TISSUE-SPECIFIC STEM CELLS

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1 TISSUE-SPECIFIC STEM CELLS Expression of Tlx in Both Stem Cells and Transit Amplifying Progenitors Regulates Stem Cell Activation and Differentiation in the Neonatal Lateral Subependymal Zone KIRSTEN OBERNIER, a INA SIMEONOVA, a TATIANA FILA, a CLAUDIA MANDL, a GABRIELE HÖLZL-WENIG, a PAULA MONAGHAN-NICHOLS, b FRANCESCA CICCOLINI a a Department of Neurobiology, Interdisciplinary Center for Neurosciences, University of Heidelberg; Heidelberg, Germany; b Department of Neurobiology, University of Pittsburgh; Pittsburgh, Pennsylvania, USA Key Words. Tlx Subependymal zone neural stem cells Transit-amplifying progenitors Flow cytometry Epidermal growth factor receptor Gliogenesis ABSTRACT Niche homeostasis in the postnatal subependymal zone of the lateral ventricle (lsez) requires coordinated proliferation and differentiation of neural progenitor cells. The mechanisms regulating this balance are scarcely known. Recent observations indicate that the orphan nuclear receptor Tlx is an intrinsic factor essential in maintaining this balance. However, the effect of Tlx on gene expression depends on age and cell-type cues. Therefore, it is essential to establish its expression pattern at different developmental ages. Here, we show for the first time that in the neonatal lsez activated neural stem cells (NSCs) and especially transit-amplifying progenitors (TAPs) express Tlx and that its expression may be regulated at the posttranscriptional level. We also provide evidence that in Disclosure of potential conflicts of interest is found at the end of this article. both cell types Tlx affects gene expression in a positive and negative manner. In activated NSCs, but not in TAPs, absence of Tlx leads to overexpression of negative cell cycle regulators and impairment of proliferation. Moreover, in both cell types, the homeobox transcription factor Dlx2 is downregulated in the absence of Tlx. This is paralleled by increased expression of Olig2 in activated NSCs and glial fibrillary acidic protein in TAPs, indicating that in both populations Tlx decreases gliogenesis. Consistent with this, we found a higher proportion of cells expressing glial makers in the neonatal lsez of mutant mice than in the wild type counterpart. Thus, Tlx playing a dual role affects the expression of distinct genes in these two lsez cell types. STEM CELLS 2011;29: INTRODUCTION In the adult subependymal zone of the lateral ventricle (lsez), quiescent neural stem cells (NSCs), with characteristics of astrocytes and apical-basal polarity [1 4], sustain a lifelong process of neurogenesis through the generation of a population of rapidly dividing cells. The latter, in contrast to lineage restricted cells, display some properties of NSCs such as clone formation and multipotency and are referred to as transit-amplifying progenitors (TAPs) [2, 3, 5, 6]. TAPs do not display immunoreactivity for the neuroglia proteoglycan 2 (NG2) and within 2 days give rise to neuroblasts by undergoing differentiation into the intermediate stage of preneuroblasts [7, 8]. From late stages of embryonic development onwards, the epidermal growth factor (EGF) is the main mitogen for NSCs and TAPs inducing clone formation in vitro [9] and massive proliferation upon infusion into the lateral ventricle [10 17]. Consistent with its importance in regulating proliferation, in the postnatal niche, the expression of EGF receptor (EGFR) is tightly controlled. A subset of both NSCs and TAPs expresses high levels of EGFR (EGFR high ) at the cell surface; however, EGFR is rapidly downregulated during neuronal differentiation [7]. As during development, also in the postnatal lsez EGFR high progenitor cells are derived from NSCs expressing low EGFR levels (EGFR low ) [9, 18 20]. At this age, the transition from EGFR low to EGFR high NSCs coincides with activation and cell cycle entry; hence, it involves a profound rearrangement of the transcriptional machinery. The forebrain specific gene Tlx (Nr2e1), the vertebrate homolog of the Drosophila tailless gene, is a key regulator of gene transcription and proliferation in adult NSCs [21, 22]. The Tlx gene encodes an orphan nuclear receptor, which mostly acts as a transcriptional repressor recruiting histone deacetylases to the promoters of its target genes [23]. However, Tlx can also activate the transcription of Sirt1 and Mash-1 genes [24, 25]. In culture, Tlx is expressed during Author contributions: K.O.: collection of data and analysis, data interpretation, manuscript writing; I.S. and T.F.: collection of data and analysis; C.M. and G.H.-W.: collection of the data; P.M.-N.: mice generation; F.C.: conception and design, data analysis and interpretation, manuscript writing. Correspondence: Francesca Ciccolini, Ph.D., Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Im Neuenheimer Feld 345, Heidelberg, Germany. Telephone: þ ; Fax: þ ; Ciccolini@nbio.uni-heidelberg.de Received December 8, 2010; accepted for publication June 7, 2011; first published online in STEM CELLS EXPRESS June 28, 2011; available online without subscription through the open access option. VC AlphaMed Press /2009/ $30.00/0 doi: /stem.682 STEM CELLS 2011;29:

