TISSUE-SPECIFIC STEM CELLS

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1 TISSUE-SPECIFIC STEM CELLS Modification of Pax6 and Olig2 Expression in Adult Hippocampal Neurogenesis Selectively Induces Stem Cell Fate and Alters Both Neuronal and Glial Populations FRIEDERIKE KLEMPIN, ROBERT A. MARR, DANIEL A. PETERSON Department of Neuroscience, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, USA Key Words. Neural stem cell Neurogenic niche Astrocyte Bromodeoxyuridine NG2 ABSTRACT The generation of new neurons in the mammalian hippocampus continues throughout life, and lineage progression is regulated by transcription factors, local cues, and environmental influences. The ability to direct stem/progenitor cell fate in situ may have therapeutic potential. Using an in vivo retroviral delivery and lineage tracing approach, we compare the lineage-instruction factors, Pax6 and Olig2, and demonstrate that both participate in regulation of adult hippocampal neurogenesis in adult rats. We show that overexpression of the proneuronal factor Pax6 pushes neuronal precursor cells to early maturation and increases the frequency of neuronal phenotypes. However, Pax6 overexpression results in no net increase in neurogenesis at 3 weeks. Blocking of Olig2 function reduces and slows neuronal commitment and differentiation and decreases net neurogenesis. Altering expression of both factors also Disclosure of potential conflicts of interest is found at the end of this article. changes gliogenesis. Our results establish that Pax6 decreases the number of Neuron-Glia 2 progenitor cells and prevents oligodendrocytic lineage commitment, while repression of Olig2 results in an expanded astrocytic lineage. We conclude that selectively modifying transcriptional cues within hippocampal progenitor cells is sufficient to induce a cell fate switch, thus altering the neurogenesis gliogenesis ratio. In addition, our data show the competence of multiple progenitor lineages to respond divergently to the same signal. Therefore, directing instructive cues to select phenotype and developmental stage could be critical to achieve precise outcomes in cell genesis. Further understanding the regulation of lineage progression in all progenitor populations within the target region will be important for developing therapeutic strategies to direct cell fate for brain repair. STEM CELLS 2012; 30: INTRODUCTION Adult hippocampal neurogenesis is a dynamic process within the dentate gyrus (DG), where neural stem cells (NSCs) retain fate plasticity and can adapt to external stimuli [1, 2], injury, or pathology [3, 4]. The hippocampal subgranular zone (SGZ) contains radial glia-like stem cells (type-1 cells) that give rise to amplifying progenitor cells (type-2 and type-3 cells), which mostly become neurons [5, 6]. In addition, NSCs are multipotent and can generate glia [7 9]. Neuron-Glia 2 (NG2) progenitor cells also reside in the DG as a potentially multipotent subpopulation [10] that makes no apparent contribution to adult neurogenesis [11]. As in development, the progression from type-1 cells to mature neurons is regulated by soluble intrinsic factors that regulate stem cell maintenance (e.g., Pax6, Tbr2) and direct cell fate (e.g., Olig2, Sox2, Pax6, Tbr2) [5, 12, 13]. Transcription factors controlling cell differentiation have been elucidated for the developing central nervous system (CNS) [14] (reviewed in [15, 16]) and to some extent for the postnatal brain [5, 12, 17]. Although adult neurogenesis can be modulated by many factors, the regulation of cell fate decisions in the adult DG is less well understood. Here, we investigate the role of the lineage-related genes Pax6 and Olig2 in directing cell fate of stem/progenitor cells in the adult SGZ in vivo and asked if manipulating their activity in situ could direct the outcome of neurogenesis. The transcription factor Pax6 essentially regulates the balance between stem cell maintenance and neuronal lineage progression in development [15, 18, 19] and postnatal neurogenesis [17, 19, 20]. Its expression is confined to type-1 stem cells and early progenitor cells of the SGZ, where Pax6 positively influences neuronal differentiation [5, 17, 21]. So far, it has not been investigated how Pax6 regulates lineage progression in the adult DG. In contrast, the transcription factor Olig2 is required in glial fate commitment and has been studied in various brain regions and injury models [20, 22, 23]. Olig2 directs neuronal-glial cell fate decision in development [24, 25] and postnatal subventricular zone [20, 26]. In this study, we retrovirally overexpressed the neurogenic factor Pax6 and blocked the pan-gliogenic factor Olig2, to Author contributions: F.K.: concept and design, data collection and analysis, interpretation, and manuscript writing; R.A.M.: producing retroviral vectors and manuscript writing; D.A.P.: financial support, concept and design, writing, and final approval of manuscript. Correspondence: Daniel A. Peterson, Ph.D., Department of Neuroscience, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, Illinois 60064, USA. Telephone: ; Fax: ; daniel.peterson@rosalindfranklin.edu Received September 14, 2011; accepted for publication November 19, 2011; first published online in STEM CELLS EXPRESS December 7, VC AlphaMed Press /2011/$30.00/0 doi: /stem.1005 STEM CELLS 2012;30:

