3938 RESEARCH ARTICLE DEVELOPMENT AND STEM CELLS
Development 139, 3938-3949 (2012) doi:10.1242/dev.081133 © 2012. Published by The Company of Biologists Ltd Sox1 marks an activated neural stem/progenitor cell in the hippocampus Monica Venere1,2,*, Young-Goo Han2,3,‡, Robert Bell2,4, Jun S. Song2,4, Arturo Alvarez-Buylla2,3 and Robert Blelloch1,2,§
SUMMARY The dentate gyrus of the hippocampus continues generating new neurons throughout life. These neurons originate from radial astrocytes within the subgranular zone (SGZ). Here, we find that Sox1, a member of the SoxB1 family of transcription factors, is expressed in a subset of radial astrocytes. Lineage tracing using Sox1-tTA;tetO-Cre;Rosa26 reporter mice shows that the Sox1- expressing cells represent an activated neural stem/progenitor population that gives rise to most if not all newly born granular neurons, as well as a small number of mature hilar astrocytes. Furthermore, a subpopulation of Sox1-marked cells have long-term neurogenic potential, producing new neurons 3 months after inactivation of tetracycline transactivator. Remarkably, after 8 weeks of labeling and a 12-week chase, as much as 44% of all granular neurons in the dentate gyrus were derived from Sox1 lineage- traced adult neural stem/progenitor cells. The fraction of Sox1-positive cells within the radial astrocyte population decreases with age, correlating with a decrease in neurogenesis. However, expression profiling shows that these cells are transcriptionally stable throughout the lifespan of the mouse. These results demonstrate that Sox1 is expressed in an activated stem/progenitor population whose numbers decrease with age while maintaining a stable molecular program.
KEY WORDS: Sox1, Neural stem cells, Hippocampus, Subgranular zone
INTRODUCTION The Sox transcription factors are central regulators of Neurogenesis continues in the adult brains of mammals in two neurodevelopment (Pevny and Placzek, 2005; Wegner and Stolt, regions: the subgranular zone (SGZ) of the dentate gyrus in the 2005; Favaro et al., 2009; Scott et al., 2010). They share homology hippocampus and along the lateral walls of the lateral ventricles in with the testis determining factor SRY through their HMG-box the ventricular-subventricular zone (V-SVZ) (Altman and Das, DNA-binding domain (Sinclair et al., 1990; Gubbay et al., 1990; 1965a; Altman and Das, 1965b; Kaplan and Bell, 1984; Cameron Denny et al., 1992; Coriat et al., 1993; Wright et al., 1993). et al., 1993; Lois and Alvarez-Buylla, 1994; Kempermann et al., Members of the SoxB1 subfamily, comprising of Sox1, Sox2 and 1997; Eriksson et al., 1998; Gage et al., 1998; Doetsch et al., 1999; Sox3, show distinct and overlapping expression patterns during Ming and Song, 2005). Putative neural stem cells (NSCs) in the embryogenesis (Uwanogho et al., 1995; Collignon et al., 1996; SGZ give rise to mature neurons of the granular layer, which have Pevny and Lovell-Badge, 1997; Rex et al., 1997; Streit et al., 1997; been suggested to be involved in learning and memory (Shors et Pevny et al., 1998; Wood and Episkopou, 1999). Sox1 is the al., 2001; Schmidt-Hieber et al., 2004; Tashiro et al., 2006). NSCs earliest known specific marker of the neuroectoderm lineage, being in the V-SVZ contribute to migrating neuroblasts along the rostral activated during gastrulation. Sox2 and Sox3 show broader migratory stream that go on to become interneurons within the expression patterns turning on at the pre-implantation and epiblast olfactory bulb (Luskin, 1993; Lois and Alvarez-Buylla, 1994; stages, respectively (Collignon et al., 1996; Pevny et al., 1998; Petreanu and Alvarez-Buylla, 2002; Carleton et al., 2003). Wood and Episkopou, 1999). The only non-neural role described Although much evidence exists to support the presence of a for SOX1 is in the developing eye lens, where SOX1 has been population of cells with stem/progenitor properties in the adult shown to regulate the -crystallin genes (Kamachi et al., 1998; brain, there is not a consistent understanding regarding the full Nishiguchi et al., 1998). Contradictory reports claim a role for potential and self-renewal properties of this cell population. SOX1 in neural lineage commitment and differentiation (Pevny et Furthermore, additional markers are needed to identify al., 1998; Kan et al., 2004; Ekonomou et al., 2005) versus stem/progenitor cells actively involved in neurogenesis. maintenance of the progenitor pool (Bylund et al., 2003; Zhao et al., 2004; Suter et al., 2009; Elkouris et al., 2011). A model reconciling both scenarios has been proposed whereby SOX1 plays 1Department of Urology, University of California, San Francisco, CA 94143, USA. an initial role in maintenance of division in the progenitor pool, but, 2The Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell upon continued expression, leads to neuronal differentiation (Kan 3 Research, University of California, San Francisco, CA 94143, USA. Department of et al., 2007). Loss of SOX1 is largely compensated by SOX2 and Neurosurgery, University of California, San Francisco, CA 94143, USA. 4Department of Epidemiology and Biostatistics, University of California, San Francisco, CA 94143, SOX3 during development, but leads to epilepsy and eventual USA. lethality (Wegner, 1999; Malas et al., 2003; Graham et al., 2003; Kan et al., 2007). *Present address: Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA In the adult, there have been reports that SOX1 is localized to ‡Present address: Department of Developmental Neurobiology, St. Jude Children’s the germinal regions as well as a putative stem cell pool in the Research Hospital, Memphis, TN 38105, USA postnatal cerebellum (Aubert et al., 2003; Pevny and Rao, 2003; §Author for correspondence ([email protected]) Barraud et al., 2005; Sottile et al., 2006; Kan et al., 2007). These
Accepted 3 August 2012 studies, however, did not fully characterize the cell type(s) positive DEVELOPMENT Sox1 NSCs of the hippocampus RESEARCH ARTICLE 3939 for SOX1 within these adult germinal regions. By contrast, SOX2 Immunocytochemistry and imaging expression has been well characterized in adult neurogenic zones Sections were postfixed for 15 minutes at room temperature in 4% PFA (Ellis et al., 2004; Ferri et al., 2004; Komitova and Eriksson, 2004; followed by PBS washes then incubated in blocking solution (5% goat Brazel et al., 2005; Episkopou, 2005; Sottile et al., 2006; Suh et al., serum, 0.1% Triton X-100 in PBS). Primary antibodies were diluted in 2007). SOX2 marks both radial astrocytes (also known as type 1 blocking solution and used as follows overnight at 4°C: GFP (chicken, 1:500, Aves Labs, Tigard, OR, USA), SOX1 (kind gift from Dr Yusuke cells) and early progenitor cells (also known as type 2a cells) Kamachi and Dr Hisato Kondoh, Osaka University, Japan), SOX2 (rabbit, within the adult dentate gyrus (Steiner et al., 2006). Radial 1:500, Abcam, Cambridge, MA, USA; mouse, 1:100, R&D Systems, astrocytes are positive for the astrocyte marker GFAP and are Minneapolis, MN, USA), GFAP (rabbit, 1:300, Dako, Carpinteria, CA, morphologically identified by having a cell body residing in the USA; mouse, 1:200, Millipore, Billerica, MA, USA), DCX (rabbit, 1:500, SGZ with a radial process extending into the granular layer. They Cell Signaling, Danvers, MA, USA), PSA-NCAM (mouse, 1:400, are well established as the relatively quiescent NSCs of the SGZ. Chemicon), TUJ1 (mouse, Covance, 1:500), NeuN (mouse, 1:500, Early progenitors represent a nonradial, intermediate proliferating Millipore) and BrdU (rat, Abcam, 1:200). All secondary antibodies were cell population within the subgranular zone (Yamaguchi et al., Alexa Fluor (1:500, Invitrogen, Grand Island, NY, USA) labeled with 488, 546 or 633, mixed in blocking solution and incubated for 2 hours at room 2000; Seri et al., 2001; Kempermann et al., 2003; Fukuda et al., temperature. DNA was stained using 