2 1416 Role of Tlx in Postnatal Neurogenesis proliferation and downregulated upon induction of differentiation, indicating its involvement in maintaining proliferation and inhibiting differentiation [21]. Indeed, Tlx downregulates genes associated to glial differentiation such as glial fibrillary acidic protein (GFAP), S100b, and Aqp4 [21]. In addition, Tlx also represses cell cycle inhibitors. For example, lack of Tlx results in upregulation of the cyclin dependent kinase inhibitor p21 [26] and in increased levels of the tumor suppressor phosphatase Pten [23, 27]. However, in vivo studies have shown contrasting effects of Tlx on progenitor proliferation. Whereas, at day 10 of embryonic development (E10), lack of Tlx leads to extra proliferation [28], and at later developmental ages and in the adult telencephalon, it causes a marked reduction of cell proliferation [21, 26, 27]. Thus, Tlx exerts a multiple and wide-ranging regulation depending on the age and progenitor type. To understand its role in the regulation of the adult NSC lineage, it is therefore essential to establish in which progenitor type Tlx is expressed. Although its expression in adult NSCs has been established, it is still unknown whether Tlx is also present in their progeny. Here, we provide first evidence that in the postnatal lsez Tlx is expressed also in TAPs. Moreover, we show that Tlx licenses activation and proliferation of NSCs but not of TAPs, and in both cell types, it represses the expression of genes associated to glial differentiation. MATERIALS AND METHODS Tissue Dissection and Cell Culture In accordance with the local ethical guidelines for the care and use of laboratory animals (Karlsruhe, Germany), time-mated pregnant (plug day ¼ 1) dames and young adult mice, between 4 and 6 weeks after birth, were killed by CO 2 followedbyneck dislocation, whereas neonatal, day 7 after birth (P7), mice were sacrificed by decapitation. Striata and lsez dissection and dissociation was done as described earlier [7, 29]. After dissociation, cells were labeled and sorted. Alternatively, they were grown for 1 day at a density of 10 5 cells per milliliter in culture medium consisting of NS-A (Euroclone, Milano, Italy complete media supplemented with 2% B27 (Gibco Invitrogen, Carlsbad, CA, invitrogen.com/site/us/en/home.html) and 10 ng/ml fibroblast growth factor (FGF)-2 (R&D System, Minneapolis, MN, (henceforth referred to as F medium). Fluorescence Activated Cell Sorting (FACS) Analysis and Clonal Analysis Dissociated cells suspended in ice-cold sorting medium were stained with either mouse-anti-prominin conjugated to phycoerythrin (PE) (1:200) or rat-anti-ng2 and secondary antibody goat-anti-rat IgG A647 (1:1,000; Invitrogen, Carlsbad, CA, and/or 20 ng/ml of EGF fluorescently tagged with Alexa488 or Alexa647 fluorochromes (Molecular Probes, Invitrogen, Carlsbad, CA, and sorted on a FACSAria cytometer (Becton-Dickinson, Franklin Lakes, NJ, at single cell precision as described earlier [20, 30]. For clonal analysis 1, EGFR high cell or 10 EGFR low cells per well were plated by FACS-automated cell deposition in 96-well plates (Nunc, Thermo Fisher Scientific Waltham, MA, nuncbrand.com/us/default.aspx) in 50 ll of culture medium supplemented with 10 ng/ml FGF-2 and 20 ng/ml EGF (proliferation medium). Primary clones were scored after 7 10 days. Clone size was analyzed by determining the number of cells per clone. Approximately 10 clones were analyzed for each experiment. Analyses of differentiation and secondary clone formation were performed as described earlier [20]. Analysis of RNA Expression Cells (500 10,000 cells depending on sorting procedure) were sorted into lysis buffer containing b-mercaptoethanol and total RNA was extracted by RNeasy Mini Kit (Qiagen, Valencia, CA, according to manufacturer s instructions. For reverse transcription and quantitative real time-polymerase chain reaction (qrt-pcr), oligo dt primers and M-MLV reverse transcriptase, RNase H Minus (Promega, Madison, WI, were used to retrotranscribe the total mrna into first-strand cdna. For microrna (mir) analysis, total RNA was retrotranscribed using the TaqMan mir reverse transcription kit (Applied Biosystems, Carlsbad, CA, home.html). For reverse transcription PCR, the following Tlx primers were used: forward 5 0 -ATGCCCCGTAGACAAGA- CAC-3 0 ; reverse5 0 -TTCAGGAGTGGCAGACACAG-3 0. After 25 amplification cycles, the DNA bands were analyzed by ethidium bromide-staining. For mir quantitative analysis, stem loop primers specific for mir-9 (ID:000583), let-7b (ID:002619), and snorna202 (ID:001232) (Applied Biosystems, Carlsbad, CA, were used. For qrt-pcr, the following TaqMan gene expression assays (Applied Biosystems) were used: Mash1 (ID: Mm _m1), Tlx (ID:Mm _m1), p21 (ID:Mm _m1), Dlx2 (ID:Mm _m1), Gfap (ID: Mm _m1), Olig2 (ID: Mm _m1), b-2 microglobulin (ID: Mm _m1), and b-actin (ID:Mm _s1). Cycle threshold values were obtained from the logarithmic phase of the amplification plot for genes or mirs of interest and were normalized against the average of B2m and bactin or snorna202, respectively. Genotyping Tail biopsies of newborn mice from Tlx heterozygous matings were enzymatically digested overnight by ProteinaseK at 56 C. The next day, genomic DNA was extracted by saturated NaCl and isopropanol/ethanol precipitation. Amplification of the genomic DNA was done using three primers: GCCTGCTCTTTACTGAAGGCTCTT-3 0, 5 0 ATTGGGTCC AGACATGGCCCTAGTTG-3 0, and 5 0 -GTTCATGTTGACT TCCAAACACTTCTTC-3 0. The PCR reaction was performed with GoTaq flexi Polymerase (Promega, Madison, WI, Cell Counts and Statistical Analysis For each quantitative analysis, the mean 6 SEs of at least three independent experiments and statistical significance tests (t-test or analysis of variance (ANOVA) with post hoc Newman- Keuls) were calculated using a statistical package (Graphpad, Prism, Graphpad-Innotech Schönaich, Germany). To quantify the percentages of immunopositive cells in brain slices, positive cells were counted relative to total cell numbers as revealed by 4 0,6-diamidine-2 0 -phenylindole-dihydrochloride (DAPI) nuclear counterstain. Double positive cells were calculated as a percentage of total cells or as subpopulation of cells positive for one antigen. The total number of Olig2 immunopositive cells per coronal section was obtained by counting the number of Olig2 positive cells within a region of interest of 100 lm 2 and normalizing this number to the total surface of the lsez calculated after the analysis of the coronal sections with ImageJ.