2 Klempin, Marr, Peterson 501 better define how multiple signals dynamically regulate the neurogenic niche. By examining both early events in cell lineage decision and the outcome of cell fate following differentiation, we were able to ascertain the effect of altering these cell lineage-instruction signals on the population of differentiated cells in the adult hippocampal niche. This is the first study to characterize the role of a gliogenic signal in the DG. Understanding the process of lineage commitment in the context of balancing the production of all cell populations derived from type-1 stem cells provides fundamental insight into both the normal process of adult neurogenesis and the way in which the population balance is altered in response to environmental cues and injury. Our data revealed that overexpression of Pax6 acts on the neuronal and NG2 progenitor population and pushed maturation of newly born neurons. The absence of Olig2 signaling led to delayed neuronal maturation of hippocampal precursors and induced infected cells to become astrocytes. NSC therapies for the diseased brain will require methods to selectively and efficiently drive cell fate of endogenous stem/progenitor cells. It has been shown that altered expression of a single gene can direct cell fate of adult hippocampal progenitors toward oligodendrocytes [27]. Our results show how both neuronal and glial lineage-instruction factors regulate the generation of new neurons, and that their modulation disrupts neurogenic homeostasis and changes the composition of the neurogenic niche. MATERIALS AND METHODS Animals and Surgical Procedure Young adult female Fisher 344 rats (7 8 week old) were purchased from Harlan Sprague Dawley (Indiana). Rats were housed two to three per cage under standard laboratory conditions with a light/dark cycle of 12 hours each and free access to food and water. All experiments were performed according to and approved by the National Institutes of Health Guide and the Institutional Animal Care and Use Committee. At day 0, rats (n ¼ 32, distributed across three groups for two time points) were deeply anesthetized with a mixture of ketamine (75 mg/kg), xylazine (4 mg/kg), and acepromazine (5.6 mg/kg), and 1 ll of a retroviral suspension containing (a) green fluorescent protein (GFP), (b) Olig2-VP16-IRES-GFP (Olig2-VP16), or (c) Pax6-IRES-GFP (Pax6) was infused into the left hemisphere of the DG via a stereotaxic system. DG coordinates from Bregma in mm are: A/P 3.6, M/L þ2.5, D/V 4.0. The retroviral vectors used have been described elsewhere [28] and were produced by transient transfection of the construct plasmids (kind gifts from M. Goetz) with gag/pol and vsv-g packaging plasmids as previously described [29]. Viral titers typically were approximately transducing units per milliliter. Retrovirally mediated transgene expression is a useful tool to determine the effect of specific genes in single cells. Because insertion of the transgene is random, retroviral GFP expression may be variable and may be reduced over time. We have designed our experimental approaches to minimize variability in delivery and detection, including using antibodies to GFP to compensate for reduced GFP protein and for any quenching of native GFP fluorescence following pretreatment for thymidine analog detection. Experimental Design and Halogenated Thymidine Analog Injections Retrovirally labeled cells and their progeny were investigated 5 days and 21 days following gene delivery. In addition, two thymidine analog injection paradigms were used to determine the effect of the transgenes on a broader population of progenitor cells. For analyzing proliferation (short-term, group A), animals received three i.p. injections of bromodeoxyuridine (BrdU, 50 mg/kg b.wt.; Sigma, St. Louis, MO, 2 hours apart on day 5 following gene delivery and were killed 2 hours after the last injection. Cell survival and long-term cell division activity of progenitor cells 3 weeks following gene delivery were determined using the combination of two other halogenated thymidine analogs, 5-chloro-2 0 deoxyuridine (CldU) and 5- iodo-2 0 deoxyuridine (IdU) [30]. First, on day 1, animals received three i.p. injections of CldU (42.5 mg/kg b.wt.; Sigma) 2 hours apart and were left 3 weeks to assess survival of the labeled cohort (group C). Prior to tissue collection at 3 weeks, IdU (57.5 mg/kg b.wt.; MP Biomedicals, Solon, OH, was administered three times 2 hours apart to label cell proliferation, and animals were killed on the next day (day 22, group B). Immunohistochemistry Rats were deeply anesthetized and perfused transcardially with 0.9% sodium chloride followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Brains were removed from the skulls, postfixed in 4% PFA at 4 C over night, and transferred into 30% sucrose. Brains were cut on a freezing sliding microtome (Leica SM2000R, Deerfield, IL, in the horizontal plane into 40-lm-thick sections and cryoprotected. Sections were stained free floating with all antibodies diluted in Tris-buffered saline containing 3% donkey serum and 0.1% Triton X-100. For BrdU, CldU, and IdU staining, DNA was denatured in 2 N HCl for 30 minutes at 37 C. Primary antibodies were applied in the following concentrations: anti-brdu (rat, 1:500; Accurate, Westbury, NY, www. accuratechemical.com), anti-cldu (rat anti-brdu, 1:500; Accurate), anti-idu (mouse anti-brdu, 1:500; Becton Dickinson, anti-gfp (chicken, 1:5,000; Aves Labs, Tigard, OR, aveslab.com), anti-ng2 (rabbit, 1:500; Millipore, Billerica, MA, anti-ng2 (mouse, 1:2,000; Millipore), anti-rip (mouse, 1:250; Millipore), anti-s100b (rabbit, 1:2,500; Swant, Belinzona, Switzerland, anti-s100b (mouse, 1:5,000; Abcam, Cambridge, MA, anti-glial fibrillary acidic protein (GFAP) (rabbit, 1:2,500; Dako, Carpinteria, CA, anti-doublecortin (DCX) (goat, 1:200; Santa Cruz Biotechnologies, Santa Cruz, CA, anti-neun (mouse, 1:1,000; Millipore). For immunofluorescence, Alexa488-, Cy3-, or Cy5-conjugated secondary antibodies were all used at a