built by the

Fig. 4 – Transgene expressions of Ngn2 and Mash1 using retroviral infection. Neural precursor cells, derived from the forebrain, midbrain, hindbrain, and spinal cord at E13, were transduced with retroviral constructs of Ngn2, Mash1 or LacZ (control) 1–2 days after in vitro proliferation. Transgene expression was determined at day 2 post infection by immunocytochemical (A), Western blot (B), and semi-quantitative RT–PCR (C) analyses. Note that endogenous levels of Mash1 expression were lower in the cultures transduced with Ngn2, compared with those of LacZ-transudced control cultures, regardless of the origin of the cultures (B and C), suggesting an inhibitory action of Ngn2 on endogenous Mash1 expression. Scale bar: 20 μm.

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inhibitory role of Ngn in Mash1 expression [14,15,48], but Ngn1/2 expression is unaltered in Mash1 mutant embryos[14]. General neuron differentiation, estimated by the number of cells positive for neuron-specific markers TuJ1 and MAP2 (Figs. 5A–C) and the level of TuJ1 proteins (Fig. 5D), was strikingly enhanced by forced expression of Ngn2 and Mash1, regardless of the regions from which the precursors were derived. The pan-neurogenic effects of Ngn2 and Mash1 from the cultured precursors were not significantly different. We have previously demonstrated that precursor cell differentiation into neurons and astrocytes is enhanced in those cultures with higher cell density, and that the cell density effect is mediated by diffusible factors secreted by differentiating and differentiated neural precursor cells [49]. Similarly GABA neuron differentiation, estimated by the percentage of cells immunoreactive for GABA, was greatly influenced by cell density, with the GABA+ cell yield being greater in the differentiated cultures plated at higher cell densities. Use of cultures with lower cell density is helpful in estimating the cell-autonomous effects of Mash1 and Ngn2 by reducing the cell density effects. However, GABA+ cells were not readily detected in differentiated cultures with too low or clonal cell densities. Thus all the following studies were done in the cultures where cells were 30–40% confluent by inducing cell proliferation only for 1–2 days after plating at 45,000 cells/ cm2 (see Materials and methods). In contrast to the indistinguishable common roles of Mash1 and Ngn2 in general

neurogenesis, the effects of Mash1 and Ngn2 overexpression in GABA neuron differentiation were divergent in cultured neural precursor cells. GABA+ cell yield was significantly enhanced by exogenous Mash1 expression in the differentiated cultures of forebrain neural precursor cells. By contrast, Ngn2 exerted an inhibitory effect on GABA neuron formation from the forebrain precursors. For instance, GABA+ cells out of 1,000 DAPI+ cells in cortical cultures (dF) at differentiation day 3 were 13.68 ± 3.77 (LacZ-control), 2.25 ± 0.95 (Ngn2), 22.09 ± 4.22 (Mash1), p < 0.05, n = 90 microscopic fields for each group (Figs. 6A–D). These findings are consistent with the previous in vivo studies demonstrating Mash1- and Ngn2-induced positive and negative regulation of GABA neuron formation, respectively, in the developing forebrain [14,15]. Similar to the Mash1 role in forebrain GABA neuron formation, a previous study has demonstrated generation of a GABA neuron population in the Mash1-expressing domain of the developing midbrain and a significant reduction of GABA+ neuron numbers in the midbrains of Mash1-deletion mice [6]. Consistent to the in vivo findings, Mash1-induced increase of GABA neuron yield was also observed in the midbrain precursor cultures (Figs. 6B–D). GABA neuron numbers, however, were not significantly different in Ngn2-transduced midbrain cultures. Interestingly and surprisingly, the effects of Mash1/Ngn2 overexpression were opposite to those of the forebrain precursors in the cultures of the precursors derived from the brain regions posterior to the isthmus, such as the hindbrain and spinal cord.

Fig. 5 – Enhanced neuronal differentiation from cultured neural precursor cells by Ngn2 and Mash1 overexpression. Neural precursor cells from the brain regions were cultured and transduced with Ngn2, Mash1, or LacZ (control) as described. Three days after differentiation, general neurogenic effects of Ngn2 and Mash1 were determined by evaluating the % cells positive for the neuron markers TuJ1, MAP2(A and B) and TuJ1 protein levels (C). Shown in A and B are the representative images of TuJ1+/MAP2+ cells and % of these immunoreacitve cells out of total cells (counterstained with DAPI) in the cultures derived from the dorsal forebrain (upper) and hindbrain (lower). Proportions are significantly different from LacZ control at **p < 0.05 and *p < 0.001. Scale bar, 40 μm.