3 Obernier, Simeonova, Fila et al Immunohistofluorescence. After i.p. injection (400 mg/kg body weight) of sodium-pentobarbital (Narcoren, Merial, Germany, and perfusion with a solution of 4% paraformaldehyde (PFA), brains were removed and fixed in 4% PFA at 4 C. After washes with phosphate buffered saline (PBS), brains were embedded in 4% low melt agarose. Coronal vibratome sections were cut at a thickness of 40 lm andthen processed for immunohistofluorescence as described previously [20]. DAPI (Roche Basel Switzerland, index.htm) was used for nuclear counterstain. Images were acquired using a laser scanning confocal microscope (TCS SP2 scanning head and inverted DMIRBE microscope, 40 oil immersion HCX PL APO objective, Leica confocal scan software; Leica, Mannheim, Germany, Immunocytofluorescence. Cells were fixed and processed for immunostaining as described previously [30]. To quantify the number of immunopositive cells, images were acquired using a Zeiss-Axiophot inverted microscope. For each antigen, cells were counted in four to seven visual fields (approximately cells per field). Antibodies. The following antibodies were used at the indicated dilution: sheep polyclonal to EGFR, 1:200 (Upstate, Upstate-Chemicon Millipore Billerica, MA, millipore.com/index.do); rabbit polyclonal to Ki67, 1:200 (Abcam, Cambridge, UK, to phosphohistone H3 (PH3), 1:500 (Upstate, Upstate-Chemicon Millipore Billerica, MA, to GFAP, 1:500 (DAKO, Dako Glostrup, Denmark, to Olig2, 1:500 (Chemicon, Chemicon Millipore Billerica, MA, and to cleaved Caspase-3, 1:400 (Cell Signaling, Invitrogen, Carlsbad, CA, mouse monoclonal to GFAP, 1:600 (Sigma-Aldrich, St. Louis, MO, rat monoclonal to NG2, 1:100 (a kind gift from J. Trotter), or mouse monoclonal to NG2, 1:200 (Chemicon, Chemicon Millipore, Billerica, MA, RESULTS Tlx Is Expressed in NSCs and TAPs It was previously shown that in the adult lsez, Tlx is expressed in GFAP þ NSCs. However, whether Tlx is also expressed in their progeny is still unclear. To address this issue, we have used flow cytometry to sort cells expressing high (EGFR high ) and low levels (EGFR low ) of EGFR. In both populations, NSCs and TAPs can be distinguished from preneuroblasts and neuroblasts by their ability to form multipotent and self-renewing clones. Moreover, we have previously shown that the first population is highly enriched in clonogenic cells and preneuroblasts; whereas, most EGFR low cells represent neuroblasts and only a small subset of these cells (approximately 1% 2%) display NSC properties [7]. As commercial antibodies are not available, we have investigated the expression of Tlx mrna within the sorted EGFR high and EGFR low cells isolated from the E18 ganglionic eminence (GE), the neonatal (P7), and the adult (6 8 weeks old) lsez of wild type (WT) mice (supporting information Fig. S1A). Reverse transcription PCR revealed the presence of Tlx transcripts in both cellular subsets at all ages analyzed (supporting information Fig. S1B) and qrt-pcr showed, at both postnatal ages, significantly higher levels of Tlx transcripts in EGFR high than in EGFR low cells (Fig. 1A). Confirming that they are enriched in TAPs, at both ages, EGFR high cells displayed the highest levels of Mash1 mrna (Fig. 1A). These differences in expression levels were also detected independently by affymetrix gene chip analysis of EGFR high and EGFR low cells isolated from the neonatal lsez, which revealed a 2.66 average fold increase of Tlx transcripts in EGFR high cells when compared with the age-matched EGFRlow population (K.O. and F.C., unpublished observation). As EGFR high cells include activated NSCs and TAPs [7, 30], we next investigated whether Tlx is expressed exclusively in the first or also in the latter subset. To this end, we sorted cells dissociated from the neonatal lsez on the basis of both EGFR and Prominin expression (Fig. 1B, 1C). This approach allows the isolation of Prominin þ /EGFR high activated NSCs and Prominin /EGFR high TAPs to a similar high degree of purity, as in both populations approximately 40% of EGFR high cells are clonogenic [30]. Instead, 4% of the neonatal Prominin þ /EGFR low cells display NSC activity, which represents more than a twofold increase when compared with agematched EGFR low and Prominin /EGFR low cells [30]. The size of the primary clones was not significantly affected by the antigenic characteristics of the cell of origin (supporting information Fig. S2B), indicating that, at this age, clonogenic cells have a similar proliferation potential. However, the analyses of the self-renewal and differentiation potential revealed differences among the clone groups (supporting information Fig. S2). In particular, around one fourth (20.5% % and 15.53% %, respectively) of the cells in Prominin þ / EGFR low and Prominin þ /EGFR high primary clones underwent secondary clone formation. In stark contrast, only 5.16% % of the cells from Prominin /EGFR high primary clones generated secondary clones (supporting information Fig. S2A), consistent with the fact that TAPs are less self-renewing than NSCs. qrt-pcr revealed the lowest levels of Tlx mrna in Prominin þ /EGFR low cells, likely reflecting the fact that ependymal cells, which represent the main cell type in this subset [7], express low Tlx levels. However, Tlx expression increased in activated NSCs and especially in TAPs (Fig. 1B). Moreover, within the four populations, there was an inverse correlation between Tlx and p21 transcript levels. As the latter is a potential target of Tlx repression [26], this observation also suggests that the expression of Tlx protein differs in the four populations. However, due to the lack of effective commercial antibodies against Tlx, we could not directly test this hypothesis. Thus, Tlx is not a NSC marker, as it is expressed at the highest levels within TAPs. The mirs mir-9 and let-7b can target the 3 0 untranslated region (UTR) of the Tlx mrna thereby destabilizing it [31, 32]. Therefore, we next quantified levels of mir-9 and let-7b in the four populations. Although both mirs were highly expressed in activated NSCs (mir and let-7b when compared with expression in neuroblasts), their expression was robustly downregulated in TAPs (mir and let-7b when compared with neuroblasts) (Fig. 2A), showing an inverse correlation between Tlx mrna and its negative regulators mir-9 and let-7b in Prominin þ /EGFRhigh activated NSCs and Prominin /EGFR high TAPs. It has been recently suggested that expression of Tlx and mir-9 are regulated by a negative feedback loop consistent with the existence of Tlx consensus binding sites in the mir- 9 locus and the observation of increased levels of pre-mir-9 in cells derived from adult Tlx null mice [31]. As individual mirs can affect stabilization and/or translation of a variety of transcripts, their upregulation in the absence of Tlx would lead to a broad range of effects. Therefore, we next compared the expression levels of both mature mir-9 and let-7b in cells