concentration of 1:500 (Jackson ImmunoResearch Laboratories, West Grove, PA, with the exception of DyLight488 (anti-chicken, 1:1,000). Fluorescent sections were coverslipped in polyvinyl alcohol with diazabicyclooctane as antifading agent (prepared with glycerol, 0.2 M Tris-HCl, and double-distilled water) [31]. Immunohistochemistry followed the peroxidase method with biotinylated secondary antibodies (all: 1:500; Jackson ImmunoResearch Laboratories), ABC Elite reagent (Vector Laboratories, Burlingame, CA) and diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO, sigmaaldrich.com) as chromogen. Quantification and Imaging For thymidine labeling, one-in-six series of sections of each brain were DAB stained, and immunoreactive cells were counted throughout the rostrocaudal extent of the DG. BrdU-, CldU-, or IdU-positive cells were quantified by the optical fractionator approach using the Stereo-Investigator software (MBF Bioscience Williston, VT, with an unbiased counting frame and the optical dissector principle (grid size: , frame size: , mean contour area: approximately 900 lm 2, estimated mean Schmitz-Hoff CE values were between 0.09 and 0.16 with a group mean of 0.11). For retrovirally labeled cells, onein-twelve series of sections were labeled for multiple immunofluorescence as described above for phenotypic analysis and evaluated for three-dimensional colocalization by examining orthogonal

3 502 Pax6 and Olig2 in Adult Hippocampal Neurogenesis views from a series of confocal microscope focal planes (FV500, Olympus America, Center Valley, PA, com). To quantify cell number, the following steps were used: (a) for each staining combination, the phenotype of GFP positive (GFPþ) cells was recorded as a percentage of all GFPþ cells detected within that animal. From these data, the mean frequency of each phenotype was determined for each experimental condition. (b) To determine differences in the number of GFPþ cells between conditions, the total number of GFPþ cells was estimated from the fractionated sampling for each condition. (c) The frequency distribution determined above (step a) was then normalized against this total GFP cell number (step b) to estimate actual population differences between phenotypes. For qualitative imaging, appropriate gain and black level settings were determined on control tissues stained with secondary antibodies alone to discriminate true signal from background. All images were taken in sequential scanning mode and minimally processed and composed in Adobe Photoshop. Only general adjustments to signal distribution were made and figures were not otherwise manipulated. Statistical Analysis Statistical tests to detect differences between group means were performed by an analysis of variance, followed by Tukey s post hoc tests, in cases where a significant F statistic was obtained (PRISM software). Student s t test was used for individual pairwise comparisons. All values are expressed as mean 6 SEM. p values of.05 were considered statistically significant. RESULTS Phenotypic Outcome of Hippocampal Precursor Cells Following GFP-Only Delivery We used retroviral vectors to target dividing cells, and to overexpress or repress, respectively, the genes Pax6 and Olig2 to determine changes in cell fate of hippocampal stem/progenitor cells. To discriminate the effect of transgene expression, we first assessed the phenotypic identity of GFP-only transduced cells. Newly committed neurons were identified by expression of the transient immature neuronal marker DCX and the mature neuronal marker NeuN. Approximately half of newly generated, GFPþ cells showed commitment to a neuronal phenotype whether assessed shortly after retroviral transduction into the DG (5 days, Fig. 1A), or when the progeny of cells were analyzed at 21 days. Confocal microscopic analysis of cell phenotype revealed the overall distribution of infected cells in the DG. After 5 days of gene delivery with the GFP-only construct, the majority of retroviral-labeled cells with neuronal lineage commitment were horizontal type-2 cells with medium processes (Fig. 1B). At 3 weeks following GFP delivery, lineage-traced cells displayed a mature neuronal morphology as seen by long apical dendritic branching into the molecular layer (ML; Fig. 1C) and migration into the inner granule cell layer (GCL; Fig. 1E). These results are consistent with the developmental progression and morphological changes of newly generated neurons that have been described previously [32, 33]. Besides becoming neurons, some retroviral-infected cells within the SGZ and GCL also adopted glial fates, demonstrated by morphology and marker coexpression of the proteoglycan NG2 (Fig. 2A, 2C), the oligodendrocytic lineage marker RIP (Fig. 2B), and S100b at both time points. Adoption of oligodendrocytic fate was confirmed by coexpression of 2 0,3 0 -cyclic nucleotide 3 0 -phospho-diesterase (CNPase) and adenomatous polyposis coli (APC); however, RIP was chosen as the most effective marker for quantifying colocalization of oligodendrocytes in the adult rat DG. S100bþ cells represent postmitotic astrocytes with many short and highly branched, bushy processes (Fig. 2D). After 5 days of gene delivery, a few cells also expressed GFAP (Fig. 2E), often colabeled with the sex-determining region Y related HMG-box gene 2 (Sox2). As NSCs enter cell cycle with low frequency, there is a low probability of their inclusion within the GFP-expressing cell population. GFAP and Sox2 mark both stem and nonstem astrocytes distinguished by radial and stellate morphology [34] and the characteristic radial morphology of type 1 NSCs was not observed in cells coexpressing GFP, GFAP, and Sox2. A portion of the