gfap-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse...

A R T I C L E S

Compelling evidence indicates that neurogenesis occurs continuallyin two sites in the adult mammalian forebrain. Neural stem or pro-genitor cells (referred to here as ‘neural progenitors’) that reside inperiventricular tissue known as the subventricular or subependymalzone (SEZ) of the lateral ventricle, and in the subgranular zone(SGZ) of the hippocampal dentate gyrus, give rise to migratory neu-roblasts that become neurons in granule cell layers of the olfactorybulb and the dentate gyrus, respectively1–5. The identity and biologyof neural progenitors in the adult brain are of considerable interest,reflecting their possible roles in health and disease and their poten-tial for treating neurological illness.

Conflicting evidence has been presented regarding the cellular iden-tity of adult neural progenitors. A report that some ependymal cells areneural progenitors6 has not been confirmed by multiple in vivo and invitro studies7–10. A proposal that adult neural progenitors express theintermediate filament GFAP and share ultrastructural characteristicswith astroglia9 has found experimental support, but has also met withcontroversy. Single cell lineage analyses using retrovirally introducedtransgenes indicate that at least a portion of neural progenitors in latepostnatal and adult SEZ and SGZ express GFAP in vivo and in vitro9–11.Using transgenically targeted cell ablation, we found that the predomi-nant population of adult forebrain neural progenitors that forms mul-tipotent neurospheres in vitro expresses GFAP12,13. In contrast, usingflow cytometry, one study found no immunohistochemical evidencefor GFAP expression14, whereas another found GFAP immunoreactiv-ity in only a portion7 of partially purified populations of adult SEZ cellsthat show characteristics of neural progenitors in vitro.

The neurogenic potential of cells can be reprogrammed by in vitroconditions15, and there is uncertainty over which types of cells have

the potential to form neurospheres in vitro. For this reason, the rela-tionship between the neurogenic potential of a cell type in vitro andits contribution to constitutive neurogenesis in vivo is uncertain. Wetherefore investigated the cellular identity of adult multipotent neuralprogenitors directly in vivo using two transgenic targeting strategies.One strategy combined the targeted expression of herpes simplexvirus thymidine kinase (HSV-TK) with delivery of the antiviral agentganciclovir to achieve the specific and inducible ablation of dividingGFAP-expressing cells. The other strategy allowed fate mapping ofprogeny cells derived from GFAP-expressing cells by using the tar-geted expression of Cre recombinase (Cre) to excise a loxP-flankedstop signal and activate reporter gene expression from an independ-ent ubiquitous promoter. Our findings show that regularly dividingGFAP-expressing cells with bipolar or unipolar morphology in theSEZ and SGZ are the predominant neural progenitors responsible forconstitutive neurogenesis in the adult mouse forebrain.

ResultsSpecificity of transgenically targeted TK expressionTo determine the effects on adult neurogenesis of ablating putativeGFAP-expressing progenitors, we used transgenic mice in whichHSV-TK is expressed from the GFAP promoter using a large pro-moter cassette (15 kb) comprising the full sequence of the mouseGFAP gene16. In these GFAP-TK mice, the antiviral agent ganciclovir(GCV) selectively ablates dividing cells expressing both GFAP and TKin vivo and in vitro12,16,17. Expression of TK alone does not cause celldeath. The ablation of dividing transgene-expressing cells is inducedin normally developed adult mice by delivery of GCV. Only dividingcells are killed by the combination of TK and GCV; nondividing

Department of Neurobiology and Brain Research Institute, University of California, Los Angeles, California 90095-1763, USA. Correspondence should be addressedto M.V.S. ([email protected]).

Published online 24 October 2004; doi:10.1038/nn1340

GFAP-expressing progenitors are the principal sourceof constitutive neurogenesis in adult mouse forebrainA Denise R Garcia, Ngan B Doan, Tetsuya Imura, Toby G Bush & Michael V Sofroniew

Establishing the cellular identity in vivo of adult multipotent neural progenitors is fundamental to understanding their biology. We used two transgenic strategies to determine the relative contribution of glial fibrillary acidic protein (GFAP)-expressingprogenitors to constitutive neurogenesis in the adult forebrain. Transgenically targeted ablation of dividing GFAP-expressing cells in the adult mouse subependymal and subgranular zones stopped the generation of immunohistochemically identifiedneuroblasts and new neurons in the olfactory bulb and the hippocampal dentate gyrus. Transgenically targeted cell fate mapping showed that essentially all neuroblasts and neurons newly generated in the adult mouse forebrain in vivo, and in adult multipotent neurospheres in vitro, derived from progenitors that expressed GFAP. Constitutively dividing GFAP-expressingprogenitors showed predominantly bipolar or unipolar morphologies with significantly fewer processes than non-neurogenicmultipolar astrocytes. These findings identify morphologically distinctive GFAP-expressing progenitor cells as the predominantsources of constitutive adult neurogenesis, and provide new methods for manipulating and investigating these cells.