4 1418 Role of Tlx in Postnatal Neurogenesis Figure 1. Tlx is expressed in neural stem cells (NSCs) and transit-amplifying progenitors (TAPs). (A): Analysis of Mash1 and Tlx transcript levels on cells isolated by FACS from neonatal and adult subependymal zone of the lateral ventricle (lsez) according to epidermal growth factor receptor (EGFR) expression. Transcript levels are shown as fold induction in high levels of EGFR (EGFR high ) cells versus low EGFR levels (EGFR low ) cells. (B): Quantitative real time-polymerase chain reaction for Tlx and p21 transcript levels on cells isolated from the neonatal lsez by FACS according to Prominin and EGFR expression. For each population, mrna levels are shown as fold change relative to the Prominin / EGFR low population. Asterisks indicate a significant change from the preceding population. Values are the means of RQ from ddct 6 SEM. *, p <.05; **, p <.01; ***, p <.001; n 3 per group. (C): Scheme indicating antigenically distinct cell types. The populations of Prominin þ / EGFR low, Prominin þ /EGFR high and Prominin þ /EGFR high, and Prominin /EGFR low cells will be here referred to as primitive NSCs, activated NSCs TAPs, and neuroblasts, respectively. This reflects the enrichment in clonogenic cells in the first three populations and in more differentiated neuronal cells in the latter. Abbreviations: ddct, delta-delta threshold cycle; EGFR high, high levels of epidermal growth factor receptor; EGFR low, low epidermal growth factor receptor levels; FACS, fluorescence activated cell sorting; NSCs, neural stem cells; RQ, relative quantification value; TAPs, transit-amplifying progenitors.

5 Obernier, Simeonova, Fila et al Figure 2. MicroRNA (mir) expression pattern in neural stem cells and transit-amplifying progenitors. (A): Quantitative analysis of mir expression in cells isolated from the neonatal subependymal zone of the lateral ventricle (lsez) by FACS according to Prominin and epidermal growth factor receptor (EGFR) expression. For each population, mir levels are shown as fold change relative to the Prominin /low EGFR levels population. For comparative purposes, expression levels of Tlx are included as shown in Figure 1B. (B): Expression levels of mir-9 in Tlx / cells isolated from the neonatal lsez by FACS according to Prominin and EGFR expression levels relative to the WT counterpart. Values are the means of RQ from ddct 6 SEM. Asterisks indicate a significant change from the preceding population. *, p <.05; **, p <.01; ***, p <.001; n 3 per group. Abbreviations: ddct, delta-delta threshold cycle; EGFR high, high levels of epidermal growth factor receptor; EGFR low, low epidermal growth factor receptor levels; FACS, fluorescence activated cell sorting; RQ, relative quantification values; WT, wild type. isolated from the Tlx / and WT neonatal lsez. This analysis revealed that the expression of either mature mirs was not altered in the absence of Tlx (Fig. 2B and data not shown). Therefore, the effects observed in neonatal Tlx / mice described below are not a consequence of changes in the levels of these mirs. Lack of Tlx Impairs Proliferation in the Neonatal lsez Lack of Tlx causes a severe impairment of proliferation in the neurogenic regions of the adult murine telencephalon. However, the effect of Tlx absence in the neonatal niche is not known. To investigate this issue, we have quantified proliferation in the lsez of WT and Tlx / neonatal mice. First, we have analyzed EGFR expression in combination with either Ki67 or PH3, which recognize cycling and mitotic cells, respectively. Quantitative analysis revealed that the genotype did not affect EGFR expression in the neonatal lsez. Independent of EGFR expression, fewer Ki67 þ cells were present in the neonatal mutant lsez than in the WT counterpart (Fig. 3A 3C and supporting information Table S1). The genotype also affected mitosis. However, a statistically significant difference between the genotypes was only observed within the population of EGFR þ cells (Fig. 3B, 3C and supporting information Table. S1). Thus, consistent with the pattern of mrna expression described above, lack of Tlx has a greater impact on the proliferation of EGFR þ than EGFR progenitor cells. As this population includes activated NSCs and TAPs, these data indicate that absence of Tlx affects proliferation already in the neonatal niche. In line with this, the reduction in proliferation was accompanied by anatomical changes in the germinal epithelium. Analysis of DAPI stained coronal sections revealed a thinning in the lsez of neonatal Tlx / mice especially evident at the dorsolateral corner (Fig. 3D, 3E). Immunostaining of brain slices with Caspase-3 antibodies indicated no significant variation in the number of apoptotic cells between WT and mutant animals (supporting information Fig. S3B) because independent of the genotype, only rare Caspase-3 þ cells were observed. This is consistent with previous studies showing that absence of Tlx does not lead to increased apoptosis in the embryonic and adult brain [21, 26]. Taken together, these data indicate that when compared with WT animals, neonatal mice lacking Tlx display a decrease in proliferation and not an increase in cell death. Age-Dependent Effect of Tlx on Neural Progenitor Cell Proliferation Our previous analysis indicates that lack of Tlx impairs the proliferation of activated NSCs and TAPs in the neonatal lsez with a consequent reduction of the area of the germinal epithelium. To further investigate this issue, we next used clonal analysis to directly compare the capacity of WT and mutant progenitor cells, including NSCs and TAPs, to proliferate. First, we investigated the clonogenic ability of EGFR high and EGFR low cells prospectively isolated from the mutant and WT telencephalon at various ages. Adult cells were not included in this analysis as, independent of EGFR expression, they failed to proliferate in vitro (data not shown). Neonatal mutant EGFR low cells,