4Ј,6-diamidino-2-phenylindole 2003; Seri et al., 2004; Encinas et al., 2011; Bonaguidi et al., 2011). (DAPI, 10 g/ml, Sigma, St Louis, MO, USA) or TO-PRO-3 iodide In this study, we investigate the nature of Sox1-positive cells in (Invitrogen) diluted in PBS for 5 minutes at room temperature. For SGZ of the adult hippocampus using real-time reporters and detection of BrdU, sequential IHC was performed. Following initial lineage tracing. We find that Sox1 marks a subset of the radial primary antibody staining, antibody antigen complexes were stabilized by astrocytes and early progenitors that have been activated to produce fixation using 4% PFA for 15 minutes at room temperature. The BrdU new neurons and astrocytes. antigen was retrieved by treatment of the tissue with 2 N HCl for 30 minutes at 37°C followed by PBS washes. Primary antibody staining was MATERIALS AND METHODS then performed for BrdU followed by secondary detection for all antibodies used. All images from stained brain sections were captured using a Zeiss Gene targeting or Leica confocal microscope and stacked images were produced using the The targeting vector to generate Sox1-tTA ES cells was prepared by corresponding system software then resized and false colored in Photoshop replacing the eGFP gene used to generate Sox1-GFP mice with the gene (Adobe, San Jose, CA, USA). expressing the tetracycline transactivator (tTA) as well as replacing the R loxP flanked hygromycin -thymidine kinase dual selection cassette with a X-gal staining neomycin resistance cassette also loxP flanked (Sox1-GFP construct kindly Sections were postfixed with 4% PFA for 15 minutes at room temperature. provided by Austin Smith, University of Cambridge, Cambridge, UK). They were then washed twice in 100 mM phosphate buffer (pH 7.4), 2 mM Sox1 knockout ES cells were generated by electroporation of SfiI linearized MgCl2 and 5 mM EGTA, followed by a wash in 100 mM phosphate buffer vector into V6.5 ES cells. Targeted clones were selected in neomycin (0.25 (pH 7.4), 2 mM MgCl2, 0.01% sodium desoxycholate and 0.02% NP40. mg/ml) and verified by Southern analysis with flanking 5Ј and 3Ј probes. Sections were then stained in X-gal staining solution [5 mM K3Fe(CN)6, The expected band size with the 5Ј probe is 10 kb for the wild-type locus 5 mM K4Fe(CN)6, 1 mg/ml X-gal] at 37°C overnight. Sections were then and 6.5 kb for the targeted locus using XbaI for genomic DNA digestion. mounted and imaged using an Olympus upright microscope. The expected band size with the 3Ј probe is 12 kb for the wild-type locus and 6.5 kb for the targeted vector using EcoRI for genomic DNA digestion. Cell counting/quantification The selection cassette was removed by transiently transfecting a Cre At least three coronal sections representing more ventral, medial or dorsal recombinase expression vector. Clones that had undergone excision were areas of the dentate gyrus were stained with the appropriate antibodies and verified by Southern as well as negative selection on G418. The expected confocal z-stacks were taken of the dentate gyrus from each hemisphere ϫ band size with the 3Ј probe is 8.8 kb for targeted locus with the selection using the 63 objective. Cells positive for each antibody were counted first in individual channels followed by counting colocalization in the merged cassette and 7.1 kb for targeted locus with the selection cassette removed image. Colocalization was then verified in the serial scans comprising the by Cre using NotI for genomic DNA digestion. One clone was injected into 3D projection of the z-stack. P-values were calculated using a two-tailed blastocysts and germline chimeras were identified. Sox1-tTA mice were unpaired t-test. outcrossed into a C57BL/6 background and maintained in a heterozygous state. Dissociation, FACS, RNA preparation and profiling Hippocampi were isolated from Sox1-GFP mice at 5-7 months (n4) or 1 Animals and tissue processing year 9 months (n4) using a Leica dissecting scope. Cells were All animals were maintained according to institutional regulations. LC-1, immediately dissociated using papain (Worthington Biochemical R26R, R26eYFP and Z/EG mice were previously published (Soriano, Corporation. Lakewood, NJ, USA) and sorted for the GFP high population 1999; Novak et al., 2000; Srinivas et al., 2001; Schönig et al., 2002). All using a FACSAria Flow Cytometer (BD Biosciences, San Diego, CA, crosses involving Sox1-tTA and LC-1 mice were fed doxycycline (20 mg/l) USA). RNA was harvested using the Arcturus PicoPure RNA isolation kit in their drinking water and experimental mice were maintained on (Life Technologies, Carlsbad, CA, USA) and the quality and quantity of doxycycline until 6 weeks of age. Long-term BrdU labeling was achieved each sample was checked using Agilent BioAnalyzer (Santa Clara, CA, by introducing BrdU in the drinking water (0.8 mg/ml) for 1 week prior to USA) and Nanodrop spectrophotometer (Wilmington, DE, USA). Samples sacrificing the mice. BrdU pulse labeling was accomplished with a single were amplified using the NuGEN FFPE kit (San Carlos, CA, USA) and IP injection of BrdU (0.05 mg/g) 1 hour before sacrificing the animal. cDNA was prepared using an oligo(dT)-T7 RNA polymerase primer before Genotypes were confirmed by PCR using genomic DNA prepared from tail biotinylation by in vitro transcription, partial hydrolysis and final clippings. For tissue collection, mice were deeply anesthetized and hybridization to the Affymetrix (Santa Clara, CA, USA) GeneChip Mouse perfused first with phosphate-buffered saline (PBS) followed by 4% Gene 1.0 ST Array (Gladstone Genomics Core). Arrays were normalized paraformaldehyde (PFA). Brains were postfixed overnight in 4% PFA at using Robust Multichip Average (RMA) and the latest probe map to MM9 4°C, washed in PBS, cryoprotected in 30% sucrose, then embedded in RefSeq genes (Irizarry et al., 2003; Dai et al., 2005). To detect potential optimal cutting temperature compound (OCT) and stored at –80°C. outlier arrays, the distributions of fold changes between all possible pairs Coronal sections were cut using a Leica cryostat at 12 m directly onto within each group were analyzed. One sample from a young mouse was slides and stored at –80°C or as 50 m floating sections into PBS and found to have a skewed distribution when compared with other biological stored at 4°C. replicate samples and was removed from further analysis. The statistical DEVELOPMENT 3940 RESEARCH ARTICLE Development 139 (21)
Fig. 1. Sox1 is expressed in a subset of putative NSCs of the SGZ. Sox1-GFP mice were sacrificed at 4 months of age and brains were harvested and sectioned into 50m slices for antibody staining and imaging by confocal microscopy. (A)GFP (green), Sox1 (red) and DNA (ToPro3, blue) staining of the dentate gyrus. (B)GFP (green), Sox1 (red) and DNA (ToPro3, blue) staining of a representative radial astrocyte in the SGZ. (C)GFP (green), Sox2 (red) and GFAP (blue) staining of the dentate gyrus. Arrowheads mark cells where all three antibodies colocalize. Arrows mark cells where Sox2 and GFP colocalize. (D)GFP (green), GFAP (red) and DNA (ToPro3, blue) staining of the dentate gyrus. Arrowheads mark radial astrocytes positive for GFP and GFAP. Arrow marks radial process that are only GFAP positive. (E)Quantification of Sox1-GFP cells or anti-SOX1 cells that are GFAP positive radial (blue bar), Sox2- positive non-radial (red bar) or uncharacterized (gray bar) in the dentate gyrus (n3). (F)Quantification of the percentage of all GFAP-positive radial astrocytes that are also GFP (n3, each circle represents an individual Sox1-GFP mouse) or Sox1 (n5, each triangle represents an individual Sox1-tTA mouse) positive in the dentate gyrus. (G)Quantification of the percentage of Sox2-positive non- radial cells that are also GFP positive (n3, each circle represents an individual Sox1-GFP mouse) or anti- SOX1 positive (n3, each triangle represents an individual Sox1-tTA