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GABA neuron yields were decreased by the forced expression of Mash1 in the cultures derived from ventral and dorsal regions of the hindbrain and spinal cord. In contrast, Ngn2 expression resulted in a significant increase of GABA neuron yield in the cultures derived from these posterior brain regions. GABA+ cells in dH were 4.66 ± 0.9 (LacZ-control), 15.59 ± 1.34 (Ngn2), 3.46 ± 0.55 (Mash1); vH: 4.86 ± 0.78 (LacZ), 12.2 ± 1.17 (Ngn2), 3.58 ±0.54 (Mash1); dS: 2.73 ± 0.43 (LacZ), 6.28 ± 0.91 (Ngn2), 1.78 ± 0.25 (Mash1); vS: 1.11 ± 0.44 (LacZ), 2.89 ± 0.75 (Ngn2), 0.68 ± 0.2 (Mash1), p < 0.05, n = 75–90 (Figs. 6B–D). No significant differences in precursor-derived GABA neuron formation were shown in the respective ventral- and dorsalbrain regions (Figs. 6B–D). The contrasting and brain regionspecific actions of Ngn2 and Mash1 were further confirmed in the RT–PCR and immunoblot analyses for GAD67, an enzyme specific to GABA synthesis (Figs. 6C, D). These findings not only further support the existing data of the regulatory roles of Mash1 and Ngn2 in forebrain GABA neuron formation, but also

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extend our knowledge to their roles in other brain regions, especially the opposing effects of the bHLHs in the regions anterior and posterior to the isthmus.

Structures responsible for the distinct roles of Ngn2 and Mash1 in forebrain GABA neuron differentiation The repeated Ngn2 and Mash1 effects in forebrain GABA neuron differentiation in in vitro cultures further support the rationale for using cultured neural precursor cells as an appropriate system for the study of neuronal subtype differentiation. As an initial mechanistic study for the Ngn2 and Mash1 activities, the forebrain cultures were utilized to define the structures of Ngn2 and Mash1 proteins responsible for the regulatory roles of forebrain GABA neuron differentiation. The basic domains of proneural bHLH transcriptional factors are the structures for binding to DNA, while the HLH portion is responsible for the protein–protein interaction between bHLH

Fig. 6 – Brain region-specific activities of Ngn2 and Mash1 in precursor-derived GABA neuron differentiation. Neural precursor cells, derived from dorsal and ventral regions of the forebrain, midbrain, hindbrain, and spinal cord at E13, were cultured and infected with retroviral constructs of Ngn2 and Mash1. The cultures were subjected to GABA− immunostaining for GABA+ cells (A and B), RT–PCR (C), and immunoblot (D) analyses against GAD67, an enzyme for GABA neurotransmitter biosynthesis, 3 days after in vitro differentiation. Shown in panel A are the representative images for GABA+ cells in the differentiated LacZ- (control; left), Ngn2- (middle), and Mash1- (right) transduced cultures of ventral forebrain (upper) and hindbrain (low) precursor cells. Scale bar: 80 μm. Graph B depicts numbers of GABA+ cells out of 1000 cells. Significantly different from LacZ-control at *p < 0.001 and at **p < 0.05.

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proteins or with other proteins [50,51]. To determine whether the regulatory roles of the bHLHs in GABA neuron differentiation are mediated through their DNA binding, the mutants Ngn2/AQ and Mash1/AQ [20,52], the DNA binding capacity of which is abolished, were constructed (Fig. 7A). These mutations of Ngn2 and Mash1 resulted in the loss of general neuronal differentiation activity as estimated by TuJ1 expression (Fig. 7B), consistent with the notion that bHLH-mediated neurogenesis occurs through direct binding to target promoters and by promoting transcriptional initiation. Similarly the forebrain precursor cells expressing Ngn1/AQ did not display the inhibitory effect on the GABA neuron formation seen with the wild-type Ngn2 (Fig. 7B) suggesting that DNA binding is required for the inhibitory activity of Ngn2. To our surprise, the DNA binding mutation of Mash1, however, did not abolish the regulatory activity of Mash1 in GABAergic differentiation. The enhanced GABA neuron yield seen in the forebrain precursors transduced by WT-Mash1 was also observed by Mash1/AQ overexpression without exception in numerous independent experiments (LacZ: 10.98 ± 3.1, Mash1: 23.47 ± 4.83, Mash1/AQ: 29.51 ± 7.7, n = 80–110, Fig. 7). These findings indicate that the Mash1-mediated regulation of GABA neuron differentiation does not occur through direct DNA binding, which is indispensable for its proneural activity, and that the molecular mechanism of Mash1-mediated control of GABAergic differentiation is distinct from that of Ngn2. To identify the important domains of the bHLH factors that impart the distinct activities of Ngn2 and Mash1 in forebrain GABAergic differentiation, we generated chimeric proteins by interchanging basic and HLH domains between Ngn2 and Mash1 (Fig. 7A). General neurogenic roles of the chimeric- and WT-bHLHs were indistinguishable (Fig. 7C). A reduction in the number of GABA+ cells, comparable to the degree seen with the wild-type Ngn2, was observed by introducing chimeric protein MBNHLH, which is composed of the Mash1 basic region and the Ngn2 HLH domain (Fig. 7C). By contrast, the chimera expressing NBMHLH exerted an increase in GABA+ cell yield, to level similar to those of WT-Mash1 (Fig. 7C). Such data indicate that the HLH domains of Ngn2 and Mash1 contain the information that specifies the distinct roles of each bHLH gene in GABA neuron differentiation in the developing forebrain. A reduction of endogenous Mash1 expression, as much as that in WT-Ngn2-transduced cultures, was shown in the cultures transduced with MBNHLH, but not in NBMHLHtransduced cultures (data not shown). Thus, the decrease of endogenous Mash1 expressions may possibly contribute partially to the reduction of GABA neuron numbers in the cultures transduced with the chimera MBNHLH or WT-Ngn2.