GFP-expressing cells did not label with any of the phenotypic markers used for stem or progenitor cells and may represent a population that becomes arrested in regard to lineage commitment following exit from cell cycle. Pax6 and Olig2 Differentially Regulate Neuronal Morphology After 3 weeks of GFP-only gene delivery, newly generated neurons in the SGZ adopted a characteristic morphology and extended an apical dendrite that branched within the molecular layer (Fig. 3A). Pax6-transduced newly generated neurons exhibited a morphology, typical of GFP-only cells, but showed somewhat abnormal arborization, having smaller dendrites in any direction, and no branching within the ML detectable by GFP expression (Fig. 3B). Furthermore, a transitional phenotype of cells coexpressing both DCX and NeuN primarily in the perikaryal cytoplasm was observed; this phenotype is not seen in noninfected tissue. These novel DCX/NeuN double-labeled cells were also found in the hilus (Fig. 3C). After 3 weeks of gene delivery of the dominant-negative Olig2-VP16, a disruption of normal neuronal maturation was observed. The majority of newly generated neurons still appear immature, with stunted, less elaborate dendritic arbor. Those cells revealed only one strong dendrite reaching into the ML without branching (Fig. 3D). Blocking Olig2 resulted in fewer transduced neurons expressing NeuN, with those detected possessing a single apical dendrite with little additional dendritic arbor, a morphology consistent with an immature neuroblast phenotype (Fig. 3E). Blocking Olig2 also resulted in GFPþ cells that exhibited small processes and appeared to have a neuronal morphology but were DCX and NeuN (Fig. 3F). Quantitative Distribution of Neurogenic and Gliogenic Lineage Commitment in the Adult Hippocampus Quantitative assessment of lineage commitment soon after transgene delivery revealed that GFP-only cells had a neuronal distribution of 43% (DCX %, NeuN %; Fig. 4A) and a smaller glial distribution of 30% (NG2 9%, RIP 8%, S100b 6%, GFAP 6.6%) with the remaining GFPþ population expressing no detectable phenotype (Table 1). Population estimates reflect the frequency distribution with 109 of 248 GFPþ cells expressing either DCX or NeuN, 73 cells that followed a glial lineage, and 66 cells remaining with no detectable phenotype (Table 1). When the progeny of GFP-infected cells was analyzed at 3 weeks, an increase in neuronal maturation was observed (to 60% or 239 of 402 GFPþ cells detected; Fig. 4B; Table 1) accompanied by a shift from primarily early to primarily mature neuronal marker expression (DCX %, NeuN %, Fig. 4B). The population of GFP/NG2þ cells doubled, accounting for 19% of observed cells. Of the remaining glial phenotypes, 6% expressed RIP and 5% were S100bþ. No GFAP labeling was found at 21 days. The number of GFPþ

4 Klempin, Marr, Peterson 503 Figure 1. Most GFP-only transduced cells adopted a neuronal lineage. (A): The number of lineage-traced cells, their progeny and phenotypes were quantified either 5 days or 21 days following gene delivery into the DG. (B): At 5 days, immunohistochemistry reveals newly generated cells (GFP-green) mainly express the immature neuronal marker DCX (red), and extended primarily horizontal and bipolar processes (arrows) within the SGZ. More mature DCXþ cells not expressing GFP have apical processes entering the NeuNþ (blue) GCL. Scale bar ¼ 40 lm. (C): By 3 weeks, the morphology of DCXþ cells changed with maturation. Newly generated neurons extended an apical dendrite through the GCL that branched within the ML (arrows) (panel C1 GFP; panel C2 DCX in red; cell at arrow is negative for NeuN detection at this time). Scale bar ¼ 40 lm. (D): Retroviral delivery infected dividing cells within all layers of the DG, resulting in morphologically distinct neurons and glia. Quantification of phenotypes was performed on cells found within the GCL and SGZ. Scale bar ¼ 150 lm. (E): NeuN expression (blue) was prominent in GFPþ cells by 3 weeks following transduction; many newly generated neurons had migrated into the inner GCL (panel E1 GFP; panel E2 NeuN). Scale bar ¼ 40 lm. Abbreviations: DCX, doublecortin; DG, dendate gyrus; GCL, granule cell layer; GFP, green fluorescent protein; ML, molecular layer; SGZ, subgranular zone. cells with no detectable phenotype had fallen to <10% by 21 days post-transduction (Table 1). Pax6 Expression Accelerates Neuronal Maturation but with Decreased Survival of Viral-Infected Cells Next, we assessed overexpression of the proneuronal transcription factor Pax6 on lineage commitment of infected cells and their progeny in the DG. Retrovirus-mediated overexpression of Pax6 led to a significant increase in the ratio of cells committed to a neuronal lineage as determined by expression of DCX and NeuN at both time points (Fig. 4; Table 1). After 5 days of infection, already more than a third of Pax6-transduced cells colabeled with NeuN, a significantly higher amount compared with the other groups (NeuN %, p vs. GFP-only ¼.0054, p vs. Olig2-VP16 ¼.0025; Fig. 4A), but only 8% expressed DCX ( %, p vs. GFP-only ¼.0207, p vs. Olig2-VP16 ¼.024). Interestingly, we observed the emergence of a novel population of cells coexpressing DCX/NeuN that accounted for 17% of the GFP population. Thus, when combined with the NeuNþ population, 55% of cells already expressed the mature neuronal marker 5 days following Pax6 overexpression. When the progeny of Pax6-infected cells was assessed at 21 days, the ratio of cells committed to a neuronal lineage has increased to approximately 80% with 21% expressing DCX/NeuN, 58% expressing NeuN-only (total NeuN p vs. GFP-only ¼.0202), and 2.8% expressing DCX-only (Fig. 4B).