mouse) in the dentate gyrus. Scale bars: 10m. NS, no statistical significance. Averages of 200 cells per Sox1-GFP mouse and 300 cells per Sox1-tTA mouse were counted. Black bars indicate average; error bars indicate s.d. package R was used to perform average linkage hierarchical clustering of astrocytes (GFAP), early progenitor cells (SOX2 positive non- the remaining samples. Significance analysis of microarrays (SAM) was radial cells), late progenitor/neuroblasts (PSA-NCAM, DCX) performed to determine significant differentially expressed genes between and neurons (TUJ1, NeuN). For SOX1, we used both an the old and young mice with a false discovery rate cutoff of 5% (Tusher et antibody to endogenous SOX1 and a Sox1-GFP reporter mouse al., 2001). (Aubert et al., 2003). SOX1 and Sox1-GFP staining overlapped both in the dentate gyrus and V-SVZ (Fig. 1A,B; supplementary RESULTS material Fig. S1A,B). Specificity of the SOX1 antibody was SOX1 is expressed in a subset of radial astrocytes confirmed by the lack of staining in Sox1-null mice (Sox1-tTA and early progenitor cells in the SGZ of the homozygous) (supplementary material Fig. S2A,B). As Sox1- hippocampus GFP served as a more robust cellular marker, we used this SOX1 is found in the neurogenic zones of the adult mouse brain reporter in most experiments. In the V-SVZ, Sox1-GFP only (Aubert et al., 2003; Barraud et al., 2005; Kan et al., 2007). To colocalized with a marker for late progenitors (PSA-NCAM), determine the specific cell types expressing SOX1 within these although we cannot rule out rare GFAP-positive cells
zones, we performed co-staining with markers for radial (supplementary material Fig. S1C). By contrast, in the dentate DEVELOPMENT Sox1 NSCs of the hippocampus RESEARCH ARTICLE 3941
Fig. 2. Production of a Sox1 driven inducible mouse model. (A)The genomic locus, targeting vector, targeted locus and targeted locus after Cre treatment to remove the selection cassette are shown. The Sox1 open reading frame (ORF) is shown as a red box. The 5Ј and 3Ј probes for Southern blotting are shown as black bars. Restriction sites are represented by X (XbaI), E (EcoRI) and N (NotI). Expected sizes of fragments resulting from the digest of the genomic locus are represented below (X to X10 kb, E to E12 kb, N to N 8.2 kb). The arms for homologous recombination are represented by the regions between the vertical lines. The knock-in construct consists of: the tetracycline trasactivator gene (tTA, orange box), polyA sequence (pA, purple boxes), lox sequence (black triangles) and pGK-neo gene (blue and yellow boxes). The targeted locus is shown with the resulting restriction fragment size changes represented below (X to X5 kb, E to E6.5 kb, N to N8.8 kb). The targeted locus following Cre treatment is shown with the resulting restriction fragment size change for NotI shown below (N to N7.1 kb). (B)Representative Southern blot of genomic DNA from a Sox1 wild-type, a Sox1-tTA heterozygous and a Sox1-tTA homozygous mouse digested with XbaI and probed with the 5Ј probe. (C)Diagram of the alleles used for lineage tracing of Sox1 cells. Sox1-tTA; LC-1; reporter mice are either kept in the presence of doxycycline to silence the system or removed from doxycycline to allow for system activation. In the absence of doxycycline, tTA can act on the bi-directional tet-responsive promoter at the LC-1 transgenic locus (Ptetbi-1). This allows for expression of Cre as well as luciferase (luc) (red boxes). Cre can then act on loxP sites within the reporter alleles. The R26R reporter will then express -gal (blue box), the Z/EG reporter will express eGFP (green box) and the R26eYFP reporter will express eYFP (green box). The promoter for each of the reporters is shown. (D)E17.5 mice were harvested from mothers
on dox (left embryo) or not on dox (right embryo) and stained with Eosin (pink) and X-gal (blue). DEVELOPMENT 3942 RESEARCH ARTICLE Development 139 (21) gyrus, Sox1-GFP did not co-stain with markers of late X-gal as expected (Fig. 2D, right). The system appeared to be progenitors/neuroblasts (PSA-NCAM or DCX) or neurons (Tuj1 specific to the neural tissue, although expression in small subsets or NeuN), with the exception of rare dim GFP-positive late of cells in other tissues could not be ruled out (Fig. 2D, right and progenitors (data not shown), which probably reflects GFP data not shown). The introduction of doxycycline into the drinking perdurance, as similar cells were not seen with the SOX1 water of mothers effectively silenced the tTA system, as shown by antibody. Co-staining with GFAP and SOX2 identified two the lack of reporter expression in similarly aged embryos (Fig. 2D, independent SOX1 populations in the dentate gyrus: (1) GFAP- left). positive, Sox2-positive radial astrocytes (Fig. 1C,D; To trace the lineage of Sox1-expressing cells in the hippocampus supplementary material Fig. S2C); and (2) SOX2 positive non- of adult mice, triple transgenic mice were kept on doxycycline radial cells (Fig. 1C). For simplicity, we call the former GFAP- throughout embryonic development and postnatally until 6 weeks positive radial astrocytes and the latter SOX2-positive non-radial of age at which time doxycycline was removed to activate tTA and cells throughout the remainder of the text. From an average of remove the lox-stop-lox cassette of the reporters. Mice were kept 200 GFP-positive cells counted per Sox1-GFP mouse, 35.30% off doxycycline for 8 weeks before being sacrificed for analysis (±3.62%) were GFAP-positive radial astrocytes and 30.20% (Fig. 3A). X-gal staining of sections from the hippocampus of mice (±1.99%) were SOX2-positive non-radial cells (Fig. 1E). From kept off doxycycline for 8 weeks showed a distinct pattern of an average of 300 SOX1 antibody-positive cells per Sox1-tTA clusters of cells extending from the SGZ into the granular layer of heterozygous mouse, 23.20% (±0.56%) were GFAP-positive the dentate gyrus consistent with the Sox1-expressing cells giving radial astrocytes and 37.00% (±1.61%) were SOX2-positive non- rise to neuroblasts and granular neurons (Fig. 3B). Two additional radial cells (Fig. 1E). Notably, unlike SOX1, SOX2 antibody reporter systems were crossed to the Sox1-tTA; LC-1 mice: a also stained astrocytes outside the SGZ zone (Fig. 1C). The CAGGS;lox-gal-stop-lox;GFP (Z/EG) and a Rosa26;lox-stop- remaining Sox1-GFP and SOX1-positive cells did not colocalize lox;eYFP (R26R-eYFP) reporter (Fig. 2C) (Novak et al., 2000; with any of the markers used in these studies and may represent Srinivas et al., 2001). Both systems produced a similar pattern of SOX1 positivity during a transition between cell types or a labeled cells into the granular layer as seen with the R26R system distinct, unidentified, population of cells within the SGZ. (Fig. 3C,D). Importantly, no reporter activity was seen in control Evaluation of the number of SOX1-positive cells within the mice kept on doxycycline for the 8-week labeling period radial astrocytes and non-radial cells revealed that SOX1 was (supplementary material Fig. S3). As the GFP and YFP reporters expressed in only a subset of each of the two cell populations. provided better resolution at the cellular level, they were used in Sox1-GFP and SOX1 antibody marked 39.29% (±7.06%) and co-staining experiments. 