NATURE NEUROSCIENCE VOLUME 7 | NUMBER 11 | NOVEMBER 2004 1233

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transgene-expressing cells survive GCV exposure in vivo and invitro12,17. Our previous studies showed that in GFAP-TK mice of the7.1 line used here, transgene-derived TK was detectable only inGFAP-expressing cells from various parts of the forebrain and periph-eral nervous system in vivo and in vitro12,16,17, including cells in theSEZ12,13. To validate this transgenic model for the study presentedhere, we used double-labeling immunohistochemistry to compareexpression of TK with that of GFAP specifically in cells of both theSEZ and SGZ in mice from line 7.1 (Fig. 1a–h). Three-dimensionalanalysis of several hundred single cells using scanning confocal lasermicroscopy showed that 100% of TK-expressing cells also expressedGFAP in both the SEZ and SGZ (Fig. 1g–h,k). Cells that were TK pos-itive and GFAP negative were not found; GFAP-positive cell bodiesthat did not appear to contain detectable levels of TK were rare (<1%)in the SEZ and SGZ (Fig. 1k). These findings show at the single-celllevel that TK expression is restricted to GFAP-expressing cells in theSEZ and SGZ, and that these GFAP-TK mice are appropriate for the

selective ablation of dividing GFAP-expressing putative neural pro-genitors in the SEZ and SGZ.

Some dividing cells in SEZ and SGZ express GFAP and TKThe hypothesis that adult neural progenitors express GFAP predictsthe presence in the SEZ and SGZ of regularly dividing GFAP- and TK-expressing cells in GFAP-TK mice. To test this prediction, GFAP-TKmice were injected with bromodeoxyuridine (BrdU) to label cells inthe S phase of cell division and killed 2 h later. Several hundred cells inthe SEZ and SGZ were analyzed individually by three-dimensionalscanning confocal microscopy after double- and triple-labelingimmunohistochemistry (Fig. 1i–k). BrdU labeling was largely con-fined to the nucleus, whereas GFAP was present predominantly in cellprocesses and TK was often present in both cell body and proximalcell processes (Fig. 1c,f,h,j). Three-dimensional analysis of triple-stained cells showed that many BrdU-labeled cells that had little or noGFAP in the cell body nonetheless had unequivocally GFAP-positivefilaments in proximal processes identified by TK staining, and were,in fact, GFAP-expressing cells. Thus, double staining for BrdU andGFAP alone might underestimate the true number of dividing GFAP-positive cells. Because our analysis had shown that all TK-expressingcells expressed GFAP in the SEZ and SGZ, we quantified the numberof dividing TK-expressing cells that were labeled with BrdU and viceversa (Fig. 1k). In the SEZ, 11.5% of BrdU-labeled cells were positivefor TK and 9.4% of TK-positive cells were positive for BrdU (Fig. 1k).In the SGZ, 68.1% of BrdU-labeled cells were positive for TK, and12.8% of TK-positive cells were positive for BrdU (Fig. 1k). Thesefindings show that in both the SEZ and SGZ only about 10–12% ofthe total population of TK- and GFAP-expressing cells were in the S phase at a given time point. It is also of interest that whereas only11.5% of dividing cells expressed TK and GFAP in the SEZ, 68% didso in the SGZ, indicating that differences exist in the proportions ofdifferent types of cells undergoing cell division in these two neuro-genic regions. These relative percentages of dividing cells that expressGFAP in the SEZ and SGZ agree well with previous findings obtainedusing retroviral labeling of dividing GFAP-expressing progenitors inthe SEZ and SGZ9,11.

Effects of ablating dividing GFAP-TK cells in SEZ and SGZWe next determined the effect of ablating dividing GFAP-expressingputative neural progenitors on the total population of actively divid-ing cells in the SEZ and SGZ, and on the population of chemically

1234 VOLUME 7 | NUMBER 11 | NOVEMBER 2004 NATURE NEUROSCIENCE

Figure 1 Expression of transgene-derived HSV-TK and GFAP and labeling byBrdU in the SEZ and SGZ of adult GFAP-TK mice. (a,b,d,e) Surveys (a,d) anddetails (b,e) of coronal sections through the lateral periventricular region (a) and the hippocampal dentate gyrus (d) stained with two-color bright-fieldimmunohistochemistry for GFAP (blue-gray) and TK (brown). All TK-positivecell bodies are associated with GFAP-positive cell processes, including cellsin the SEZ (b) and the SGZ (e). (c,f–j) Confocal micrographs of single opticalslices through cells in the SEZ (c,g,i) and the SGZ (f,h,j) that are double-stained (c,f–h) or triple-stained(i,j) by immunofluorescence for GFAP, TK andBrdU. (g–j) Individual channels, merged images and orthogonal analysis showthat all TK-expressing cells in the SEZ (g,i) and the SGZ (h,j) also expressGFAP, and that some of these cells also label with BrdU (i,j) administered 2 hbefore tissue harvest. Orthogonal images (ortho) show three-dimensionalanalysis of individual cells at specific sites marked by intersecting lines in thex, y and z axes, as used for quantitative analysis of double or triple labeling.Note that TK is often present in proximal cell processes (arrows, c,f,h). (k) Quantitative analysis of cells double labeled for TK and GFAP or BrdU; n = 6 mice analyzed for each value. GCL, granule cell layer; Hil, hilus; LV,lateral ventricle; SEZ, subependymal zone; SGZ, subgranular zone.