5 504 Pax6 and Olig2 in Adult Hippocampal Neurogenesis Figure 2. Gliogenesis of lineage-traced hippocampal progenitors. (A): Following GFP-only gene delivery, immunohistochemistry shows GFPþ cells in the hippocampal dentate gyrus committed to both a neuronal fate (large arrow) and also costained for NG2, a marker of glial progenitor cells (small arrows, NG2 in red, and in C) near or within the SGZ. Some GFPþ cells also displayed an astrocytic morphology (arrowhead, NG2 ). Through-projection of an image stack; signal colocalization confirmed by analyses of orthogonal projections (data not shown); Scale bar ¼ 30 lm. (B): GFPþ cells also showed glial commitment by coexpression of the oligodendrocytic lineage marker RIP. The lack of coexpression with S100b indicates that GFP expression is truly colocalized with RIP as evidenced by the orthogonal projections (panel B1 GFP, panel B2 RIP, panel B3 S100b). Scale bar ¼ 20 lm. (C): GFP/NG2þ precursor cell indicated by small arrows in panel (A) with symmetrically branching fine processes. Scale bar ¼ 10 lm. (D): GFP/S100bþ mature astroglia in the SGZ. Scale bar ¼ 15 lm. (E): After 3 weeks of Olig2-VP16 gene delivery, a significant increase in GFAP coexpression (red) was observed (panel E1 GFP, panel E2 GFAP). Scale bar ¼ 20 lm. Abbreviations: GFP, green fluorescent protein; GFAP, glial fibrillary acidic protein; NG2, Neuron-Glia 2; SGZ, subgranular zone. At 5 days, overexpression of Pax6 increased both the proportion of newly generated neurons as a function of the GFP-labeled population and their total number (Fig. 4A), which resulted in a significant net increase of neurogenesis (Pax6: 157 cells vs. GFP-only: 11 cells, p ¼.002; Table 1). At 21 days following Pax6 overexpression, it may appear that neurogenesis was also increased as the percentage of NeuNpositive cells was enhanced (Fig. 4B). However, this percentage increase was due to a reduced number of GFPþ cells detected at 21 days following Pax6 (Table 1). Comparison of the actual number of NeuN-positive cells reveals no difference between Pax6 and control-gfp conditions at this time (Pax6: 215 cells vs. GFP-only: 207 cells, p ¼.7740; Table 1). Thus, there was no net increase in neurogenesis following Pax6 gene delivery at 3 weeks. To evaluate the effect of Pax6 overexpression on the fate of glial progenitors, we examined relevant phenotypic markers. The distribution pattern of S100bþ astrocytes did not differ from that seen with GFP-transduction alone at both early and later times of differentiation. Pax6-infected cells expressing GFAP were detected at 5 days ( %; Fig. 4A) following gene delivery, but this phenotype was not detected in the progeny at 21 days (Fig. 4B; Table 1). Overexpression of Pax6 also led to a decrease in the number of NG2þ cells over time (from cells at 5 days to cells at 3 weeks) and in comparison to control at 21 days (GFP-only cells, p ¼.05; Table 1). Furthermore, no cells committed to an oligodendrocytic lineage (RIP-expression), normally detected in the GFP-only group, at both time points (Fig. 4; Table 1). Blockade of Olig2 Signaling Slows Neuronal Differentiation of Transduced Cells This study is the first to investigate the role of Olig2 in adult hippocampal neurogenesis by retroviral gene delivery of the dominant-negative Olig2-VP16. By fusion to Olig2, the transcriptional activator VP16 replaces the Olig2 repressor domain and thus interferes with the normal function of Olig2. The viral constructs used have been described elsewhere [25, 28]. Olig2-VP16 gene delivery produced changes in neurogenesis, slowing maturation of cells and reducing overall neurogenesis. At 5 days, 38% were DCXþ cells indicating that early neuronal commitment was similar to that seen in the GFP-only group (Fig. 4A). Unlike the GFP-only group, expression of Olig2-VP16 resulted in the appearance of a transition population coexpressing DCX/NeuN (9%), and in the absence of

6 Klempin, Marr, Peterson 505 Figure 3. Engineering fate changes neuronal maturation of transduced cells. (A): Immunohistochemistry showing typical neuronal cytoarchitecture of green fluorescent protein (GFP)-only transfected cells with a strong apical dendrite that branched within the ML. Scale bar ¼ 60 lm. (B): Following Pax6 overexpression, transduced cells that coexpressed NeuN showed abnormal arborization and a persistence of basal processes (arrows). Scale bar ¼ 30 lm. (C): Pax6 overexpression resulted in a novel transitional phenotype where DCX and NeuN were coexpressed. These cells could be found in the hilus near the CA3 region (arrows). Scale bar ¼ 50 lm. (D): Following Olig2-VP16 transgene expression, newly committed neurons (GFP-green) appeared morphologically immature, with fewer and smaller processes reaching into the ML (arrows). Scale bar ¼ 25 lm. (E): Blocking Olig2 expression resulted in fewer transduced neurons expressing NeuN with no dendritic arborization. Scale bar ¼ 20 lm. (F): Blocking Olig2 expression also produced cells that exhibited small processes and appeared to have a neuronal morphology but did not express neuronal markers (DCX /NeuN ; arrows). Scale bar ¼ 50 lm. Abbreviations: DCX, doublecortin; GFAP, glial fibrillary acidic protein; ML, molecular layer; SGZ, subgranular zone. NeuN-only positive cells. By 3 weeks, the number of lineagetraced cells expressing NeuN slightly increased to 18.5% but was still significantly lower compared with GFP-only conditions with 51.6% (p ¼.0217; Fig. 4B). Corresponding to the increase in NeuN-only expression, the frequency of DCX/ NeuN (4%) and DCXþ cells (16%) decreased but still was more prominent compared with control virus (Fig. 4B). Taking all DCX and NeuN expression together as an indicator of neuronal lineage commitment at 3 weeks, only 39% of retrovirally infected cells adopted a neuronal fate when Olig2-VP16 was expressed, compared with 60% in the GFP-only condition and approximately 80% in the Pax6 condition. In