6 1420 Role of Tlx in Postnatal Neurogenesis Figure 3. Proliferation is impaired in the neonatal Tlx / subependymal zone of the lateral ventricle (lsez). Representative confocal photomicrographs of day 7 after birth (P7) WT (left panel) and Tlx / (right panel) coronal lsez sections immunostained with antibodies against epidermal growth factor receptor [red in (A), (B)] and Ki67 [green in (A)] or phosphohistone H3 (PH3) [green in (B)] with 4 0,6-diamidine phenylindole-dihydrochloride (DAPI) nuclear counterstain [blue in (A), (B)]. (C): Quantification of Ki67 þ and PH3 þ cells as percentage of DAPI in WT and Tlx / lsez (supporting information Table S1). (D): DAPI staining of coronal lsez sections from neonatal WT and Tlx / mice showing a reduction of the germinal area in the mutant animals. Quantification of the area of the dorsolateral corner is shown in (E). Scale bars ¼ 100 lm. Values are the means 6 SEM. Asterisks indicate significantly different from the WT counterpart. *, p <.05; **, p <.01; ***, p <.001. Abbreviations: DAPI, 4 0,6-diamidine-2 0 -phenylindole-dihydrochloride; EGFR, epidermal growth factor receptor; LV, lateral ventricle; PH3, phosphohistone H3; SEZ, subependymal zone; Str, striatum; WT, wild type. but not EGFR high cells, contained significantly less clone-forming cells than the WT counterpart (Fig. 4A, 4B shows percentage values normalized to WT; original values in supporting information Table S2). Instead, the genotype did not affect the clonogenic activity of cells isolated from the E18 GE (Fig. 4A, 4B), consistent with the fact that the effect of Tlx on cell proliferation is not stereotypical but age-dependent. Moreover, there was no significant difference in cell viability between WT and Tlx / mice as determined by propidium iodide exclusion (supporting information Fig. S3A). Taken together, these data show that during development, the first alteration in cell proliferation is detected within the population of EGFR low clonogenic NSCs. Treatment with FGF-2 promotes the transition of clonogenic cells from EGFR low to EGFR high [18 20, 29]. Therefore, we reasoned that if lack of Tlx affected the proliferative ability of EGFR low NSCs, the number and the clonogenic activity of EGFR high cells obtained from dissociated neonatal lsez at day in vitro 1 (DIV1), after overnight treatment with FGF-2, should be both reduced in mutant mice. Indeed, at DIV1, EGFR high cells derived from mutant mice were less clonogenic and fewer than in the WT counterpart (Fig. 4C, 4D shows percentage values normalized to WT; original values in supporting information Tables S3 and S4). Instead, at E18, the genotype affected the number but not the clonogenic activity of striatal EGFR high cells sorted at DIV1, suggesting that lack of Tlx hampers the activation of EGFR low clonogenic cells already before birth (Fig. 4C, 4D). Indeed, this analysis across different developmental ages revealed a progressive impairment of the transition from EGFR low to EGFRhigh cells in the absence of Tlx (Fig. 4D). Confirming this, we found that upon overnight treatment with FGF-2 mutant neonatal EGFR low cells, sorted after dissection (DIV0), gave rise to 50% less EGFR high cells than their WT counterpart (Fig. 4E shows percentage values normalized to WT; original values in supporting information Table S5). Thus, consistent with our previous observation that fewer EGFR þ cells undergo mitosis in this region, in the neonatal lsez only the clonogenic activity of EGFR low NSCs is affected by the loss

7 Obernier, Simeonova, Fila et al Figure 4. Proliferation and linage transition are impaired in Tlx / subependymal zone of the lateral ventricle (lsez) cells in vitro. Clonal analysis of high levels of epidermal growth factor receptor (EGFR high )(A, C) and low EGFR levels (EGFR low )(B) cells isolated by FACS from the day 18 of embryonic development (E18) striatum and the neonatal lsez of WT and Tlx / mice after dissection day in vitro 0 (DIV0) (A, B) and after overnight exposure to FGF-2 (DIV1) (C). (D): Quantification of the numbers of EGFR high cells in bulk cultures of cells dissociated from the E18 striatum and the neonatal and adult lsez and analyzed by FACS at DIV1. (E): Quantification of EGFR high cells obtained from EGFR low cells isolated at DIV0 and analyzed at DIV1 by FACS. Values represent the relative percentage of cells undergoing clone formation normalized to the WT and are shown as means 6 SEM. Asterisks indicate significantly different from the WT counterpart. *, p <.05; **, p <.01; ***, p <.001; n 3 per group. Abbreviations: DIV0/1, day in vitro 0/1; E18, day 10 of embryonic development; EGFR high, high levels of epidermal growth factor receptor; EGFR low, low epidermal growth factor receptor levels; FACS, fluorescence activated cell sorting; FGF-2, fibroblast growth factor 2; P7, day 7 after birth; WT, wild type. of Tlx as fewer of these cells can undergo activation and upregulate EGFR expression. Tlx Enables the Activation of NSCs in the Neonatal lsez To directly investigate NSC activation, we next analyzed the number of Prominin þ /EGFR low NSCs, Prominin þ /EGFR high activated NSCs, and Prominin /EGFR high TAPs isolated from the neonatal and adult lsez. Independent of the age, absence of Tlx affected the number of both activated NSCs and TAPs (supporting information Table S6; original values in supporting information Table S7). This effect was even greater in the adult lsez, where only a few Prominin /EGFR high cells were detected (supporting information Table S6), although it is