Co-expression of Otx2 reverses the regulatory activities of Ngn2 and Mash1 in the hindbrain GABA neuron differentiation The most notable finding in this study is the opposing effects of the bHLH factors in GABA neuron formation when comparing the brain regions anterior and posterior to the isthmus. Otx1/2 factors are exclusively expressed in the regions anterior to the isthmus during brain development, and are critical for specification and regionalization of the forebrain and the midbrain. Furthermore, recent loss- and gain-of-function

studies of Otx2 in the midbrain and the hindbrain precursor cells have demonstrated clear neuronal subtype transitions of midbrain- (DA neuron) to hindbrain-specific (serotonin) neurons and vice versa, respectively [53]. These findings prompted us to test whether ectopic expression of Otx2 in the precursor cells derived from the posterior brain regions can alter the roles of Mash1 and Ngn2 in the regulation of GABA neuron differentiation. Exogene expression of Otx2 in the precursor cells derived from the embryonic hindbrain and spinal cord did not significantly altered GABA neuron yields. Interestingly coexpression of Otx2 in the hindbrain neural precursor cells completely reversed the Mash1- or Ngn2-mediated regulation of GABA neuron differentiation. As shown above (Fig. 6), the Ngn2-mediated increase and Mash1-mediated decrease of GABA+ cell yields were shown in the hindbrain precursor cultures; GABA+ cells: 5.26 ± 0.6 (LacZ), 12.37 ± 0.9 (Ngn2), 3.52 ± 0.39 (Mash1) (Figs. 8A and B). In contrast, in the Otx2transduced hindbrain cultures, Ngn2 exerted an inhibitory role, whereas Mash1 exhibited enhanced GABA+ cell yields; GABA+ cells: 5.96 ± 0.47 (LacZ + Otx2), 3.78 ± 0.35 (Ngn2 + Otx2), 13.02 ± 0.85 (Mash1 + Otx2), n = 170–180 microscopic fields from 3 to 5 independent experiments, significantly different from the control at p < 0.05 (Figs. 8A and B). Ectopic expression of Otx2 in the precursor cells from the other brain regions, however, did not elicit any significant influences on the Mash1/Ngn2-mediated control of GABAergic differentiation. These results suggest that Otx2 is a critical factor, with which the brain region-dependent differences of the bHLH activities in GABA neuron differentiation could be, at least in part, elucidated.

Discussion Generation of region-specific neuronal subtypes is a key element of brain development, but the understanding of which remains mostly unclear. It is generally accepted that different subtypes of mature neurons arise from neural precursor cells, the intrinsic identities of which had been established by local environments during early developmental processes. Previous studies have demonstrated that neural precursor cells derived from different brain regions retain their region-specific identities [54–56]. These findings suggest neural precursor cell cultures as an experimental system for cell differentiation studies. In this study, we systematically performed cultures for highly uniform populations of uncommitted neural precursor cells from various embryonic brains along the anterior–posterior and dorsal–ventral axes, and further confirmed that the naive expression profiles of regionspecific genes were largely inherited in the region-specific cultures. Using gain-of-function analyses in the region-specific cultures, the roles of bHLH transcription factors Ngn2 and Mash1 were investigated in precursor cell differentiation towards GABAergic inhibitory neurons. Important findings previously demonstrated in in vivo embryos, such as the inhibitory role of Ngn2 in endogenous Mash1 expression [14,15,48] and Ngn2/Mash1-mediated controls of forebrain GABA neuron differentiation [14,15], were exactly reproduced in our in vitro precursor cultures (Figs. 4C and 6). These findings

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further support that the rest of the findings obtained in our in vitro cultures could be reflective of real in vivo developmental phenomena.