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identifiable putative neuroblasts. To do so, we gave GFAP-TK micesingle daily injections of elaidic acid ganciclovir (eGCV). The numberof dividing cells was determined at successive time points by stereo-logical quantification of cells labeled with BrdU at 2 h after an injection of BrdU. In addition, migrating neuroblasts wereimmunohistochemically identified by staining for polysialicacid–neural cell adhesion molecule (PSA-NCAM)3,18. The numbersof BrdU-labeled cells did not differ significantly (ANOVA, P > 0.2) inthree groups of control mice (nontransgenic or GFAP-TK mice givenvehicle, or nontransgenic mice given eGCV), and control data werepooled for quantitative analysis. In contrast, in GFAP-TK mice giveneGCV the number of BrdU-labeled cells in both the SEZ and SGZdeclined substantially, falling to 18.6% and 29.6% of controls, respec-tively, after 20 d of eGCV (Fig. 2a–h). The rapid decline in the numberof BrdU-labeled cells in the SEZ was most probably caused not onlyby the ablation of resident GFAP-expressing neural progenitors,which comprise only about 12% of the population of BrdU-labeledcells, but also by the migration away from the SEZ of previously gen-erated migratory amplifying cells and neuroblasts that form themajority of cells that label with BrdU in the SEZ; these migratory cellsreach the olfactory bulb within days of being generated in the SEZ3.Because dividing migratory cells are no longer formed from progeni-tors in GFAP-TK mice given GCV, BrdU labeling would fall off rap-idly as previously formed cells migrate away. The decline seemsinitially less rapid in the SGZ, from which migratory neuroblasts donot leave. Labeling with BrdU also declined in the rostral migratorystream (RMS) and was essentially absent after 20 d of eGCV (data notshown). In agreement with the BrdU findings, the number of cellsstained for the migrating neuroblast marker, PSA-NCAM, declinedprogressively in the SEZ and SGZ with eGCV treatment, and few PSA-NCAM-stained cells remained after 20 d (Fig. 2i–n). In addition,there was no detectable damage to basic tissue structure in immuno-histochemically stained sections (Fig. 2c,h,k,n) or in neighboring sec-tions stained with a general cytological stain (data not shown).Together, these findings showed that chronic ablation of dividingGFAP-expressing cells progressively depleted the pool of activelydividing cells in forebrain germinal zones and by 20 d essentiallyeliminated the pool of immunohistochemically identifiable neurob-lasts in the SEZ and SGZ.

Figure 2 Depletion of BrdU labeling andneuroblast staining by progressive ablation ofdividing GFAP-TK cells in the SEZ and SGZ.(a–c,f–n) Survey images of coronal sectionsthrough the lateral periventricular region (a–c,i–k) and hippocampal dentate gyrus (f–h,l–n) stained by bright-fieldimmunohistochemistry for either BrdUadministered 2 h before tissue harvest (a–c,f–h), or the neuroblast marker PSA-NCAM(i–n), in control mice (a,f,i,l) and GFAP-TK micegiven eGCV) for 4 d (b,g,j,m) or 20 d (c,h,k,n).Note that in GFAP-TK mice, 20 d of eGCV hasdepleted BrdU- and PSA-NCAM–labeled cells from the SEZ and SGZ without disrupting tissue integrity (c,h,k,n). Arrows indicaterepresentative BrdU-positive or PSA-NCAM–positive cells. (d,e) Quantitative analysisshowing a statistically significant decline innumbers of cells stained for BrdU in both the SEZ(d) and the SGZ (e) in GFAP-TK mice treated witheGCV. Data points represent mean ± s.e.m.; * P < 0.001 versus controls (ANOVA with Dunnett’s test); control, n = 8; GFAP-TK mice, n = 6 each at 4 d, 8d, 12 d eGCV and n = 4 at 20 d eGCV. GCL, granule cell layer; LV, lateral ventricle; SEZ, subependymal zone; SGZ, subgranular zone; Str, striatum.

A R T I C L E S

Ablation of dividing GFAP-TK cells stops adult neurogenesisWe next determined the effect of chronic ablation of dividingGFAP- and TK-expressing cells on the generation of immunohisto-chemically identifiable new neurons in the adult olfactory bulb anddentate gyrus. To do so, GFAP-TK mice and nontransgenic micewere given chronic low-dose GCV or vehicle for 21 d via subcuta-neous osmotic minipumps. This chronic low dose of GCV wasdetermined empirically, after comparing several dosing regimens,to be effective in ablating dividing putative forebrain progenitors inGFAP-TK mice while avoiding the gut illness caused by high dosesof GCV or eGCV16. After 21 d, vehicle or low-dose GCV wasstopped; the mice were given four injections of BrdU over 2 d andwere killed 14 d after the last injection, an interval previously shownto be sufficient for neuronal maturation of cells generated in theadult forebrain in vivo3. Newly generated mature neurons wereidentified by double staining for BrdU and the neuronal markerNeuN1. In addition, newly generated migrating immature neuronswere identified by staining for doublecortin19–21.

GFAP-TK mice given chronic low-dose GCV had almost no newlygenerated migrating immature neurons in the SEZ, RMS or SGZ(Fig. 3a–f). At 14 days after BrdU treatment, these mice also showeda marked reduction in the numbers of BrdU-labeled cells in thegranule layers of both the olfactory bulb and dentate gyrus relativeto control mice (Fig. 3g–j). For quantitative comparison, BrdU-labeled cells were stereologically counted using bright-fieldmicroscopy, and scanning confocal laser microscopy was used toidentify neurons staining for BrdU and NeuN (Figs. 3k–n). Thenumbers of newly generated neurons were calculated by multiplyingthe total number of BrdU-labeled cells by the percentage of BrdU-labeled cells that were NeuN positive. Cell counts from three groupsof control mice (nontransgenic or GFAP-TK mice given vehicle, ornontransgenic mice given GCV) did not differ significantly(ANOVA, P > 0.2), and control data were pooled for quantitativeanalysis (Fig. 3o,p). In the GFAP-TK mice given GCV, the total num-ber of BrdU-labeled NeuN-positive granule neurons in the olfactorybulb was reduced to 0%, and in the dentate gyrus to 1.8%, of controlvalues (Fig. 3o,p). These findings show that ablation of dividingGFAP-expressing cells in SEZ reduced constitutive neurogenesis byover 98% in both the olfactory bulb and dentate gyrus.