unclear if they represent TAPs as they are not clonogenic (data not shown). The incidence of Prominin þ /EGFR low cells was also affected by the lack of Tlx, although in the opposite direction. When compared with the WT counterparts, mutant neonatal and adult mice displayed 2.5-fold and fivefold increase in the number of these cells, respectively. Also in the mutant neonatal and adult lsez, the number of Prominin / EGFR low cells was slightly but significantly increased when compared with the WT. As immunohistofluorescence had not revealed a significant effect of the genotype on EGFR in the neonatal lsez, these data indicate that lack of Tlx leads to a reduction of the expression of EGFR at the cell surface. The clonal activity of neonatal cells was also investigated. Despite the increase in their number, fewer mutant neonatal Prominin þ /EGFR low cells were clonogenic than the WT counterpart (Fig. 5A shows percentage values normalized to WT; original values in supporting information Table S7). The clonogenic activity of Prominin /EGFR low population was also reduced in Tlx / mice. These cells likely represent Lex/SSEA-1 þ cells, which are clonogenic [7, 33], do not express Prominin, and are capable to generate secondary clones (supporting information Fig. S2A). In contrast, lack of Tlx did not alter the incidence of clone-forming cells within the population of TAPs (Fig. 5A). However, when compared with the WT counterpart, the ability of mutant Prominin þ /EGFR high activated NSCs to form clones was significantly impaired. Most likely, this effect was not detected after sorting cells solely on the basis of EGFR expression, because the vast majority of EGFR high cells are Prominin /EGFR high TAPs whose clonal activity is not affected by Tlx deficiency. Taken together, these data are consistent with the hypothesis that lack of Tlx

8 1422 Role of Tlx in Postnatal Neurogenesis Figure 5. Absence of Tlx impairs neural stem cell activation. (A): Clonal analysis of the four populations isolated from the neonatal WT and Tlx / subependymal zone of the lateral ventricle by FACS. All data are normalized to WT and are shown as mean 6 SEM. (B): Fold change in p21 transcript levels in neonatal Tlx / cells isolated by FACS according to Prominin and epidermal growth factor receptor expression relative to transcript levels in the WT counterpart. Values are the means of RQ from ddct 6 SEM. Asterisks indicate significantly different from the WT counterpart. *, p <.05; **, p <.01; ***, p <.001; n 3 per group. Abbreviations: ddct, delta-delta threshold cycle; EGFR high, high levels of epidermal growth factor receptor; EGFR low, low epidermal growth factor receptor levels; FACS, fluorescence activated cell sorting; RQ, relative quantification values; WT, wild type. impairs NSC activation, which in turn leads to a reduction in the number of TAPs. They also show that Tlx is necessary for maintaining proliferation within the NSC compartment, whereas it does not affect the proliferative potential of TAPs. As Tlx represses p21 thereby promoting cell cycle progression, we next compared p21 mrna expression by qrt-pcr in the four cellular subsets isolated from the neonatal WT and mutant lsez. This analysis revealed that lack of Tlx affected p21 expression specifically in activated but not in Prominin þ / EGFR low NSCs (Fig. 5B). Thus, increased p21 expression may be responsible for the specific impairment of the clonogenic activity of activated NSCs and the consequent decrease in the generation of TAPs. Absence of Tlx Promotes Gliogenesis We next investigated the possibility that Tlx also regulates cell fate decision. As previous studies have indicated a strong decrease in neurogenesis already at P9 in Tlx mutant mice [27], we next quantified the expression of doublecortin (DCX) in coronal sections of the WT and mutant neonatal lsez. Underscoring the age-progressive impairment of neurogenesis consequent to the loss of Tlx, P7 mutant mice displayed only a nonsignificanttrendreductionindcx þ cells in the dorsolateral corner (WT 30.34% % vs. Tlx / 23.1% % DCXþ cells, p ¼.09 and data not shown). Instead, the number of GFAP þ cells was greatly increased in mutant neonatal mice (Fig. 6A and supporting information Fig. S4). Independent of the genotype, most GFAP þ cells within the germinal region had morphology of bipolar/tripolar astroglial progenitors and only rare cells, mostly localized outside of the lsez, displayed the stellate shape of astrocytes (Fig. 6A3, 6A4; arrows indicate bipolar/tripolar cells, arrowheads stellate morphology). However, in the mutant lsez fewer GFAP þ cells were also EGFR þ than in the WT counterpart, supporting our conclusion that Tlx regulates NSC activation (supporting information Fig. S4). We then analyzed the expression of NG2 and the transcription factor Olig2 to investigate oligodendrogenesis. Immunostaining revealed an increase in the expression of both antigens (Fig. 6 and supporting information Fig. S5). As their quantification is problematic on brain slices (supporting information Fig. S5), NG2 þ cells were analyzed by FACS after dissociation of the lsez and immunostaining with specific antibodies. This analysis also revealed an increase in the number of NG2 þ cells in mutant mice (WT 2.19% % vs. Tlx / 8.64% %; Fig. 6D). Moreover, as in the WT [7], also in Tlx / mice, all NG2 þ cells were EGFR low and nonclonogenic (data not shown). Quantitative analysis of the number of Olig2 þ cells both as percentage of DAPI (Fig. 6B, 6C) and per region of interest (WT Olig2 þ cells/mm 2 vs. Tlx / Olig2 þ cells/mm 2, p ¼.00008) on coronal sections showed an increase in the neonatal Tlx / lsez versus the WT counterpart. However, when the difference in the size of the germinal area was taken into account (WT mm 2 ; Tlx / mm 2, p ¼.00002; and Fig. 3E), then the number of Olig2 þ cells in WT and mutant animals was not significantly different (total Olig2 þ cells per coronal section: WT vs. Tlx / , p ¼.168). This likely reflects the decrease in proliferation and therefore in cell generation in the mutant niche. We next investigated the expression levels of GFAP and Olig2 transcripts by qrt-pcr in the four populations sorted on the basis of EGFR and Prominin expression (Fig. 7). In this analysis, we also investigated Dlx2 and Mash1, as they have been involved in neuronal differentiation. We have previously shown that in WT cells, Dlx2 and especially Mash1 transcripts are highly