34.62% (±4.28%), respectively, of the GFAP positive radial To determine which cell types were being marked in the astrocytes (Fig. 1F). Sox1-GFP and SOX1 marked 42.36% lineage tracing experiments, we co-stained sections from the (±3.72%) and 38.85% (±2.10%), respectively, of the SOX2- hippocampus of triple transgenic mice with anti-GFP and positive non-radial cells (Fig. 1G). BrdU incorporation studies markers for late progenitors/neuroblasts, neurons and astrocytes. supported the more proliferative nature of the Sox1-GFP, SOX2- The mice carrying the Z/EG reporter showed GFP stained PSA- positive non-radial cells (supplementary material Fig. S2D). NCAM late progenitors, DCX neuroblasts, TUJ1 immature Therefore, SOX1 specifically marks two populations of cells that neurons and NeuN mature granular neurons of the dentate gyrus have anatomical, immunohistochemical and morphological (Fig. 3E-H; supplementary material Fig. S4). However, the Z/EG patterns consistent with previously described putative stem cells reporter did not show GFP staining in the radial astrocytes (data and early progenitors. not shown), even though Sox1-GFP was expressed in a subset of these cells (Fig. 1). We considered two possibilities: (1) Sox1 is SOX1-positive cells of the SGZ produce granular expressed in a subset of radial astrocytes that are already neurons and hilar astrocytes committed to the neuroblast lineage and have transformed into To determine the fate of SOX1-positive cells in the adult SGZ, we this lineage by the time we analyzed the mice; and/or (2) the performed lineage-tracing experiments. Specifically, we developed Z/EG reporter is poorly expressed in the Sox1-positive radial an inducible system based on the tetracycline transactivator (tTA) astrocytes. To test the later possibility, we examined mice (Fig. 2A). The inducible system was crucial as Sox1 is expressed carrying the R26R-eYFP reporter instead of Z/EG. Similar to throughout the neuroectoderm during embryogenesis; therefore, in Z/EG mice, the R26R-eYFP was activated and expressed order to trace Sox1-derived cells in the adult mouse, it was throughout neuronal differentiation (Fig. 3I-L; supplementary essential to keep the system off during this earlier developmental material Fig. S5). In contrast to the Z/EG reporter, the R26R- time period. Using the Sox1-GFP targeting construct, a tTA cDNA eYFP reporter was also expressed in a subset of GFAP-positive was exchanged for GFP (Fig. 2A) so that tTA expression would radial astrocytes and SOX2-positive non-radial SGZ cells (Fig. recapitulate Sox1-GFP expression. Targeted embryonic stem cell 4A,B). A 1-week pulse of BrdU showed that these cells as well clones were confirmed by Southern blotting (Fig. 2B) and used to as PSA-NCAM-positive late progenitors were actively cycling produce Sox1-tTA transgenic mice. To initially test effectiveness of (Fig. 4C; supplementary material Fig. S6). Non-radial GFAP- the Sox1-tTA system, the mice were crossed to mice carrying a tetO positive astrocytes within the hilar region of the dentate gyrus promoter driving Cre (LC-1) and a Rosa26;lox-stop-lox;lacZ also expressed the reporter (Fig. 4D). These cells, which had reporter (R26R) (Soriano, 1999; Schönig et al., 2002). In the triple morphology consistent with mature parenchymal astrocytes, did transgenic mice (Sox1-tTA; LC-1; R26R), the tTA drives not express Sox1-GFP or stain positive with the SOX1 antibody. expression of Cre, resulting in removal of the lox-stop-lox cassette Therefore, we infer they were derived from the Sox1-expressing hence permanently activating the lacZ gene (Fig. 2C). Therefore, progenitors. Together, these lineage-tracing experiments show all Sox1-expressing cells along with downstream progeny should that Sox1-expressing cells, including radial astrocytes and non- be marked. Indeed, in the absence of doxycycline, the entire radial early progenitors, give rise to both granular neurons and
developing nervous system of E17.5 embryos stained positively for mature astrocytes. DEVELOPMENT Sox1 NSCs of the hippocampus RESEARCH ARTICLE 3943
Fig. 3. Sox1-expressing cells give rise to new neurons. (A)Schematic of the timeline for lineage-tracing experiments. Sox1-tTA; LC-1; reporter mice were taken off doxycycline at 6 weeks of age (system on) for 8 weeks then sacrificed at 14 weeks of age, except for R26R mice which were kept off doxycyline for 26 weeks. (B)X-gal staining of a 50m section of the dentate gyrus from an 8-month-old Sox1-tTA; LC-1; R26R mouse. (C)GFP (green) and DNA (ToPro3, blue) staining of a 50m section of the dentate gyrus from a 3.5-month-old Sox1-tTA; LC-1; Z/EG mouse. (D)GFP (green) and DNA (ToPro3, blue) staining of a 50m section of the dentate gyrus from a 3.5- month-old Sox1-tTA; LC-1; R26eYFP mouse. (E-H)GFP (green), DCX (red) and DNA (Topro3, blue) staining (E); GFP (green), PSA- NCAM (red) and DNA (Topro3, blue) staining (F); GFP (green), Tuj1 (red) and DNA (Topro3, blue) staining (G); and GFP (green) and NeuN (red) staining (H) of a 50m section of the dentate gyrus from a 3.5-month-old Sox1-tTA; LC-1; Z/EG mouse. (I-L)GFP (green), DCX (red) and DNA (Topro3, blue) staining (I); GFP (green), PSA-NCAM (red) and DNA (Topro3, blue) staining (J); GFP (green), Tuj1 (red) and DNA (Topro3, blue) staining (K); and GFP (green) and NeuN (red) (L) staining of a 50m section of the dentate gyrus from a 3.5-month-old Sox1- tTA; LC-1; R26eYFP mouse. All mice were taken off of doxycycline at 1.5 months. Arrowheads in E-L indicate cells that are double positive for GFP and described marker. Arrows in H and L indicate cells marked only by GFP. Scale bars: 10m.
SOX1 marks cells with long-term capacity to distribution of GFP positive, lineage-traced cells at the 8-week produce new neurons pulse versus 12-week chase timepoints showed: GFAP-positive Although activation of Sox1-tTA for 8 weeks showed that Sox1- radial astrocytes [4.96% (±0.83%) versus 3.41% (±0.68%)], SOX2- expressing cells could give rise to granular neurons and mature positive non-radial cells [20.33% (±5.22%) versus 8.40% astrocytes, it was unclear whether the lineage marked radial (±0.81%)], DCX-positive cells [19.93% (±5.45%) versus 1.56% astrocytes were a stable population within the stem cell (±0.51%)] and NeuN positive granular neurons [5.65% (±0.91%) compartment that could give rise to new neurons at a later time versus 25.38% (±5.39%)] (Fig. 5B). Overall, this distribution of point. To test this possibility, we took mice that had been off Sox1-traced cells showed a shift from the progenitor to the mature doxycycline for 8 weeks (pulse; tTA system on) and put them back neuron pool during the chase period, although a population of on doxycycline for 12 weeks (chase; tTA system off) (Fig. 5A). labeled radial astrocytes and neuroblasts remained, consistent with This enabled the initial marking of Sox1-expressing cells followed ongoing neurogenesis from cells marked 12 weeks earlier. by a 12-week chase. Marked cells at the end of the 12-week chase Next, we compared the fraction of GFAP-positive radial represent cells or the progeny of cells that had expressed Sox1 at astrocytes, SOX2-positive non-radial cells, DCX neuroblasts and some point during the 8-week pulse of tTA activity. If Sox1 marks NeuN neurons marked by GFP at the end of the 8-week pulse a stable population of stem/progenitor cells, there should be versus after the 12-week chase (Fig. 5C). The percentage of ongoing reporter expression in these cells as well as downstream GFAP-positive radial astrocytes decreased [24.10% (±4.81%)
neuroblasts at the end of the chase. Quantification of the versus 10.54% (±1.74%)] whereas the Sox2-positive non-radial DEVELOPMENT 3944 RESEARCH ARTICLE Development 139 (21)
Fig. 4. Sox1 lineage tracing marks a subset of putative stem cells, as well as newly born neuroblasts, granule neurons and astrocytes. Sox1-tTA; LC-1; R26eYFP mice were sacrificed at 3.5 months of age and brains were harvested and sectioned into 50m slices for antibody staining and imaging by confocal microscopy. (A)GFAP (red), GFP (green) and Sox1 (blue) staining of the dentate gyrus. (B)Sox2 (red), GFP (green) and DNA (ToPro3, blue) staining of the dentate gyrus. Note that grainy GFP staining above Sox2- positive cells is background. (C)GFAP (red), GFP (green) and BrdU (blue) staining of the dentate gyrus. (D)GFAP (red), GFP (green) and DNA (ToPro3, blue) staining of an astrocyte within the hilus. Arrowheads mark cells that are positive for all three antibodies. All mice were taken off of doxycycline at 1.5 months. Scale bars: 10m.