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Taken together, the findings presented thus far show that regularlydividing GFAP-expressing cells are required for constitutive neuroge-nesis in the adult forebrain. The findings are consistent with, but do not prove, the hypothesis that these regularly dividing GFAP-expressing cells are the predominant adult neural progenitors. Thesedeletion experiments cannot rule out the possibility that dividingGFAP-expressing cells may merely provide induction signals or sup-port that are essential but not sufficient for neurogenesis22.Transgenically targeted fate mapping of progeny of GFAP-expressingcells provided a means to address this question further.

Transgenically targeted fate mapping of progenitorsTo map the fate of progeny of GFAP-expressing cells in vivo and invitro we used Cre/loxP technology23. We generated transgenic micethat express Cre recombinase (Cre) from the mouse GFAP promoterand crossed these mice with two strains of reporter mice carryinggenes for either β-galactosidase (β-gal) or enhanced green fluores-cent protein (GFP) under the regulation of ubiquitous promotersthat contain a loxP-flanked stop sequence24,25. In the resulting GFAP-

Cre reporter mice, cells that express Cre activate independent andconstitutive expression of the reporter molecule. Once activated,reporter is expressed even if GFAP and Cre are downregulated, andcontinues to be expressed in all progeny cells (Fig. 4a). Several linesof GFAP-Cre mice were generated and screened. All data presentedhere derived from line 73.12, in which qualitative analysis showedappropriate transgene expression (Fig. 4b–m). Quantitative analysisof line 73.12 using scanning confocal laser microscopy showed that100% of Cre-expressing cells also expressed GFAP in both the SEZand SGZ (Figs. 4i,j and 5m), and that no Cre-positive cells expressedNeuN in either the olfactory bulb or the dentate gyrus (Figs. 4k and5m). These findings show that these GFAP-Cre mice are appropriatefor the fate mapping of dividing putative GFAP-expressing neuralprogenitors in the SEZ and SGZ.

Adult multipotent neurospheres express reporter proteinPutative adult neural progenitors can be isolated from the adult fore-brain and grown in vitro as sphere-forming cell colonies26,27. Althoughthe exact relationship between neurosphere-forming cells in vitro andneural progenitor cells in vivo is not fully understood, the ability toform multipotent neurospheres is currently the best in vitro assay forthe presence of putative neural progenitor cells7. We therefore deter-mined whether neurosphere-forming cells in the adult SEZ derivedfrom a GFAP-expressing lineage. Single cell analysis conducted as pre-viously described for GFAP-TK mice12 showed that in GFAP-Cre miceof line 73.12, Cre was expressed only by GFAP-expressing cells in vitro.Examination of over 30 neurospheres per mouse prepared under bothclonal and nonclonal conditions from the SEZ of adult GFAP-Crereporter mice (n = 3) showed robust expression of reporter protein inall multipotent neurospheres (Fig. 5a–c). After differentiation of thesespheres, essentially all cells that expressed markers of neurons, oligo-dendrocytes and glia also expressed reporter protein (Fig. 5d–f). Thesefindings show that multipotent neural progenitor cells isolated fromthe adult SEZ and propagated in vitro derive from lineages of GFAP-expressing progenitors.

Adult glia and some neurons express reporter protein in vivoThe hypothesis that adult neural progenitor cells express GFAP pre-dicts that in GFAP-Cre reporter mice, reporter protein should be


Figure 3 Chronic ablation of dividing GFAP-TK cells stops the generation of new neurons in the adult olfactory bulb (OB) and dentate gyrus (DG). (a–j) Bright-field immunohistochemistry for the immature neuron markerdoublecortin (DCX, a–f) and for BrdU administered 14 d before tissue harvest(g–j) of control (Con) mice (a,c,e,g,i) and GFAP-TK mice given continuouslow-dose GCV for 21 d (b,d,f,h,j). In GFAP-TK mice, chronic GCV hasdepleted cells labeled for DCX from the SEZ (b), RMS (d) and SGZ (f), as wellas depleted BrdU labeling in the GCL of the OB (h) and the DG (j), withoutdisrupting tissue integrity. Arrows in i,j indicate representative BrdU-positivecells. (k–n) Confocal micrographs of single optical slices through cells in the OB (k,l) and the DG (m,n) after double-staining immunofluorescence forNeuN (green), and for BrdU (red) administered 14 d before tissue harvest. (l) is a detail of (k). Orthogonal images (l–n) show three-dimensional analysisof individual cells in x, y and z axes, as used for quantitative analysis ofdouble labeling; arrows indicate cells positive for both BrdU and NeuN,arrowheads indicate BrdU-positive/NeuN-negative cells. (o,p) Quantitativeanalysis showing a pronounced and statistically significant decline in thenumbers of new neurons (cells stained for both BrdU and NeuN) generated in the OB (o) and DG (p) of GFAP-TK mice treated continuously for 21 d withlow-dose GCV. Data points represent mean ± s.e.m.; * P < 0.01 versus control(t-test); control mice, n = 8; GFAP-TK mice, n = 6. GCL, granule cell layer; LV, lateral ventricle; RMS, rostral migratory stream; SEZ, subependymal zone;SGZ, subgranular zone; Str, striatum.