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The regulatory roles of Mash1 and Ngn2 in forebrain GABA neuron differentiation have been previously demonstrated and further confirmed in this study. However, the

Fig. 7 – Analyses of the structural components of Ngn2 and Mash1 required for regulatory roles in forebrain GABA neuron differentiation. Schematic diagrams for the structures of the DNA binding mutants and chimeric proteins of Ngn2 and Mash1 (A). B–C: Effects of the mutants of Ngn2 and Mash1 on GABA+ and TuJ1+ cell yields from forebrain precursor cells. Wild-type and the mutants of Ngn2 and Mash1 were introduced into forebrain precursors, and the numbers of cells positive for GABA and TuJ1 were determined at day 3 of differentiation. Effects of the abolishment of DNA binding capacity of Ngn2 and Mash1 in GABA+ (right column, red) and TuJ1+ (left column, green) cell yields. Note that the abolishment of DNA binding capacity in Ngn2 (Ngn2/AQ) abrogates not only general neurogenesis (TuJ+ neuronal yield), but also the inhibitory activity on GABA+ cell yield. By contrast, the regulatory action of Mash1 on forebrain GABA+ cell yield is preserved in the DNA binding mutant of Mash1 (Mash1/ AQ). Analyses of the chimeric proteins of Ngn2 and Mash1 reveal that the HLH domains are responsible for the regulatory actions of Ngn2 and Mash1 on forebrain GABA neuron differentiation. Scale bar: 30 μm. The graphs in C depict the proportions of GABA+ out of 1000 cells and TuJ1+ cells out of 100 DAPI+ cells. p < 0.001, compared with the value of LacZ-expressing control* and wild-type of Ngn2 or Mash1#.

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Fig. 8 – Regulatory activities of Ngn2 and Mash1 in GABA+ cell yields from the hindbrain precursor cells are reversed by ectopic expression of Otx2. Effects of Ngn2 and Mash1 in GABA+ cell yields were compared 3 days after differentiation in the hindbrain cultures untransduced and transduced with Otx2. Coexpressions of Ngn2+ Otx2, Mash1+ Otx2, and LacZ+ Otx2 were induced by infecting cells with the mixtures of the individual viral constructs (1:1, v:v). Shown in panel A are representative images for GABA+ cells of the control LacZ- (left), Ngn2- (middle), and Mash1- (right) transduced hindbrain cultures in the condition of Otx2-coexpression (lower) or not (upper). Scale bar: 20 μm. Graph B depicts numbers of GABA+ cells out of 1000 cells in untransduced (left) and Otx2-transduced (right) hindbrain cultures.

mechanisms involved in this phenomenon are completely unknown. As an initial mechanistic study, we tried to define structures encompassing the regulatory functions of Ngn2/ Mash1 by analyzing the functions of various Ngn2/Mash1 mutants in the forebrain precursor cell cultures. Domain swapping experiments demonstrated that the HLH domains of Ngn2 and Mash1, structures for protein–protein interactions of Ngn2 and Mash1, but not their DNA binding basic domains, are the structures responsible for the contrasting specificities of Ngn2 and Mash1 in forebrain GABA differentiation (Fig. 7). These findings are consistent with previous studies demonstrating the importance of the helix domains, rather than the basic domain of proneural bHLH factors, in the specification of midbrain dopamine neurons [21] and interneurons in the dorsal neural tube [52]. By contrast, the basic domains of scute and atonal proneural bHLH transcription factors, which promote external sensory organs and chordotonal organs of Drosophila, respectively, have been proposed to be also required for the functional specificities [57]. However, the residues that differ between the basic regions of scute and atonal are predicted not to directly contact the DNA, but to be involved in interactions with cofactors. Together, these data suggest that protein interactions with other cofactors are critical for the subtype specification role of each proneural bHLH factor, although