assessing the effect of blocking Olig2 on net neurogenesis at 3 weeks, the significant decrease in the frequency of newly generated neurons (Fig. 4B) corresponded to a significant decrease in the actual number (total NeuN in Olig2-VP16: 102 cells vs. GFP-only: 207 cells, p ¼.038; Table 1). Blocking Olig2 also resulted in an increase in the population of GFPþ cells for which no phenotype could be determined using the panel of markers in this analysis (145 of 450 cells, n ¼ 4, p vs. GFP-only ¼.043, Table 1). Disruption of Olig2 Signaling Increases Astrocytic While Blocking Oligodendrocytic Lineage Specification Similar to the alteration of neurogenic maturation, blocking of Olig2 effected gliogenesis and resulted in a higher frequency of astrocytes (Fig. 4). The percentage of retrovirus-infected cells coexpressing GFAP doubled at 5 days when compared with GFP-only (Olig2-VP % vs. GFP %; Fig. 4A) but was lower at 21 days (6.9% that account for 31 cells of 450 cells detected; Fig. 4B; Table 1). This is in contrast to the GFP-only and Pax6 conditions, where no GFAPþ cells were detected at 21 days. Although some costaining with S100b was found (GFAP-negative cells; Fig. 4, Table 1), it did not significantly differ between conditions or time points. Hence, Olig2-VP16 increases the ratio of GFAPþ but not S100bþ astrocytes. Expression of the dominant-negative Olig2-VP16 did not alter the frequency and number of NG2 relative to control (Fig. 4; Table 1), suggesting no disruption of glial precursor cells. However, coexpression of RIP was only detected at 5 days and was not observed in the progeny at 3 weeks. Therefore, the dominant-negative Olig2-VP16 appears to inhibit the relatively infrequent oligodendrocytic maturation of proliferating cells in the SGZ, while increasing frequency and number of retrovirally labeled cells that produced no detectable phenotype (Table 1). Delivery of Lineage-Instruction Factors Stimulates Proliferation As Detected with Thymidine Analogs Changing the distribution of cell lineage specification in the hippocampal neurogenic niche could produce a response to re-establish homeostasis in cell production. To determine if overexpression or repression, respectively, of the lineage-

7 506 Pax6 and Olig2 in Adult Hippocampal Neurogenesis Figure 4. Altered phenotypic distribution of lineage-traced cells. (A): Following Pax6 overexpression, the majority of transduced cells showed ultimate neuronal lineage commitment by coexpression of NeuN, which significantly differs from the other conditions. (B): At 3 weeks, more than 60% of GFP-only transduced cells and 80% of Pax6-transduced cells followed a neuronal lineage commitment. The increase in NeuN expression in Pax6-transduced cells was accompanied with a decrease in the number of NG2þ cells and overall gliogenesis. The distribution of phenotypes in Olig2-VP16 conditions revealed cells committed to a glial and neuronal fate in almost equal parts. Notably, the number of NeuNþ newly generated neurons was significantly decreased at 3 weeks compared with control virus and Pax6 conditions, whereas GFAPþ cells were solely detected in Olig2-VP16. The oligodendrocytic marker RIP was never detected in Pax6 conditions and solely expressed in GFP-only conditions at 3 weeks. *, p.05 indicates statistical significance relative to control; n ¼ 4. Abbreviations: DCX, doublecortin; GFP, green fluorescent protein; GFAP, glial fibrillary acidic protein; N.D., no detectable phenotype, cells do not coexpress any of the tested markers. Table 1. Distribution of GFP-expressing cells 5 days and 21 days following gene delivery by cell number (mean 6 SEM; n 5 4) Sum of GFPþ cells N.D. GFAP S100b NG2 RIP DCX DCX/NeuN NeuN 5 days GFP Pax * ** * ** Olig2-VP * 0 6 0* 21 days GFP Pax * 0 6 0* * Olig2-VP * * * * * *, p vs. GFP-only **, p vs. GFP-only Abbreviations: DCX, doublecortin; GFP, green fluorescent protein; GFAP, glial fibrillary acidic protein; NG2, Neuron-Glia 2; N.D.: no detectable phenotype, cells do not coexpress any of the tested markers. instruction factors Pax6 and Olig2 influenced proliferative activity in the hippocampal DG through a secondary effect of the environmental niche, subjects were probed with halogenated thymidine markers at 5, 21, and 22 days following gene delivery (Fig. 5A). The distribution of thymidine analoglabeled cells (that includes both virally transduced and noninfected progenitor cells) is illustrated in Figure 5B. The number of BrdUþ cells (group A at 5 days) following retroviral-gfp delivery into the DG of the left hemisphere did not differ from the noninjected hemisphere (control-gfp vs. noninjected site ; Fig. 5C), revealing no effect due to gene delivery. The expression of the Pax6 or Olig2-VP16 transgenes produced a significant elevation in proliferation relative to GFP alone (control-gfp vs. Olig2-VP16 6, , p ¼.0045 and control-gfp vs. Pax6 5, ; p ¼.0224; n ¼ 5; Fig. 5C). Here also, the number of BrdU-positive cells in the contralateral, noninjected DG of those animals was not effected (data not shown), suggesting that elevated proliferation was solely due to local transgene expression. However, no difference was found in the ultimate survival of these newly generated cells detected by CldU labeling (group C) irrespective of which transgene was expressed (Fig. 5C). The stimulation of proliferation observed with expression of Pax6 and Olig2-VP16 was largely transitory as the number of newly generated cells at 3 weeks following gene delivery (detected by IdU labeling; group B) had returned to normal levels with the exception of Olig2-VP16, which still produced a significant elevation by 3, cells (p ¼.0247; n ¼ 7, Fig. 5C). DISCUSSION Our data demonstrate that both neuronal and glial lineageinstruction factors, known to shape the developing CNS, are active in adult hippocampal neurogenesis. We show a differential effect of Pax6 and Olig2 on distinct progenitor cell populations and their lineage progression. The results establish that it is possible to manipulate the stem cell niche to direct cell fate outcome. We found that retroviral-labeled progenitor cells in the adult DG predominantly differentiate into neurons. Yet, another population of lineage-traced NG2