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expressed not only in astrocytes, but also in GFAP-expressing progen-itors and their progeny neuroblasts and neurons in adult neurogenicregions, the SEZ and SGZ. To test this prediction, we examined thephenotype of reporter-expressing cells at the single-cell level usingmultiple-labeling immunohistochemistry and confocal scanninglaser microscopy. In GFAP-Cre reporter mice, the overwhelmingmajority of cells that expressed reporter protein throughout the adultforebrain expressed GFAP and had the multipolar shape typical ofstellate astrocytes (for examples see Fig. 4c inset, 4l). Nevertheless,consistent with the hypothesis, both Cre and reporter protein wereexpressed in both the SEZ and SGZ (Fig. 4b,c,g,h) by both GFAP-pos-itive (Fig. 4i,j,m) and GFAP-negative cells (Fig. 4m). Reporter proteinwas also expressed in the RMS (Fig. 4d) and in the granule cell layersof both the olfactory bulb (Fig. 4e,f) and the hippocampal dentategyrus (Fig. 4h). Double labeling showed that reporter protein wasexpressed by neuroblasts positive for PSA-NCAM in the adult SGZ(Fig. 5g) and the RMS (data not shown), and by subpopulations of

NeuN-positive neurons in the granule cell layers of both the olfactorybulb and the hippocampal dentate gyrus (see Fig. 5i,k andSupplementary Fig. 1 online). In contrast, nearby regions for whichconstitutive adult neurogenesis has not been reported, such as thestriatum, did not contain any reporter-positive neurons (Fig. 5h,Supplementary Fig. 1), and the overwhelming majority of neuronsthroughout the forebrain did not express reporter protein (data notshown). These findings show that immunohistochemically identifiedadult neuroblasts and subpopulations of neurons in the two regionsknown to show neurogenesis in the adult forebrain derive from line-ages of GFAP-expressing progenitors, whereas the vast majority offorebrain neurons do not.

Newly generated adult neurons express reporter proteinTo determine whether newly generated neurons in adult mice derivedfrom GFAP-expressing cell lineages, we looked for expression ofreporter protein by cells that expressed mature neuronal markers andlabeled with BrdU. Adult GFAP-Cre reporter mice were given fourinjections of BrdU over 2 d and were killed 14 d after the last BrdUinjection. The granule cell layers of both the olfactory bulb and den-tate gyrus contained newly generated neurons that also expressedreporter protein, as shown by triple labeling for BrdU, NeuN andreporter (Fig. 5j,l), and these newly generated neurons were intermin-gled with NeuN-positive neurons that did not express reporter pro-tein and did not label with BrdU (Fig. 5j,l). Quantitative analysis ofsingle cells by scanning confocal laser microscopy after triple-labelingimmunohistochemistry showed that in the granule cell layers of theolfactory and the hippocampal dentate gyrus, 97.1% and 91.3%,respectively, of the cells that were double labeled for both BrdU andNeuN also expressed reporter protein (Fig. 5m). These findings showthat the vast majority of new neurons generated constitutively inthese two regions of the adult forebrain derive from lineages of GFAP-expressing progenitors, and that GFAP-expressing cells are not merelyproviding essential support for adult neurogenesis.

Dividing GFAP-expressing progenitors are bipolar or unipolarIn the adult CNS, GFAP expression is commonly associated withastrocytes, a differentiated multipolar cell type (see Fig. 4l). Our qual-


Figure 4 Cre/loxP-mediated fate mapping of GFAP-expressing progenitors.(a) Schematic of transgenically targeted fate mapping strategy. GFAP-Cremice are crossed with reporter mice. In GFAP-Cre reporter offspring, only cells that express Cre excise a loxP flanked stop signal and activateconstitutive reporter protein expression (Reporter*) by the cell and all of itsprogeny. (b–h) Survey images of coronal sections of the lateral ventricularregion (b,c), RMS (d), OB (f) and hippocampal dentate gyrus (g,h) stainedby bright-field immunohistochemistry. Both Cre (b,g) and the reporterprotein β-gal (c,d,f,h) are expressed by cells in the SEZ, RMS, and SGZ ofadult GFAP-Cre reporter mice. Arrows in b,c indicate representative Cre- or β-gal–positive cells in SEZ. Box and insert in c show a multipolar β-gal–positive cell with the morphology typical of astrocytes in the striatumadjacent to the SEZ. Box in e indicates region shown in f. (i–m) Confocalmicrographs of single optical slices through the SEZ (i) and SGZ/GCL (j–m) double stained by immunofluorescence for Cre (red) and GFAP(green) (i,j); Cre (red) and NeuN (green) (k); or reporter protein GFP (green) and GFAP (red) (l,m). Cells that express Cre also express GFAP (i,j), whereas cells that express Cre do not express NeuN or vice versa (k). (l) Typical multipolar astrocyte that expresses GFAP and reporterprotein. (m) The SGZ contains reporter-positive cells that are GFAP positive (arrows), as well as ones that are GFAP negative (arrowheads).GCL, granule cell layer; Gl, glomerular layer, LV, lateral ventricle; RMS,rostral migratory stream; SEZ, subependymal zone; SGZ, subgranular zone;Str, striatum.

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A R T I C L E S

itative observations indicated that GFAP-expressing putative neuralprogenitors in the SEZ and SGZ had fewer processes than did typicalstellate astrocytes (see Figs. 1b,c,f,h,j and 4i,j). We therefore quantita-tively compared the morphology of actively dividing, BrdU-labeledGFAP- and TK-expressing progenitors in the SEZ and SGZ (see Fig. 6a,b and Supplementary Fig. 2 online) with that of nondividingstellate astrocytes in the nearby striatum and hippocampus (Fig. 6d)by counting the number of processes arising directly from the cellsoma. Nondividing astrocytes in the striatum and hippocampal CA1had a mean of 4.3 ± 0.2 and 4.0 ± 0.2 processes, respectively, that arosedirectly from the cell soma, whereas actively dividing BrdU-labeledGFAP- and TK-expressing cells in the SEZ and SGZ had a mean of 1.6± 0.1 and 1.2 ± 0.1 processes, respectively—significantly fewer than

astrocytes (P < 0.001; Fig. 6e). These findings show that in both theSEZ and SGZ, actively dividing GFAP-expressing neural progenitorshave a predominantly bipolar or unipolar morphology that ismarkedly different from the multipolar appearance of astrocytes.These bipolar and unipolar putative adult neural progenitors also dif-fer from astrocytes in their expression of developmental markers suchas vimentin (Fig. 6c).