different domains within the bHLH transcription factors are important in different cellular contexts. The DNA binding mutant Mash1/AQ that failed to activate general neurogenesis exerted comparable or more prominent activities as a positive regulator for forebrain precursor-derived GABAergic differentiation, indicating that DNA binding of Mash1 is not required for the regulatory function of GABA neuron formation. Considering the native actions of bHLH proteins as transcription factors, the bHLH role without DNA binding may not be easily acceptable. Another example supporting a DNA-binding-independent and protein–protein interactionmediated function of proneural bHLH activities, however, has been proposed because of the actions of Ngn2 in the migration [58] and dendritic morphology of cortical neurons [59], which are completely dissociated from the novel neurogenic function of the bHLH protein. However, unlike Mash1, the regulatory action of Ngn2 in GABA differentiation requires binding of Ngn2 to DNA. A previous study has demonstrated findings indicating distinct modes of action of Mash1 and Ngn2 in neuronal subtype specification [15]. Therefore, our functional studies of the mutant proteins, taken together, suggest that the modes of action of Mash1 and Ngn2 in forebrain GABAergic differentiation are similar with regard to the HLH domain-mediated functional specificities, but different in requirement for DNA binding in the

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regulatory activities. Considering the nuclear localization of proneural bHLH factors and the proposed mechanism of Ngn2-mediated cell migration through protein interaction with transcriptional modulators [58,59], a possible model for the bHLH-mediated control of forebrain GABA neuron formation is that Mash1 and Ngn2 proteins interact with the modulators regulating the transcription of genes that are required for GABA neuron specification, for instance, the interactions converting transcription repressors into transcriptional activators or vice versa. A possible explanation for the abolishment of the regulatory activity in the DNA binding mutant of Ngn2 is that the mutation has a critical influence on the HLH domain-mediated protein interactions [59]. Otherwise, Ngn2, but not Mash1, may act with the transcriptional modulators in a form bound to DNA at the promoter regions and the DNA binding of Ngn2 is a prerequisite for the protein interaction. Intriguingly, the regulatory effects of the bHLHs in the precursor-derived GABA neuron differentiation were clearly distinct in the cultures derived from the brain regions anterior and posterior to the isthmus, suggesting that the bHLHmediated controls of the subtype differentiation are not fixed, but can be varied in different molecular contexts. We further demonstrated that Otx2, expression of which is exclusively confined to the anterior regions, is at least partially responsible for the brain region-dependent difference of the bHLH activities. These findings are quite interesting but not as surprising, because many examples of context-dependent alterations of proneural bHLH activities have been previously reported in neuronal subtype determinations [60]. For example, in the vertebrate peripheral nervous system (PNS), Ngn1/2 induces the differentiation of sensory neurons from PNS precursors, and Mash1 is required for that of autonomic neurons. At high concentrations of BMP2, however, Ngns like MASH1 promote autonomic neuron differentiation [61]. In addition, Ngn2 induces a motor neuron program in the context of Olig2 expression in the developing spinal cord [62,63]. The findings shown here provide a broad spectrum of information for generating further insights into the roles of Mash1 and Ngn2 in GABA neuron differentiation, but molecular mechanisms for the functional specificities are largely unclear. As a proposed mode of action, proneural bHLH factors has been implicated to interact with cofactors and/or induce expressions of the other determinant factors, probably more directly involved in the specification process [10]. For instance, it has been shown that Mash1 and Heslike cooperatively specify GABA neurons in the midbrain region [6]. In this regard, it is of import to examine if Ngn2/Mash1 acts through the interactions with other known GABA phenotype determinants in specific brain regions such as Dlx1, Ptf1a, and Lbx1 in a combinatorial manner, and/or through activating the expressions of the determinants. As described earlier, the in vitro data of the bHLH effects in forebrain and midbrain GABA neuron differentiation (Fig. 6) are consistent with previous in vivo function studies [14,15]. In addition, interneuron populations in the ventral spinal cord, the neurotransmitter subtype of which is mostly GABA, are generated in the Ngn2-expression domain [60], suggesting an

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Ngn2 role in the spinal cord GABA neuron formation observed in our in vitro study. It remains to be further confirmed whether the findings observed in the in vitro cultures are biologically relevant to bHLHs' roles in GABA neuron formation in the regions of the developing brain. This study is an example of the exploitation of in vitro cultures to study the developmental biology of region-specific neural precursor cells. Beside the application of neural precursor cells as an experimental tool for neurodevelopmental studies, culture systems for neural precursor cells offer a desirable cell source in cell transplantation approaches for intractable brain disorders. Recent studies have demonstrated that transplantation of GABA neurons allows reconstitution of distorted neural circuits or restoration of behavioral deficits in experimental models of epilepsy, ischemia, and Huntington's disease [64–69]. Information obtained in this study could be utilized to supply transplantable region-specific GABA neurons in GABA neuron-based transplantation approaches.

Acknowledgments This work was supported by SC2150 (Stem Cell Research Center of the 21st Century Frontier Research Program) funded by the Ministry of Science and Technology, Republic of Korea.

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