9 Obernier, Simeonova, Fila et al Figure 6. Absence of Tlx leads to enhanced gliogenesis in vivo. Representative confocal photomicrographs of coronal subependymal zone of the lateral ventricle (lsez) sections from day 7 after birth (P7) WT and Tlx / mice immunostained for (A) glial fibrillary acidic protein (GFAP) (in green) and (B) Olig2 (in green) and 4 0,6-diamidine-2 0 -phenylindole-dihydrochloride (in blue), as nuclear counterstain. Magnifications (A1 A4) correspond to indicate boxes. In (A), arrows indicate elongated bipolar GFAP þ cells, arrowheads point out stellate GFAP þ cells. Scale bars ¼ 100 lm and 50 lm in magnified photomicrographs in (A) and 100 lm in(b). (C): Quantification of Olig2 þ cells shown in (B). (D): Quantification of neuroglia proteoglycan 2 (NG2 þ ) cells in Tlx / lsez cells normalized to WT revealed by FACS analysis. Values are the means 6 SEM. Asterisks indicate significantly different from the WT counterpart. *, p <.05; n 3 per group. Abbreviations: cc, corpus callosum; FACS, fluorescence activated cell sorting; GFAP, glial fibrillary acidic protein; LV, lateral ventricle; Str, striatum; WT, wild type. expressed in TAPs; whereas in comparison, activated NSCs express moderate and low levels of Mash1 and Dlx2, respectively [30]. Although we found that TAPs express elevated levels of Olig2, they do not express appreciable levels of GFAP transcripts in contrast to activated NSCs and Prominin þ /EGFR low cells (Obernier et al. unpublished observations). Direct comparison of the transcript levels in neonatal Tlx / cells and respective WT counterparts revealed a downregulation of Dlx2 expression across all populations (Fig. 7A). In contrast, lack of Tlx affected more specifically the expression of the other genes in the various populations. In particular, Mash1 levels were decreased specifically in activated NSCs (Fig. 7D), whereas the expression of Olig2 was upregulated in NSCs but not in TAPs (Fig. 7C). Finally, GFAP was overexpressed in both Prominin þ /EGFR low cells and Prominin /EGFR high TAPs (Fig. 7B), suggesting that Tlx regulates GFAP expression not only in NSCs but also in TAPs. Taken together, these data provide evidences that neuronal differentiation is already affected in activated NSCs as indicated by decreased Mash1 and Dlx2 and increased Olig2 expression. An increase in glial cells is also detected in mutant TAPs as indicated by increased GFAP and decreased Dlx2 transcript levels. DISCUSSION We here show that Tlx is expressed not only in NSCs but also in TAPs, and it differentially affects gene expression in the two populations. Tlx Is Expressed in Activated NSCs and TAPs Most effects of Tlx on NSC and TAP proliferation are cellautonomous underscoring the importance of establishing its expression pattern. The expression of Tlx mrna has been

10 1424 Role of Tlx in Postnatal Neurogenesis Figure 7. Absence of Tlx promotes gliogenesis and represses neurogenesis. Quantitative analysis of fold change in Dlx2 (A), glial fibrillary acidic protein (GFAP) (B), Olig2 (C), and Mash1 (D) expression levels in neonatal Tlx / cells isolated by FACS based on Prominin and epidermal growth factor receptor expression relative to the transcript levels in the WT counterpart. Values are the means of RQ from ddct 6 SEM. Asterisks indicate significantly different from the WT counterpart. *, p <.05; **, p <.01; ***, p <.001; n 3 per group. Abbreviations: ddct, delta-delta threshold cycle; EGFR high, high levels of epidermal growth factor receptor; EGFR low, low epidermal growth factor receptor levels; GFAP, glial fibrillary acidic protein; RQ, relative quantification values; WT, wild type. extensively investigated in the developing murine brain [22]. Although high levels of Tlx transcripts are also present in the adult brain, at this age the expression of Tlx has been analyzed only by indirect means. Using cultures of adult neural progenitor cells it has been shown that Tlx is expressed during proliferation and downregulated after induction of differentiation [21]. Our data support these previous findings as activated NSCs and TAPs represent the most proliferative compartments of the neural lineage. Expression of Tlx in the adult lsez in vivo has been investigated by monitoring Tlx transcriptional activity using a bacterial artificial chromosome (BAC)-based reporter construct [27]. This approach revealed Tlx transcription only in GFAP expressing cells within the lsez and the rostromigratory stream. This study detected no expression overlap between Tlx and EGFR, suggesting that Tlx is not expressed in activated NSCs. Such a restricted pattern of Tlx expression in the adult neural lineage contrasts with the one observed in the developing forebrain where Tlx is expressed at high levels both in ventricular and subventricular zone cells [26, 28]. The restriction of Tlx expression only to NSCs is also not consistent with the fact that similar levels of Tlx expression were found in the adult and embryonic