DISCUSSIONWe used two different transgenic targeting experimental strategies toinvestigate the cellular identity of adult neural progenitors in vivo.Our combined findings show that GFAP-expressing cells with a bipo-lar or unipolar morphology in the SEZ and SGZ are the principleneural progenitors responsible for constitutive neurogenesis in theadult olfactory bulb and hippocampal dentate gyrus. In contrast, thevast majority of developmentally generated forebrain neurons,including those in the olfactory bulb, hippocampus and cerebral cor-tex, do not derive from GFAP-expressing cells.

Technical considerationsTransgenes insert randomly into the genome, so that transgene expres-sion cannot be assumed to mimic endogenous promoter activity.Nevertheless, transgene expression is stable within transgenic breedinglines and, once characterized, is a useful tool28. Transgene expressionshould be characterized at the single cell level in every tissue region stud-ied. We used a large promoter cassette containing the full sequence ofthe mouse GFAP gene to enhance the specificity of transgenic targeting.Our quantitative analyses showed that in the SEZ and SGZ of the trans-genic breeding lines used, transgene-derived TK and Cre expressionwere confined to GFAP-expressing cells, and that no NeuN-expressingneurons expressed Cre in the olfactory bulb or dentate gyrus.

Cell ablation using transgenically targeted HSV-TK and GCV hasbeen validated in many previous studies from various laboratories,


Figure 5 Adult neuroblasts and adult-born neurons express reporter protein in GFAP-Cre reporter mice in vitro and in vivo. (a,b) Live floatingneurosphere derived from the periventricular germinal zone of an adultGFAP-Cre reporter mouse and illuminated by phase contrast (a) orfluorescence (b) shows that the entire sphere expresses reporter protein GFP(green). Scale bars, 60µm. (c) Survey of differentiated neurosphere showingthat all cells express GFP (green). Scale bar, 40µm. (d–f) Double-labelingimmunofluorescence of a differentiated neurosphere shows that all cellsthatexpress markers of neurons (Tuj1, d), oligodendrocytes (O4, e) or astrocytes(GFAP, f) also express reporter protein (GFP, green). Scale bars, 20µm. (g) Confocal orthogonal three-dimensional analysis of a PSA-NCAM–positive(red) neuroblast expressing reporter protein β-gal (green) in the SGZ.Immunoreactivity for the extracellular PSA-NCAM (red) is patchy and on thesurface of the reporter-positive (green) cell body. (h–l) Confocal micrographsof single optical slices through cells in the striatum (h), OB (i,j) and DG (k,l)that are double (i,k) or triple (j,l) stained by immunofluorescence for β-gal orGFP (green) in combination with the neuronal marker NeuN (blue) and BrdU(red) administered 14 d before tissue harvest. In the GCL of both the OB (i) and DG (k), but not in the striatum (h), a portion of NeuN-positiveneurons expresses reporter protein (arrows). See Supplementary Figure 1for individual channels of h,i,k. In both the OB and DG, adult-born neuronsin the GCL that are BrdU labeled and NeuN positive also express reporterprotein and are triple labeled and therefore appear white in mergedorthogonal (ortho) images (j,i). These triple-labeled newly born neurons areimmediately adjacent to NeuN-positive neurons that do not express reporterprotein and are not labeled with BrdU (j,i). (m) Quantitative analysis of thepercentages of cells double labeled for Cre and GFAP, or for Cre and NeuN,or triple labeled for reporter, NeuN and BrdU; n = 3 mice analyzed for eachvalue. DG, dentate gyrus; GCL, granule cell layer; OB, olfactory bulb; SGZ,subgranular zone; Str, striatum.

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including our own. These studies have shown that (i) GCV at the dosesused has no detectable effects on nontransgene-expressing cells, (ii)phosphorylated GCV blocks DNA synthesis and thereby selectivelykills transgene-expressing cells that are dividing and (iii) TK-express-ing cells do not release metabolites of GCV that are lethal to neighbor-ing, nontransgene expressing cells in vivo or in vitro12,16,17,29,30. Basedon these findings and on the extensive histological single cell analysesconducted here, we are confident that the cell ablation mediated by TKwas selective and specific for dividing GFAP-expressing cells, and thatthere was little, if any, loss of nondividing GFAP-expressing astrocytesor toxicity to other cell types in surrounding tissue.

Cell fate mapping using Cre/loxP recombination and activation ofreporter gene expression, as done here, has been validated in previousstudies from several laboratories31,32. Based on these findings and theextensive single cell analyses conducted here, we are confident that therecombination events mediated by Cre occurred selectively and specif-ically in GFAP-expressing cells, and that the reporter gene expressionwe observed was restricted to progeny of GFAP-expressing cells.