8 Klempin, Marr, Peterson 507 precursor cells differentiates into oligodendrocytes [35]. We present new estimates of gliogenesis in the adult rat DG and show that under normal conditions approximately 30% of newly generated cells are glia-committed; a frequency higher than previously reported in mice [27, 36]. Figure 5. Altered proliferative activity assessed by thymidine analog labeling. (A): Experimental design to determine proliferation and survival of the entire population of progenitor cells resulting from transgene expression. The immediate effect on proliferation was assessed 5 days following transduction by pulsing with BrdU (group A, short-term proliferation). The effect of transgene expression on long-term proliferation was examined by pulsing with IdU at 21 days following transduction (group B). To determine the effect on survival of newly generated cells, these animals had also received a pulse of CldU 21 days prior to tissue collection (group C). (B): Diaminobenzidine reaction to characterize IdU- and CldU-labeled cells. IdU injection 1 day prior to tissue collection shows the clustered distribution of proliferating cells within the SGZ while delivering CldU 3 weeks earlier permits assessment of survival and reveals migration of labeled cells into the GCL. Scale bar ¼ 30 lm. (C): The total number of proliferating BrdUþ cells 5 days following gene delivery was significantly increased after both, Olig2-VP16 and Pax6 expression (n ¼ 5). The effect was transitory since the number of proliferating IdUþ cells has returned to baseline at 22 days following gene delivery with the exception of Olig2-VP16, which still produced a significant elevation relative to control virus (n ¼ 7). For survival of previously CldUlabeled cells, no difference was seen between groups (n ¼ 5). *, p.05 and **, p.01 indicate statistical significance in relation to GFP-only. Abbreviations: BrdU, bromodeoxyuridine; CTR-CLS, control, contralateral side, noninjected; CldU, 5-chloro-2 0 deoxyuridine; GCL, granule cell layer; GFP, green fluorescent protein; IdU, 5-iodo- 2 0 deoxyuridine; SGZ, subgranular zone. Pax6 Overexpression Drives Early Neuronal Lineage Commitment Lineage-instruction factors act on different cell types and multiple developmental steps [27, 33, 37]. Pax6 is normally expressed by early neuronal progenitors [5, 12, 21], where its expression influences neuronal differentiation [17, 19, 38]. GFAPþ cells also express Pax6, but due to the low probability of targeting quiescent type-1 stem cells, our data primarily address the competence of neuronal progenitors to respond to Pax6. In this regard, overexpression of Pax6 accelerated differentiation more quickly through the DCX-expressing stage, generating both more NeuNþ cells and giving rise to a novel DCX/NeuN transitional phenotype. Pax6-transduced cells mostly displayed neuronal morphology and maturation at both time points examined; however, inducing early differentiation resulted in abnormal granule cell dendritic arborization. Potentially, this atypical granule cell morphology may represent differentiation into interneurons, as has been suggested for NG2-derived cells [10, 39]. Despite Pax6-induced rapid maturation there was no increase in net neurogenesis at 3 weeks relative to control, but rather a decrease in the number of virally transduced cells. It may be that Pax6 overexpression hastened maturation (increased NeuN expression) and reduced the time spent as a transient-amplifying cell (DCX expression), thus causing loss of transduced proliferating immature progenitor cells contributing to the lower number of newly generated cells at 3 weeks. It is also possible, that early maturation forced cells to terminally exit cell cycle prematurely so that fewer GFPþ cells were detected. In this case, a therapeutic increase of neurogenesis may require an expansion of progenitor populations prior to delivery of a lineage-instruction signal. The decrease at 3 weeks was despite a transient elevation in overall proliferation induced by Pax6 gene delivery when assessed by BrdU-only (thymidine analog-labeled cells include both virally infected and noninfected progenitor cells). Increased proliferation may be an indirect response of the niche due to forced cell cycle exit or death of progenitor cells as a result of Pax6 pressure. In turn, disruption of proliferation of Pax6-GFPþ cells may be on a background of the niche s transient increased expression of mitogens. Blocking Olig2 Function Delays Neuronal Maturation This is the first study to estimate the influence of a gliogenic factor on adult hippocampal neurogenesis. Our data demonstrate that Olig2 function is required for the proper maturation of newly generated neurons in the hippocampal niche. Although blocking Olig2 signaling increased the transient DCX progenitor population, relatively few cells expressed NeuN at either time. In contrast to Pax6, Olig2-VP16 acts negatively on the neuronal population by slowing maturation of neuronal progenitor cells resulting in a decrease in net neurogenesis. At 3 weeks, only 39% of transduced cells followed a neuronal lineage. These newly generated neurons together with a large number of marker-negative (N.D.) GFP-expressing cells exhibited characteristic morphology of type-2 and type-3 progenitor cells such as bipolarity and few processes [32, 33]. Olig2-VP16-tranduced cells thus may be arrested at a transient amplifying progenitor stage. Interestingly, the modulation of cell fate decision in the relatively small number