cells remained stable [9.65% (±1.56%) versus 9.33% (±1.12)]. Following the 12-week chase, most neuroblasts were not marked By contrast, the percentage of DCX-positive cells dropped by lineage tracing and hence were not arising from the Sox1- dramatically [75.12% (±10.25%) versus 12.80% (±5.46%)], labeled radial astrocytes (Fig. 5C). This finding may reflect a cyclic while the percentage of NeuN-positive granular neurons expression of Sox1. That is, most of the radial astrocytes that were increased [18.87% (±2.67%) versus 43.96% (±6.33%)]. BrdU or marked by Sox1 expression during the 8-week pulse have since PCNA staining confirmed proliferation of the Sox1-marked cells silenced the gene and entered a quiescent state. Meanwhile, a new following the 12-week chase (supplementary material Fig. S7). population of Sox1-expressing cells gives rise to most of the newly These pulse-chase experiments give rise to several conclusions. born neuroblasts. To test this hypothesis, we evaluated SOX1 First, the high percentage of marked neuroblasts at the end of the protein expression, using the anti-SOX1 antibody, within the 8-week pulse shows that most newly born neurons arise from lineage-traced (GFP-positive) GFAP-positive radial astroyctes after Sox1-expressing cells. Second, the decrease in marked radial the 8-week pulse and the 12-week chase (Fig. 5D). Following the astrocytes after the 12-week chase suggests that some of these 8-week pulse, only 1.23% (±0.26%) of the marked (GFP-positive) presumptive stem cells were actually committed progenitors or radial astrocytes were anti-SOX1 positive. This finding suggests went on to die. Third, the continuing production of neural that Sox1 expression is highly transitory where most of the radial progenitors after the 12-week chase is consistent with a number astrocytes that were labeled during the 8 weeks had already of the marked radial astrocytes having long-term neurogenic silenced Sox1 by the end of the pulse. Consistent with this
potential. conclusion, following the 12-week chase, only rare Sox1-traced DEVELOPMENT Sox1 NSCs of the hippocampus RESEARCH ARTICLE 3945
Fig. 5. Sox1-marked cells within the stem cell compartment are long lived and continue to give rise to new neurons. (A)Schematic of the timeline for back-on-doxycycline experiments. Sox1-tTA; LC-1; R26eYFP mice were taken off doxycycline at 6 weeks of age for 8 weeks (pulse; system on). They were then put back on doxycycline at 14 weeks of age for 12 weeks (chase; system silenced) and sacrificed at 26 weeks of age. (B)Quantification of GFP-positive lineage-traced cells that are radial GFAP positive, Sox2 non-radial positive, DCX positive or NeuN positive in mice kept off doxycycline (pulse) for 8 weeks (n4, each black circle represents an individual mouse) or put back on doxycycline (chase) for 12 weeks (n4, each red circle represents an individual mouse). Black bar represents the average. (C)Quantification of the percentage of all GFAP-positive radial astrocytes, Sox2-positive non-radial cells, Dcx-positive neuroblasts or NeuN-positive neurons that are also GFP positive in mice kept off doxycycline (pulse) for 8 weeks (n4, each black circle represents an individual mouse) or put back on doxycycline (chase) for 12 weeks (n4, each red circle represents an individual mouse). Black bar represents the average. (D)Quantification of the percentage of GFAP positive, GFP-positive radial astrocytes that are also Sox1 positive in mice kept off doxycycline (pulse) for 8 weeks (n3, each black circle represents an individual mouse) or put back on doxycycline (chase) for 12 weeks (n3, each red circle represents an individual mouse). Black bars represent the average; error bars indicate s.d. An average of 200 cells were counted per mouse. NS, no statistical significance; *P<0.05, **P<0.01. cells [0.07% (±0.07%)] were still expressing Sox1. The vast fraction of SOX1-positive cells decreased from 26.23% (±2.76%) majority of anti-SOX1-positive cells at this time point were not at 2.5 months to 21.48% (±2.39%) at 6 months to 10.75% expressing GFP and, therefore, had activated Sox1 in previously (±5.59%) at 12 months (Fig. 6A). Next, we asked whether the Sox1-negative cells. molecular constitution of the Sox1 population itself changed with age. To test this proposition, Sox1-GFP cells were sorted from SOX1-positive cells of the SGZ decrease in number dissociated hippocampi from young (4-7 month) and old (21 but are molecularly stable during aging month) mice. Purified mRNA from these cells was analyzed on It has been proposed that the molecular constitution of NSCs microarrays. Very few genes were altered as defined by a false changes with aging resulting in a decreased regenerative capacity discovery rate of less than 5% (Fig. 6B,C). Only eight probe sets (for review, see Galvan and Jin, 2007). However, considering the representing five genes were significantly up and 7 probe sets heterogeneity that we uncovered within the stem cell pool, the representing 5 genes were significantly down (Fig. 6C). Cluster change with aging could be due to changes in cellular composition analysis was unable to separate the young from old mice showing of the germinal niche and/or cell-autonomous changes in gene that their molecular constitutions were almost identical (Fig. 6D). expression within progenitors. To test these alternatives, we asked P16 and HMGA2, which have previously been shown to increase whether the fraction of radial astrocytes that is SOX1 positive and decrease, respectively, with age in the V-SVZ (Molofsky et al.,
changes with age. Indeed, within the radial astrocyte pool, the 2006), did not show significant changes at the transcript level in DEVELOPMENT 3946 RESEARCH ARTICLE Development 139 (21)
Fig. 6. The fraction of Sox1-positive cells within the stem cell compartment decreases with age, but is molecularly stable. (A)C57BL/6 mice were sacrificed at 2.5 months, 6 months or 12 months of age and brains were harvested and sectioned into 50m slices for antibody staining with GFAP and Sox1. Confocal microscopy was used for quantification of the percentage of all GFAP-positive radial astrocytes that are also Sox1 positive (n3 for each time point; each circle represents an individual mouse). Black bar represents the average. (B)SAM analysis comparing three younger mice (4-7 month old versus 21 month old). Blue dots represent downregulated genes and red dots represent upregulated genes with FDR<5. (C)Fold difference (log2) of mean expression values of individual genes, old versus young. Red and blue dots as in A. List of differential genes with FDR<5 listed. (D)Tree diagram of clusteral analysis showing relatedness of individual mice. Note that old and young mice cannot be differentiated, consistent with the very similar expression patterns. (E)Expression levels of p16 and HMGA2. the SOX1-positive cells of the SGZ (Fig. 6E). Therefore, although controversy stems from a lack of markers. Here, we show that the fraction of SOX1-positive cells within the stem cell pool SOX1 is expressed in a subset of GFAP-positive radial astrocytes declines with aging, their molecular constitution remains and SOX2-positive non-radial cells within the SGZ. SOX1 shows remarkably stable throughout adult life. a much more restricted pattern than other previously described markers of putative SGZ stem/progenitor cells (Fig. 7). That is DISCUSSION SOX1 is expressed in a fraction of GFAP radial astrocytes and Neurogenesis occurs within the hippocampus of adult mice SOX2 positive non-radial cells. By contrast, SOX2 is expressed throughout their lifespan. However, the self-renewal properties and broadly across radial astrocytes, non-radial progenitors and mature nature of the stem cells has remained controversial (Suh et al., astrocytes in the dentate gyrus (Steiner et al., 2006; Suh et al.,
2007; Bonaguidi et al., 2011; Encinas et al., 2011). Part of this 2007). Similarly, other common markers (i.e. nestin or GFAP) are DEVELOPMENT Sox1 NSCs of the hippocampus RESEARCH ARTICLE 3947
Fig. 7. Model of SGZ lineages in the adult dentate gyrus based on SOX1. SOX1 is expressed in activated stem/progenitor cells. These activated SOX1-marked stem cells can give rise to hilar astrocytes as well as to committed early neural progenitors, which then give rise to downstream lineages. Alternatively, activated SOX1 stem cells may return to a quiescent state, whereby SOX1 is silenced, with the potential to be reactivated, and re- express SOX1, at a later time.