Intermediate filaments expressed by adult neural progenitorsAlthough GFAP expression is commonly used to identify differentiatedastrocytes in vivo and in vitro, numerous other cell types in CNS, liver,gut, kidney, lung and other organs express GFAP16,33,34, and this mole-cule cannot be considered an exclusive marker for astrocytes. GFAP isan intermediate filament protein, as are nestin and vimentin35. The

dynamically regulated expression of nestin and vimentin by neuralprogenitors and astrocytes at different stages of development, at matu-rity or after CNS injury is well documented and accepted35–37. A grow-ing body of evidence, including that presented here, now indicates thatGFAP is also dynamically regulated in neural progenitors during differ-ent stages of maturation in vivo and in vitro9–12. Nevertheless, two stud-ies based on flow cytometry reported no expression or only partialexpression of immunohistochemically detectable GFAP in partiallypurified populations of adult SEZ cells that show neurogenic (that is,neurosphere-forming) potential in vitro7,14, and considerable differ-ences exist in the numbers of BrdU and GFAP double-labeled cells inthe SEZ and SGZ found by different investigators. Our findings suggestthat negative findings based solely on GFAP immunohistochemicallymay be due to undetectable levels of GFAP in cell bodies as comparedwith cell processes.

The function of GFAP in adult multipotent neural progenitors inthe SEZ and SGZ remains unclear. Deletion of the GFAP gene seemsnot to have a grossly detectable effect on neurogenesis or gliogene-sis38,39. In spite of its uncertain function, our findings indicate thatGFAP is a marker that, in combination with BrdU-labeling and mor-phological analysis, can be used to identify morphologically distinc-tive adult neural progenitors in the SEZ and SGZ in vivo, and that theGFAP promoter can be used to target transgenic manipulations thatwill help dissect the biology of these cells. Deletion experiments arepowerful tools in biology. Previous studies have used pharmacologi-cal agents or localized irradiation that kill all dividing cells to targetthe ablation of adult neural progenitors and study their functionalroles40,41. Our findings extend these procedures by providing trans-genic models that more selectively target either cellular ablation orgene deletion to adult neural progenitors.

Identity and potential origin of adult neural progenitorsPrevious studies have reported that at least a portion of adult neuralprogenitors express GFAP9–11. We extend these findings to show thatGFAP-expressing progenitors are the predominant source of consti-tutive adult neurogenesis in vivo, and that GFAP-expressing adultmultipotent neural progenitors have a predominantly bipolar orunipolar morphology with markedly fewer processes than non-neu-rogenic multipolar astrocytes. These bipolar or unipolar GFAP-expressing progenitors also express vimentin (this study) andnestin-regulated transgene42, which are not expressed by differenti-


Figure 6 Dividing, GFAP-expressing progenitors in the adult SEZ and SGZ have a bipolar or unipolar phenotype that differs from nondividingmultipolar stellate astrocytes. (a–d) Projection confocal micrographs of cells that are triple stained by immunofluorescence for GFAP, TK and BrdU(a,b) or are double stained for TK and vimentin (c) or GFAP and DAPI (d). (a,b) Actively dividing putative neural progenitor cells in the SEZ (a) andthe SGZ (b) that are labeled with BrdU (injected 2 h before tissue harvest)and are stained for both TK and GFAP show a characteristic bipolar orunipolar shape with one or two processes arising directly from the cell body.See Supplementary Figure 2 for individual channels of a,b. (c) Unipolar TK-positive cell in the SGZ coexpresses vimentin (arrow), whereas a nearbymultipolar stellate TK-expressing astrocyte does not (arrowhead). (d) Typicalnondividing stellate astrocyte in hippocampal CA1 shows multiple processesarising from the cell soma. (e) Mean ± s.e.m. number of processes arisingdirectly from the cell body of BrdU-positive GFAP- and TK-expressing cells in the SEZ and SGZ, and BrdU-negative GFAP-expressing cells in thestriatum and CA1; n = 5 mice analyzed for each value (tissue collected 2 h after a single injection of BrdU). * P < 0.001 versus striatum and CA1(ANOVA with Tukey’s test). GCL, granule cell layer; LV, lateral ventricle; SEZ, subependymal zone; SGZ, subgranular zone.

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ated stellate astrocytes. These observations add to evidence indicatingthat GFAP-expressing neural progenitors are distinct from astrocytes.Astrocytes are cells that differentiate to achieve functional specializa-tion. Differentiated astrocytes throughout the CNS are not neuro-genic in vivo, and differentiated astrocytes taken from adult tissuedistant from the SEZ are not neurogenic in vitro10. There is, at pres-ent, no evidence that functionally differentiated astrocytes can dedif-ferentiate to become neurogenic. Instead, several lines of evidenceindicate a developmental origin and distinct identity for adult GFAP-expressing neural progenitors that does not require astrocytic differ-entiation to explain the expression of GFAP by these cells. Ventricularzone radial glia are neurogenic during development throughout theCNS32,43,44. Radial glia adopt GFAP expression at later stages of devel-opment, and some seem to differentiate into GFAP-positive astrocytesafter developmental neurogenesis is completed45,46. The predominantneural progenitor isolated from the mouse forebrain does not expressGFAP in early development, but does so in late development andadulthood12. A parsimonious model for the origin of adult GFAP-expressing progenitors is that they derive from periventricular neuro-genic radial glia that begin to express GFAP in late development butdo not differentiate into astrocytes, and instead retain multipotentneural progenitor potential past development in regions of the SEZthat provide a favorable environment. GFAP-expressing progenitorsin the SGZ may also derive from radial glia, which migrate into thedentate gyrus towards the end of development47.