11 Obernier, Simeonova, Fila et al brain. As the number of NSCs decreases with age, such high levels of Tlx expression would be justified if adult NSCs expressed higher levels of Tlx than the embryonic counterpart. However, we found that at least during postnatal development, Tlx expression does not change significantly. Our analysis instead revealed that Tlx is expressed not only in neonatal activated NSCs but also in TAPs, which indeed display the highest levels of Tlx transcript. The discrepancy between our and the previous observation could be due to the fact that in the adult lsez, EGFR high cells expressing Tlx are all activated NSCs. However, in this case, it is unclear why in the previous study colocalization of EGFR and GFAP, at least in a subset of the cells expressing the reporter gene, was not detected. More likely, the difference between the previous analysis and ours reflects the complex regulation of Tlx expression, which may not be adequately reproduced by the BAC construct used to monitor Tlx expression in vivo. For example, this approach will detect only differences in transcriptional but not posttranscriptional regulation of Tlx expression [31, 32]. The expression of Tlx is negatively regulated posttranscriptionally by the mirs mir-9 and let-7b. Indeed, we found that levels of these mirs are significantly higher in activated NSCs than in TAPs, suggesting that the differences in the levels of Tlx transcripts between the two populations reflect a posttranscriptional downregulation of Tlx expression in activated NSCs. Thus, in light of the previous analysis of Tlx transcriptional activity [27], our data indicate that although its promoter is active only in the NSC compartment, Tlx is also expressed in TAPs. It has been proposed that Tlx and mir-9 reciprocally regulate their expression to ensure a rapid transition from proliferation to differentiation [31]. Supporting this conclusion, overexpression of mir-9 in cell cultures caused upregulation of p21 and reduced proliferation. However, whether mir-9 directly affects differentiation is unclear, as its overexpression accelerated NSC differentiation only after pharmacological induction of differentiation [31]. Indeed, we found that, when compared with TAPs, activated NSCs expressed more mir-9 and p21 and less Tlx. However, the expression of mir-9 did not increase with increasing stages of differentiation, as previously reported. Moreover, we did not find that lack of Tlx ledtoan increase in the expression of mature mir-9. Therefore, the significance of the feedback regulatory loop between mir-9 and Tlx in the regulation of postnatal neurogenesis remains elusive. Tlx Controls Activation of Postnatal NSCs We here provide evidence that Tlx is essential for the activation of NSCs. This conclusion is based on multiple evidences. First, we found a correlation between Tlx mrna levels and NSC activation. However, one caveat in this data is the difference in NSC enrichment between the populations of Prominin þ /EGFR low and Prominin þ /EGFR high cells. Thus, it is possible that the differences in Tlx expression between the two subsets reflect the fact that ependymal cells, which represent the majority of Prominin þ /EGFR low cells, do not express Tlx [27]. Second, our developmental analysis shows that the impairment of the transition between EGFR low and EGFR high cells is one of the earliest defects detected within the NSC compartment of mutant mice. Interestingly, this analysis revealed that despite the fact that in mutant embryos proliferation is already affected at early developmental stages, the proliferative ability of NSCs is not impaired before birth. This is consistent with previous observations showing that cell proliferation is specifically reduced in the embryonic subventricular zone but not in the ventricular zone [28]. Indeed, our findings show a progressive decrease in NSC proliferation in the absence of Tlx only in the postnatal germinal region, indicating that this process affects NSC proliferation only after birth. The strongest evidence that Tlx affects NSC activation is the increase in p21 expression and the impairment of proliferation only within the mutant NSC compartment and not in TAPs. As we also detected a decrease in the number of mitotic EGFR þ cells in the mutant lsez, it is likely that this impairment of NSC activation occurs also in vivo. Tlx Is Essential for the Differentiation Along the Neural Lineage Besides the proliferation, Tlx also affects the differentiation of neural progenitors. For example, during development, Tlx contributes to the establishment of the pallial-subpallial boundary [34]. Tlx also acts as a repressor of genes associated to astroglial differentiation [21]. Besides its role as a suppressor, Tlx also acts as an activator of gene transcription, for example, Tlx binds to the Mash1 promoter acting as transcriptional activator [24]. Indeed, we found that the levels of Mash1 transcript were lower in mutant mice, although only within the subset of activated NSCs. Also Dlx2 was downregulated within this population; however, this was observed not only in activated NSCs but also across the various populations, including neuroblasts. This is particularly relevant considering the specific role of Dlx2 in promoting neurogenesis in the postnatal niche [35]. Interestingly, although our analysis at P7 revealed not only a downregulation of Dlx2 but also a trend reduction in DCX þ cells, the number of neuroblasts is significantly decreased at P9 [27]. This is consistent with our previous findings that TAPs are giving rise to Dlx2 þ /DCX preneuroblasts and Dlx2 þ /DCX þ neuroblasts within 2 and 3 days, respectively [7]. Instead, at P7, we found an increase in the number of cells expressing oligoastroglial markers. As the genotype does not impact cell viability, these data indicate that lack of Tlx not only impairs the proliferation of progenitor cells but also leads to a prominence of cells displaying astro-oligodendroglial characteristics in the neonatal lsez. Consistent with this, we have found that sorted activated NSCs and TAPs display increased Olig2 and GFAP expression, respectively. This finding is particularly significant in the population of TAPs, where the percentage of clonogenic cells is not altered by the genotype, indicating that the differences in mrna levels are not a consequence of a change in the cellular composition of the populations isolated from WT and g mutant mice. However, as our data also show that fewer EGFR þ cells proliferate in vivo in the absence of Tlx, we cannot exclude the possibility of a selective mechanism that contributes to alter the differentiation of neural progenitor cells in the mutant lsvz. CONCLUSION Thus, Tlx is expressed in both NSCs and TAPs; in the NSCs, Tlx regulates activation, proliferation, and differentiation, while in TAPs, Tlx mainly affects differentiation. ACKNOWLEDGMENTS We thank Dr. J. Trotter for the NG2 antibody. This work was supported by Baden-Württemberg Stiftung. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conflicts of interest.