expressed in a broader range of SGZ cells, endothelial cells and/or lineage trace NSCs, also found evidence for long-term production mature astrocytes (Brenner et al., 1994; Wiese et al., 2004). of neurons (Lugert et al., 2012). In a third contrasting report, the Even though Sox1 is expressed in only a subset of stem/early authors use higher doses of tamoxifen in the nestin-creER model progenitor cells, lineage tracing shows that these are the cells along with real-time expression of nestin (nestin-GFP transgenic responsible for producing a majority of the neuroblasts. Considering mice) and sequential CIdU/IdU staining to arrive at the conclusion the likely incompleteness of removal of the lox-stop-lox cassette in that radial astrocytes have a limited lifespan (‘disposable stem cell Cre-expressing cells, it is possible that Sox1 marks all the stem/early model’) (Encinas et al., 2011). In particular, they conclude that progenitors giving rise to neuroblasts and hence newly born neurons. once radial astrocytes are activated, they undergo several rounds of However, the subset of radial astrocytes and non-radial cells that division to produce neuronally committed progenitors and then express SOX1 do not all differentiate into neurons. Lineage tracing differentiate into terminal astrocytes. Our results support aspects of shows a substantial fraction of Sox1 marked cells remain within the both models. The ongoing production of progenitors/neuroblasts two stem/progenitor populations even 12 weeks after lineage tracing from Sox1-marked cells even after 3 months of having the system has been shut off. Interestingly, most of the Sox1 marked radial off is consistent with a population of stem cells with long-term astrocytes no longer actively express Sox1. Even at the end of the 8- neurogenic potential that cycle between an activated and quiescent week pulse many traced cells are no longer expressing Sox1, state. However, the reduction of Sox1-traced radial astrocytes over suggestive of a highly transitory expression of this transcription the 12-week chase is consistent with some of these cells factor. After the 12-week chase, a small fraction of late permanently exiting the stem cell pool. progenitors/neuroblasts continue to be marked, presumably arising Our results also identify crucial variables when considering the from lineage traced radial astrocytes. Based on these findings, we NSC population of the hippocampus. First, SOX1 expression propose a model where radial astrocytes that activate Sox1 can either clearly identifies heterogeneity within the GFAP-positive radial commit to differentiate or to remain as stem cells. The latter cells astrocyte and SOX2 positive non-radial cell populations. Second, silence Sox1 and then reactivate it at a later time point to give rise to our results show the influence that reporter expression can have on future generations of neurons (Fig. 7). Although it is possible that the interpretation of data. In particular, we find that the Z/EG reporter marked late progenitors/neuroblasts after the 12-week chase were does not mark Sox1-expressing cells. The Bonaguidi et al. and somehow delayed in their differentiation rather than arising anew Ecinas et al. papers both use the Z/EG reporter in most of their from traced radial astrocytes, this interpretation seems unlikely lineage-tracing experiments and, therefore, may be visualizing a considering that the chase is three times longer than the reported 4- different subset of progenitor cells. By contrast, we find the R26R- week time span from a committed progenitor to mature neuron eYFP reporter is expressed in the Sox1 radial astrocytes, but not all (Zhao et al., 2008; Encinas et al., 2006). Furthermore, the marked of the Sox1-expressing cells when compared with either Sox1-GFP late progenitors were proliferating, inconsistent with a dormant state. or anti-SOX1 staining (Fig. 5B versus Fig. 1E). This finding may Lineage-tracing experiments using inducible nestin-CreER reflect that either many of the Sox1 radial astrocytes have transgenic mice have led to contradictory interpretations regarding transitioned into late progenitors prior to complete activation of the the lifespan of adult NSCs. In one report, the authors use low dose reporter or the failure of the reporter to be expressed in all cells. tamoxifen to mark rare cells in the stem cell pool and follow clones Another important consideration is comparisons between over time (Bonaguidi et al., 2011). Based on this approach, they transgenic lines. We use a Sox1 knock-in approach, while the find evidence that radial astrocytes can self-renew long term and nestin-GFP and nestin-creER are transgenics inserted at different can produce both neurons and astrocytes. Consistent with this loci. Therefore, comparisons may be complicated by local
conclusion, another report using a Hes5-CreER transgenic to epigenetic influences on gene expression. Resolution of these DEVELOPMENT 3948 RESEARCH ARTICLE Development 139 (21) confounding factors and others will be essential to resolve the very Competing interests statement important question of the nature of hippocampal stem cells and The authors declare no competing financial interests. their long-term self-renewal capacity. Supplementary material SOX1 appears to play multiple roles in the adult brain. Its Supplementary material available online at expression is limited to stem/progenitor cells in the dentate gyrus http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.081133/-/DC1 whereas it is expressed in mature neurons in other parts of the brain. For example, Sox1 is highly expressed in the CA1 region of References Ables, J. L., Breunig, J. J., Eisch, A. J. and Rakic, P. (2011). Not(ch) just the hippocampus (Kan et al., 2007). SOX1 is also expressed in development: Notch signalling in the adult brain. Nat. Rev. Neurosci. 12, 269- other neuron subtypes in the brain and has been shown to play an 283. important role in the migration of the ventral striatal neurons from Altman, J. and Das, G. D. (1965a). Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J. Comp. Neurol. 124, 319- the VZ during embryonic development (Ekonomou et al., 2005; 335. Kan et al., 2007). SOX1 is also expressed in the V-SVZ of the adult Altman, J. and Das, G. D. (1965b). Post-natal origin of microneurones in the rat (Kan et al., 2007). However, unlike in the SGZ, real-time brain. Nature 207, 953-956. expression (Sox1-GFP) and pulse-chase lineage tracing (Sox1-tTA Aubert, J., Stavridis, M. P., Tweedie, S., O’Reilly, M., Vierlinger, K., Li, M., Ghazal, P., Pratt, T., Mason, J. O., Roy, D. et al. (2003). Screening for system) suggests that the Sox1-expressing cells of the V-SVZ are mammalian neural genes via fluorescence-activated cell sorter purification of predominantly, if not solely, committed intermediate progenitors neural precursors from Sox1-gfp knock-in mice. Proc. Natl. Acad. Sci. USA 100 (supplementary material Figs S1, S8). Why Sox1 is expressed in Suppl. 1, 11836-11841. such distinct populations is unclear. The downstream molecular Barraud, P., Thompson, L., Kirik, D., Björklund, A. and Parmar, M. (2005). Isolation and characterization of neural precursor cells from the Sox1-GFP pathways will need to be dissected to better understand how SOX1 reporter mouse. Eur. J. Neurosci. 22, 1555-1569. is used in the different contexts. In vitro studies have suggested that Bonaguidi, M. A., Wheeler, M. A., Shapiro, J. S., Stadel, R. P., Sun, G. J., SOX1 regulates Notch signaling (Kan et al., 2004). Interestingly, Ming, G. L. and Song, H. (2011). In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics. Cell 145, 1142-1155. Notch signaling is used iteratively in stem/progenitor cells and then Brazel, C. Y., Limke, T. L., Osborne, J. K., Miura, T., Cai, J., Pevny, L. and Rao, again later in differentiated neurons (for review, see Ables et al., M. S. (2005). Sox2 expression defines a heterogeneous population of 2011). Therefore, although SOX1 may be used at different stages neurosphere-forming cells in the adult murine brain. Aging Cell 4, 197-207. leading to different outcomes, it could be doing so through Brenner, M., Kisseberth, W. C., Su, Y., Besnard, F. and Messing, A. (1994). GFAP promoter directs astrocyte-specific expression in transgenic mice. J. common molecular pathways. Neurosci. 