9 508 Pax6 and Olig2 in Adult Hippocampal Neurogenesis of infected cells stimulated a proliferative response among the general progenitor cell pool. Results from BrdU and IdU pulsing showed a significant increase in the number of proliferating cells after Olig2-VP16 transgene expression. However, no difference was found in the ultimate survival of these newly generated cells. The transient elevation of proliferation may reflect increased expression of mitogens as a homeostatic response of the niche following retroviral gene delivery. Pax6 Expression Selectively Eliminates Oligodendrocytic Lineage Under normal conditions, Pax6 is not expressed in NG2þ cells in the adult DG. However, we are able to retrovirally target dividing NG2 cells to express Pax6. NG2 glia precursor cells are multipotent ex vivo [40] and following transplantation [10]. While Pax6 expression did not change the number of NG2þ cells at 5 days, fewer cells were detected at 3 weeks, and no mature oligodendrocytes were observed relative to control at either time. Here, elevated Pax6 exerted a selective pressure on NG2þ cells, reducing cell survival and thus preventing their differentiation into oligodendrocytes. As there is no induction of alternate glial fate, it is likely that Pax6 expression leads to the loss of NG2 cells and their progeny or indirectly impairs NG2 cell survival by directing a neuronal fate. The selective drive to neuronal versus glial fate decision (a cell fate switch) appears to limit the total progenitor pool and in turn decreases the number of lineage-traced GFPþ cells found at 3 weeks. Blocking Olig2 Function Induces Astrocytic Lineage Commitment of Hippocampal Precursor Cells Olig2 is normally expressed by oligodendrocytes throughout the CNS, and precursor cells during development [24, 25], and is present in radial glia astrocytes and transient-amplifying progenitors in the postnatal and adult SVZ, where it functions as a regulator for neuronal-glial fate decisions [20, 26]. Olig2 mrna is also expressed in the adult mouse hippocampus [41] but we detected few positive cells by immunohistochemistry in the rat hippocampus. One result of delayed neuronal maturation caused by Olig2-VP16 in this study is the generation of a significant number of astrocytes. This is in line with previous studies of neurospheres cultures and injury models, where Olig2-VP16 transduction decreased the number of oligodendrocytes and neurons, suggesting a role for Olig2 in neuronal-glial fate decision by acting as a repressor of the astrocytic lineage [28]. We show that blocking Olig2 function can direct adult hippocampal precursor cells, largely committed to a neuronal lineage, toward a glial fate, consistent with a previous report where early lineage instruction with mash1 directed cell fate in the DG [27]. We suggest that blocking Olig2 in the progeny of type-1 cells results in their direction to astrocytes at very early stages of fate decision, for example, type-2a cell stage that marks a transition between glial and neuronal differentiation [42], while blocking Olig2 in cells at later stages of commitment (type-2 and type-3) has a negative effect on neuronal lineage commitment as seen by distinct changes in their morphology, maturation, and survival. Interestingly, our results showed an increase in GFAPþ cells, but not S100bþ astrocytes. One possibility is that Olig2 inhibition affects GFAPþ astrocytes that potentially could be type-1 stem cells. However, based solely on morphology, no lineage traced GFPþ type-1 stem cells were found. This may be a technical limitation due to the low probability of targeting type-1 cells by retroviral infection. Furthermore, there was no alteration of the NG2 cell population following Olig2-VP16 expression, but there was a disturbance in their commitment to an oligodendrocytic lineage over time. CONCLUSIONS Consistent with earlier studies, GFP-only transduced cells largely matured into granule neurons over time [27, 33]. However, our results establish that at least a quarter of newly generated cells showed commitment to a gliogenic lineage, which in turn was susceptible to engineered expression of the transgenes used in this study. Our findings demonstrate that inductive signals can not only positively regulate cell fate outcome but also produced a selective pressure on the niche, for example, a shift in the ratio of neurogenesis versus gliogenesis that in turn produced a homeostatic response as shown by an elevation in overall proliferation. In addition, we showed the competence of multiple progenitor cell populations to respond to the same lineage-instruction signal. Thus, targeting the expression of one lineage-instruction factor to the adult neurogenic niche may have multiple consequences. Furthermore, in the absence of other influences on the neurogenic niche, there appears to be an upper limit on neuronal production. While homeostasis is enforced in this direction, there does not seem to be an immediate compensatory response to restore phenotype ratios following redirection of cell fate or loss within progenitor cell pools. Therefore, increasing output of a particular mature cell phenotype from the neurogenic niche will likely require a combination of lineage-instruction factors with other signals favorable for cell survival and differentiation. The development of such strategies will be required to manipulate neurogenesis for therapeutic use. ACKNOWLEDGMENTS We thank Magdalena Goetz for the pmxig Pax6 and pmxig Olig2-VP16 constructs, Sarah Schuck and Carol Galioto for technical assistance, and Rupert W. Overall and Isabelle Aubert for critical reading of the manuscript. This work was supported by NIH awards AG20047 and AG22555 and DoE award DE-SC to D.A.P. F.K. is currently affiliated with ISCRM, University of Washington, Seattle, Washington. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conflicts of interest. REFERENCES 1 Kempermann G, Kuhn HG, Gage FH. More hippocampal neurons in adult mice living in an enriched environment. 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