METHODSAnimals. GFAP-TK transgenic mice (line 7.1) were generated using a 15-kbpromoter cassette containing the full sequence of the mouse GFAP gene16,17.This cassette (clone 445) contains all introns, promoter regulatory elements,exons and 2 kb of 3′ and 2.5 kb of 5′ flanking regions; expression of GFAP isprevented by the removal of a small fragment of the first exon48. GFAP-Cretransgenic mice (line 73.12) were generated using the same promoter cassette.ROSA (R26R) reporter mice24 were purchased from The Jackson Laboratory;Z/EG reporter mice25 were provided by C. Lobe (Toronto, Canada). Adultmice were used between 3 and 6 months of age. All mice were housed in a facil-ity with a 12-h light/dark cycle and allowed free access to food and water.Experiments were conducted according to protocols approved by the UCLAOffice for Protection of Research Subjects.

eGCV and GCV. Elaidic acid ganciclovir (eGCV), a potent lipophilic esterof ganciclovir that crosses the blood brain barrier efficiently49 (ClavisPharma AS), was given as single daily intraperitoneal (i.p.) injections at 100 mg/kg/d. Ganciclovir (GCV; Hoffman La Roche) was delivered subcu-taneously using osmotic minipumps at 10 mg/kg/d for 21 d. This low dosedid not cause gut illness as previously found when using 100 mg/kg16. GCVcrosses the blood brain barrier such that CSF levels are about 30% of serumlevels (Roche data).

BrdU. BrdU (Sigma) was given as either a single i.p. injection of 200 mg/kg fol-lowed by perfusion with 4% paraformaldehyde after 2 h, or as four i.p. injec-tions of 200 mg/kg every 12 h followed by perfusion after 14 d.

Immunohistochemistry of tissue sections. Frozen tissue sections were preparedof 4% paraformaldehyde-fixed brains17. Bright-field immunohistochemistrywas performed using biotinylated secondary antibodies (Vector), biotin-avidin-peroxidase complex (Vector), and diaminobenzidine (brown; Sigma) or Vectorblue (Vector) as the developing agents. Fluorescence immunohistochemistrywas performed using AlexaFluor tagged secondary antibodies Alexa 488 (green),Alexa 568 (red), Alexa 350 (blue) or Alexa 633 (pseudocolor blue) (MolecularProbes). Primary antibodies used were rabbit anti–HSV-TK (1:10,000; ref. 17);mouse anti-GFAP (1:10,000; Chemicon, MAB3402); sheep anti-BrdU (1:5000;Maine Biotechnology Services, PAB105P); mouse anti–PSA-NCAM (1:2000,Chemicon, MAB5324); goat anti-doublecortin (1:500; Santa Cruz, sc8066);mouse anti-NeuN (1:1000; Chemicon, MAB377); rabbit anti–β-galactosidase(1:1000; ICN Pharmaceuticals, 55976); rabbit anti–green fluorescent protein

(1:1000; Molecular Probes, A-21311); Tuj1 (1: 1000; Covance, MMS-435P);mouse anti-O4 (1:1000; Chemicon, MAB345). 4′,6-diamidino-2-phenylindole(DAPI; Molecular Probes, D-1306) was used as a fluorescent counterstain.Sections stained for BrdU were pretreated with 2 M HCl for 30 min and neutral-ized with PBS before incubation in primary antibody. Stained sections wereexamined and photographed using bright-field and fluorescence microscopy(Zeiss), and scanning confocal laser microscopy (Leica).

Preparation and immunocytochemistry of neurospheres. Neurosphere cul-tures were prepared from the SEZ of adult GFAP-Cre reporter mice. After 12 din vitro, spheres were differentiated by plating on coated glass coverslips inbasal media in the absence of added growth factors. Differentiated cells werefixed in 4% paraformaldehyde and stained by immunofluorescence12.

Single cell analysis of coexpression of different molecules. Expression ofdifferent proteins by the same cells was evaluated using double- and triple-labeling immunohistochemistry combined with analysis of single cells in threedimensions by scanning confocal laser microscopy. Stacks of 0.6-µm-thickoptical slices (100 ×100 µm) were collected through the z axis (15–25 µm) oftissue sections of regions to be analyzed. Only cells contained entirely withinthe three dimensions of a stack were analyzed.

Morphometric and statistical evaluation. BrdU-labeled cells were countedusing a modified optical fractionator50 and stereological image analysis software(StereoInvestigator, Microbrightfield) operating a computer-driven microscoperegulated in the x, y and z axes (Zeiss). Areas to be counted were traced at lowpower and counting frames were selected at random by the image analysis soft-ware. Cells were counted using a 100× objective and DIC optics. For the dentategyrus, both hemispheres were evaluated on every twelfth section through therostrocaudal extent of the hippocampus and final cell counts were expressed astotal cell number per hippocampal dentate gyrus. For the olfactory bulbs andSEZ, two to three sections per region were analyzed and final cell counts wereexpressed as cells per mm3. The numbers of new neurons generated in the adultolfactory bulb and dentate gyrus were calculated by counting the total numberof BrdU-labeled cells and multiplying this number by the percentage of BrdU-positive cells that were also NeuN positive as determined by confocal analysis.Statistical evaluations were performed using Prism software (GraphPad).

Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTSThis work was supported by grants from the US National Institutes of Health(NINDS) NS042693 and NS47386, and a Stein/Oppenheimer Award. ADRG issupported by a Ford Foundation predoctoral fellowship. We thank M.E. Sislak andT. Chiem for technical assistance. We thank F. Myhren and Clavis Pharma foreGCV, and P. Borgese and Hoffman La Roche for GCV.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 30 June; accepted 18 August 2004.Published online at http://www.nature.com/natureneuroscience/

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gfap-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse...

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