14, 1030-1037. A major question in the adult neurogenesis field is how aging Bylund, M., Andersson, E., Novitch, B. G. and Muhr, J. (2003). Vertebrate influences the intrinsic molecular nature of the stem cells. In the V- neurogenesis is counteracted by Sox1-3 activity. Nat. Neurosci. 6, 1162-1168. Cameron, H. A., Woolley, C. S., McEwen, B. S. and Gould, E. (1993). SVZ, it has been shown that levels of the cell cycle inhibitor p16 Differentiation of newly born neurons and glia in the dentate gyrus of the adult increase with aging, resulting in a decline in proliferation and hence rat. Neuroscience 56, 337-344. neurogenesis, consistent with intrinsic changes within these cells Carleton, A., Petreanu, L. T., Lansford, R., Alvarez-Buylla, A. and Lledo, P. M. (Molofsky et al., 2006). Therefore, it appeared likely that mRNA (2003). Becoming a new neuron in the adult olfactory bulb. Nat. Neurosci. 6, 507-518. profiles of Sox1-expressing cells from young and old mice would Collignon, J., Sockanathan, S., Hacker, A., Cohen-Tannoudji, M., Norris, D., differ. However, our profiling data suggest that these cells are Rastan, S., Stevanovic, M., Goodfellow, P. N. and Lovell-Badge, R. (1996). intrinsically stable. An interesting alternative to intrinsic A comparison of the properties of Sox-3 with Sry and two related genes, Sox-1 and Sox-2. Development 122, 509-520. modifications of gene expression profiles is that changes in the Coriat, A. M., Müller, U., Harry, J. L., Uwanogho, D. and Sharpe, P. T. (1993). composition of the germinal niche cell populations occur. PCR amplification of SRY-related gene sequences reveals evolutionary Therefore, with an increasing purification of stem cells, the more conservation of the SRY-box motif. PCR Methods Appl. 2, 218-222. stable the cells seem. To test this possibility, it will be essential to Dai, M. H., Wang, P. L., Boyd, A. D., Kostov, G., Athey, B., Jones, E. G., Bunney, W. E., Myers, R. M., Speed, T. P., Akil, H. et al. (2005). Evolving develop new markers that can subdivide the presumptive stem cells gene/transcript definitions significantly alter the interpretation of GeneChip to increasing homogeneous populations. Indeed, our data supports data. Nucleic Acids Res. 33, e175. an unexpected heterogeneity among the GFAP-positive radial Denny, P., Swift, S., Connor, F. and Ashworth, A. (1992). An SRY-related gene expressed during spermatogenesis in the mouse encodes a sequence-specific astrocytes and Sox2-positive non-radial cells of the SGZ DNA-binding protein. EMBO J. 11, 3705-3712. (Yamaguchi et al., 2000; Seri et al., 2001; Kempermann et al., Doetsch, F., Caillé, I., Lim, D. A., García-Verdugo, J. M. and Alvarez-Buylla, A. 2003; Fukuda et al., 2003; Seri et al., 2004; Encinas et al., 2011; (1999). Subventricular zone astrocytes are neural stem cells in the adult Bonaguidi et al., 2011). We propose that this heterogeneity along mammalian brain. Cell 97, 703-716. Ekonomou, A., Kazanis, I., Malas, S., Wood, H., Alifragis, P., Denaxa, M., with limitations in the tools used to trace the presumptive stem Karagogeos, D., Constanti, A., Lovell-Badge, R. and Episkopou, V. (2005). cells underlie the often-contradictory results within the adult NSC Neuronal migration and ventral subtype identity in the telencephalon depend on field. The further characterization of new markers such as Sox1 SOX1. PLoS Biol. 3, e186. will enable the refinement and hence better understanding of the Elkouris, M., Balaskas, N., Poulou, M., Politis, P. K., Panayiotou, E., Malas, S., Thomaidou, D. and Remboutsika, E. (2011). Sox1 maintains the true stem cell pool within the SGZ. undifferentiated state of cortical neural progenitor cells via the suppression of Prox1-mediated cell cycle exit and neurogenesis. Stem Cells 29, 89-98. Acknowledgements Ellis, P., Fagan, B. M., Magness, S. T., Hutton, S., Taranova, O., Hayashi, S., We appreciate critical manuscript review by J. Rich, H. Suh, J. Lathia, D. McMahon, A., Rao, M. and Pevny, L. (2004). Sox2, a persistent marker for Schonberg and D. Lim. We thank Austin Smith for the Sox1-GFP targeting multipotential neural stem cells derived from embryonic stem cells, the embryo construct and Yusuke Kamachi and Hisato Kondoh for the Sox1 antibody. or the adult. Dev. Neurosci. 26, 148-165. Encinas, J. M., Vaahtokari, A. and Enikolopov, G. (2006). Fluoxetine targets Funding early progenitor cells in the adult brain. Proc. Natl. Acad. Sci. USA 103, 8233- R.B. was supported by the National Institutes of Health (NIH) [K08 NS48118 8238. and R01 NS057221], California Institute of Regenerative Medicine (CIRM) Encinas, J. M., Michurina, T. V., Peunova, N., Park, J. H., Tordo, J., Peterson, [Seed Grant RS1-00161, New Faculty Award RN2-00906] and the Pew D. A., Fishell, G., Koulakov, A. and Enikolopov, G. (2011). Division-coupled Charitable Trust. M.V. was supported by an American Brain Tumor Association astrocytic differentiation and age-related depletion of neural stem cells in the Basic Research Fellowship and was supported by a National Service Research adult hippocampus. Cell Stem Cell 8, 566-579. Award [NINDS F32 NS058042]. Y.G.H. and A.A.B. were supported by NIH [R01 Episkopou, V. (2005). Sox2 functions in adult neural stem cells. Trends Neurosci.
NS28478]. Deposited in PMC for release after 12 months. 28, 219-221. DEVELOPMENT Sox1 NSCs of the hippocampus RESEARCH ARTICLE 3949
Eriksson, P. S., Perfilieva, E., Björk-Eriksson, T., Alborn, A. M., Nordborg, C., Pevny, L. H., Sockanathan, S., Placzek, M. and Lovell-Badge, R. (1998). A role Peterson, D. A. and Gage, F. H. (1998). Neurogenesis in the adult human for Sox1 in neural determination. Development 125, 1967-1978. hippocampus. Nat. Med. 4, 1313-1317. Rex, M., Orme, A., Uwanogho, D., Tointon, K., Wigmore, P. M., Sharpe, P. T. Favaro, R., Valotta, M., Ferri, A. L., Latorre, E., Mariani, J., Giachino, C., and Scotting, P. J. (1997). Dynamic expression of chicken Sox2 and Sox3 genes Lancini, C., Tosetti, V., Ottolenghi, S., Taylor, V. et al. (2009). Hippocampal in ectoderm induced to form neural tissue. Dev. Dyn. 209, 323-332. development and neural stem cell maintenance require Sox2-dependent Schmidt-Hieber, C., Jonas, P. and Bischofberger, J. (2004). Enhanced synaptic regulation of Shh. Nat. Neurosci. 12, 1248-1256. plasticity in newly generated granule cells of the adult hippocampus. Nature Ferri, A. L., Cavallaro, M., Braida, D., Di Cristofano, A., Canta, A., Vezzani, 429, 184-187. A., Ottolenghi, S., Pandolfi, P. P., Sala, M., DeBiasi, S. et al. (2004). Sox2 Schönig, K., Schwenk, F., Rajewsky, K. and Bujard, H. (2002). Stringent deficiency causes neurodegeneration and impaired neurogenesis in the adult doxycycline dependent control of CRE recombinase in vivo. Nucleic Acids Res. mouse brain. Development 131, 3805-3819. 30, e134. Fukuda, S., Kato, F., Tozuka, Y., Yamaguchi, M., Miyamoto, Y. and Scott, C. E., Wynn, S. L., Sesay, A., Cruz, C., Cheung, M., Gomez Gaviro, M. Hisatsune, T. (2003). Two distinct subpopulations of nestin-positive cells in V., Booth, S., Gao, B., Cheah, K. S., Lovell-Badge, R. et al. (2010). SOX9 adult mouse dentate gyrus. J. Neurosci. 23, 9357-9366. induces and maintains neural stem cells. Nat. Neurosci. 13, 1181-1189. Gage, F. H., Kempermann, G., Palmer, T. D., Peterson, D. A. and Ray, J. Seri, B., García-Verdugo, J. M., McEwen, B. S. and Alvarez-Buylla, A. (2001). (1998). Multipotent progenitor cells in the adult dentate gyrus. J. Neurobiol. 36, Astrocytes give rise to new neurons in the adult mammalian hippocampus. J. 249-266. Neurosci. 21, 7153-7160. Galvan, V. and Jin, K. (2007). Neurogenesis in the aging brain. Clin. Interv. Aging Seri, B., García-Verdugo, J. M., Collado-Morente, L., McEwen, B. S. and 2, 605-610. Alvarez-Buylla, A. (2004). Cell types, lineage, and architecture of the germinal Graham, V., Khudyakov, J., Ellis, P. and Pevny, L. (2003). Sox2 functions to zone in the adult dentate gyrus. J. Comp. Neurol. 478, 359-378. maintain neural progenitor identity. Neuron 39, 749-765. Shors, T. J., Miesegaes, G., Beylin, A., Zhao, M., Rydel, T. and Gould, E. Gubbay, J., Collignon, J., Koopman, P., Capel, B., Economou, A., Münsterberg, A., Vivian, N., Goodfellow, P. and Lovell-Badge, R. (1990). A (2001). Neurogenesis in the adult is involved in the formation of trace memories. gene mapping to the sex-determining region of the mouse Y chromosome is a Nature 410, 372-376. member of a novel family of embryonically expressed genes. Nature 346, 245- Sinclair, A. H., Berta, P., Palmer, M. S., Hawkins, J. R., Griffiths, B. L., Smith, 250. M. J., Foster, J. W., Frischauf, A. M., Lovell-Badge, R. and Goodfellow, P. Irizarry, R. A., Hobbs, B., Collin, F., Beazer-Barclay, Y. D., Antonellis, K. J., N. (1990). A gene from the human sex-determining region encodes a protein Scherf, U. and Speed, T. P. (2003). Exploration, normalization, and summaries with homology to a conserved DNA-binding motif. Nature 346, 240-244. of high density oligonucleotide array probe level data. Biostatistics 4, 249-264. Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter Kamachi, Y., Uchikawa, M., Collignon, J., Lovell-Badge, R. and Kondoh, H. strain. Nat. Genet. 21, 70-71. (1998). Involvement of Sox1, 2 and 3 in the early and subsequent molecular Sottile, V., Li, M. and Scotting, P. J. (2006). Stem cell marker expression in the events of lens induction. Development 125, 2521-2532. Bergmann glia population of the adult mouse brain. Brain Res. 1099, 8-17. Kan, L., Israsena, N., Zhang, Z., Hu, M., Zhao, L. R., Jalali, A., Sahni, V. and Srinivas, S., Watanabe, T., Lin, C. S., William, C. M., Tanabe, Y., Jessell, T. M. Kessler, J. A. (2004). Sox1 acts through multiple independent pathways to and Costantini, F. (2001). Cre reporter strains produced by targeted insertion promote neurogenesis. Dev. Biol. 269, 580-594. of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4. Kan, L., Jalali, A., Zhao, L. R., Zhou, X., McGuire, T., Kazanis, I., Episkopou, Steiner, B., Klempin, F., Wang, L., Kott, M., Kettenmann, H. and V., Bassuk, A. G. and Kessler, J. A. (2007). Dual function of Sox1 in Kempermann, G. (2006). Type-2 cells as link between glial and neuronal telencephalic progenitor cells. Dev. Biol. 310, 85-98. lineage in adult hippocampal neurogenesis. Glia 54, 805-814. Kaplan, M. S. and Bell, D. H. (1984). Mitotic neuroblasts in the 9-day-old and Streit, A., Sockanathan, S., Pérez, L., Rex, M., Scotting, P. J., Sharpe, P. T., 11-month-old rodent hippocampus. J. Neurosci. 4, 1429-1441. Lovell-Badge, R. and Stern, C. D. (1997). Preventing the loss of competence Kempermann, G., Kuhn, H. G. and Gage, F. H. (1997). More hippocampal for neural induction: HGF/SF, L5 and Sox-2. Development 124, 1191-1202. neurons in adult mice living in an enriched environment. Nature 386, 493-495. Suh, H., Consiglio, A., Ray, J., Sawai, T., D’Amour, K. A. and Gage, F. H. Kempermann, G., Gast, D., Kronenberg, G., Yamaguchi, M. and Gage, F. H. (2007). In vivo fate analysis reveals the multipotent and self-renewal capacities (2003). Early determination and long-term persistence of adult-generated new of Sox2+ neural stem cells in the adult hippocampus. Cell Stem Cell 1, 515-528. neurons in the hippocampus of mice. Development 130, 391-399. Suter, D. M., Tirefort, D., Julien, S. and Krause, K. H. (2009). A Sox1 to Pax6 Komitova, M. and Eriksson, P. S. (2004). Sox-2 is expressed by neural switch drives neuroectoderm to radial glia progression during differentiation of progenitors and astroglia in the adult rat brain. Neurosci. Lett. 369, 24-27. mouse embryonic stem cells. Stem Cells 27, 49-58. Lois, C. and Alvarez-Buylla, A. (1994). Long-distance neuronal migration in the Tashiro, A., Sandler, V. M., Toni, N., Zhao, C. and Gage, F. H. (2006). NMDA- adult mammalian brain. Science 264, 1145-1148. receptor-mediated, cell-specific integration of new neurons in adult dentate Lugert, S., Vogt, M., Tchorz, J. S., Müller, M., Giachino, C. and Taylor, V. gyrus. Nature 442, 929-933. (2012). Homeostatic neurogenesis in the adult hippocampus does not involve Tusher, V. G., Tibshirani, R. and Chu, G. (2001). Significance analysis of amplification of Ascl1(high) intermediate progenitors. Nat. Commun. 3, 670. microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA Luskin, M. B. (1993). Restricted proliferation and migration of postnatally 98, 5116-5121. generated neurons derived from the forebrain subventricular zone. Neuron 11, Uwanogho, D., Rex, M., Cartwright, E. J., Pearl, G., Healy, C., Scotting, P. J. 173-189. and Sharpe, P. T. (1995). Embryonic expression of the chicken Sox2, Sox3 and Malas, S., Postlethwaite, M., Ekonomou, A., Whalley, B., Nishiguchi, S., Sox11 genes suggests an interactive role in neuronal development. Mech. Dev. Wood, H., Meldrum, B., Constanti, A. and Episkopou, V. (2003). Sox1- 49, 23-36. deficient mice suffer from epilepsy associated with abnormal ventral forebrain Wegner, M. (1999). From head to toes: the multiple facets of Sox proteins. development and olfactory cortex hyperexcitability. Neuroscience 119, 421-432. Nucleic Acids Res. 27, 1409-1420. Ming, G. L. and Song, H. (2005). Adult neurogenesis in the mammalian central Wegner, M. and Stolt, C. C. (2005). From stem cells to neurons and glia: a nervous system. Annu. Rev. Neurosci. 28, 223-250. Molofsky, A. V., Slutsky, S. G., Joseph, N. M., He, S., Pardal, R., Soxist’s view of neural development. Trends Neurosci. 28, 583-588. Krishnamurthy, J., Sharpless, N. E. and Morrison, S. J. (2006). Increasing Wiese, C., Rolletschek, A., Kania, G., Blyszczuk, P., Tarasov, K. V., Tarasova, p16INK4a expression decreases forebrain progenitors and neurogenesis during Y., Wersto, R. P., Boheler, K. R. and Wobus, A. M. (2004). Nestin expression – ageing. Nature 443, 448-452. a property of multi-lineage progenitor cells? Cell. Mol. Life Sci. 61, 2510-2522. Nishiguchi, S., Wood, H., Kondoh, H., Lovell-Badge, R. and Episkopou, V. Wood, H. B. and Episkopou, V. (1999). Comparative expression of the mouse (1998). Sox1 directly regulates the gamma-crystallin genes and is essential for Sox1, Sox2 and Sox3 genes from pre-gastrulation to early somite stages. Mech. lens development in mice. Genes Dev. 12, 776-781. Dev. 86, 197-201. Novak, A., Guo, C., Yang, W., Nagy, A. and Lobe, C. G. (2000). Z/EG, a double Wright, E. M., Snopek, B. and Koopman, P. (1993). Seven new members of the reporter mouse line that expresses enhanced green fluorescent protein upon Sox gene family expressed during mouse development. Nucleic Acids Res. 21, Cre-mediated excision. Genesis 28, 147-155. 744. Petreanu, L. and Alvarez-Buylla, A. (2002). Maturation and death of adult-born Yamaguchi, M., Saito, H., Suzuki, M. and Mori, K. (2000). Visualization of olfactory bulb granule neurons: role of olfaction. J. Neurosci. 22, 6106-6113. neurogenesis in the central nervous system using nestin promoter-GFP Pevny, L. and Rao, M. S. (2003). The stem-cell menagerie. Trends Neurosci. 26, transgenic mice. Neuroreport 11, 1991-1996. 351-359. Zhao, C., Deng, W. and Gage, F. H. (2008). Mechanisms and functional Pevny, L. and Placzek, M. (2005). Sox genes and neural progenitor identity. Curr. implications of adult neurogenesis. Cell 132, 645-660. Opin. Neurobiol. 15, 7-13. Zhao, S., Nichols, J., Smith, A. G. and Li, M. (2004). SoxB transcription factors Pevny, L. H. and Lovell-Badge, R. (1997). Sox genes find their feet. Curr. Opin. specify neuroectodermal lineage choice in ES cells. Mol. Cell. Neurosci. 27, 332-
Genet. Dev. 7, 338-344. 342. DEVELOPMENT