In vivo analysis of quiescent adult neural stem cellsresponding to Sonic hedgehogSohyun Ahn1† & Alexandra L. Joyner1,2

Sonic hedgehog (Shh) has been implicated in the ongoingneurogenesis in postnatal rodent brains1,2. Here we adopted anin vivo genetic fate-mapping strategy, using Gli1 (GLI-Kruppelfamily member) as a sensitive readout of Shh activity, to system-atically mark and follow the fate of Shh-responding cells in theadult mouse forebrain. We show that initially, only a small popu-lation of cells (including both quiescent neural stem cells andtransit-amplifying cells) responds to Shh in regions undergoingneurogenesis. This population subsequently expands markedly tocontinuously provide new neurons in the forebrain. Our study ofthe behaviour of quiescent neural stem cells provides in vivoevidence that they can self-renew for over a year and generatemultiple cell types. Furthermore, we show that the neural stem cellniches in the subventricular zone and dentate gyrus are establishedsequentially and not until late embryonic stages.

Two regions of sustained neurogenesis in the adult mammaliannervous system are the subventricular zone (SVZ) of the lateralventricles, which provides interneurons of the olfactory bulb (OB)via the rostral migratory stream (RMS), and the subgranular zone(SGZ) of the dentate gyrus (DG) within the hippocampus, whichgenerates granule neurons (reviewed in refs 3–6). Although previousstudies have shown that the secreted protein Sonic hedgehog (Shh)increases cell proliferation in the SVZ and SGZ1,2 and is required fornormal proliferation in the SVZ2, it has not been determined whetherShh acts on putative quiescent neural stem cells and/or fast-dividingtransit-amplifying cells.

In order to investigate whether neural stem cells normally respondto Shh in the adult mouse brain, we used genetic techniques toidentify cells that respond to Shh. First, the SVZ and SGZ of adultmice (n ¼ 3) were analysed for co-expression of Gli1-nlacZ, which isa sensitive readout of positive Shh signalling7,8, and glial fibrillaryacidic protein (GFAP), which marks neural stem cells9,10 as well asmature astrocytes. Shh-responding (nuclear lacZþ) cells in theventral SVZ and SGZ co-expressed GFAP (25.3% and 18.8%,respectively), suggesting that neural stem cells respond to Shhactivity (Fig. 1b, c). In addition, 18% of Shh-responding cells inthe SVZ and 36% in the SGZ co-expressed polysialic acid-neural celladhesion molecule (PSA-NCAM, Supplementary Table 1), a neuro-blast/neural precursor marker. Co-labelling with Dlx2 (distal-lesshomeobox 2; ref. 11, data not shown) or Olig2 (oligodendrocytetranscription factor 2; ref. 12, Supplementary Fig. 1; 57% ofShh-responding cells), which are markers of transit-amplifyingcells, showed that fast-dividing transit-amplifying cells also respondto Shh signalling. However, no Shh-responding cells were found inthe RMS and OB, the target structures of the cells generated in theSVZ, suggesting that only neural stem cells and their immediateprecursor cells respond to Shh activity.

The only definitive test to show that Shh signals to neural stem cellsin vivo would be to mark the Shh-responding cells and determinewhether they self-renew and continuously give rise to matureneurons. To mark and follow Shh-responding cells in the SVZ andSGZ in vivo, we performed genetic fate mapping using Gli1-CreERT2

mice, in which an inducible Cre recombinase (CreERT2) isexpressed from Gli1 (ref. 13). Upon tamoxifen treatment ofGli1-CreERT2;R26R mice, CreERT2 is transiently activated for,30 h, resulting in permanent expression of cytoplasmic lacZ fromthe R26R locus in Gli1-expressing cells and all their progeny13,14.Initially, a small number of Shh-responding cells in the SVZ and SGZof adult Gli1-CreERT2;R26R mice (n ¼ 3) were labelled (Fig. 1e, h).Owing to the mosaic nature of our genetic fate mapping approach,not all Shh-responding cells are labelled following tamoxifen treat-ment13,14. However, marker expression analysis of the initial popu-lation revealed that the distribution of different marked cell types wassimilar to that of Gli1-nlacZ mice (Supplementary Table 1). Thus, theinitially marked population faithfully represents the Shh-respondingcells in the SVZ and SGZ.

The long-term fate of the marked cells analysed six months aftertamoxifen treatment revealed a marked increase in the number oflabelled cells in Gli1-CreERT2;R26R mice (n ¼ 5) in both the SVZand DG (Fig. 1g, i and data not shown). In addition, although nolabelled cells were detected in the RMS or OB at the initial timepoint (Fig. 1d and data not shown), long-term fate mappingshowed labelled cells throughout the RMS and OB (Fig. 1f anddata not shown), demonstrating that the small initial populationof Shh-responding cells in the SVZ was amplified to generatelabelled cells in the OB that had migrated via the RMS. Also, inthe DG of the hippocampus, many labelled cells had integratedinto deeper layers of the granular zone after six months (Fig. 1i,black bracket) compared with the initial Shh-responding popu-lation that was primarily confined to the SGZ (Fig. 1h, redbracket). Taken together, our long-term fate-mapping resultsindicate that Shh-responding cells in the SVZ and SGZ includeneural stem cells that can generate progeny over time in theirrespective target structures.

To determine whether Shh-responding cells include the quiescentneural stem cells that rarely divide, rather than only the fast-dividingtransit-amplifying cells with a limited lifespan, we used AraC, ananti-mitotic reagent that effectively kills fast-dividing cells in the SVZand SGZ while sparing quiescent neural stem cells15. Two days aftertamoxifen treatment, AraC was infused for one week into the brain ofGli1-CreERT2;R26R mice using a mini-osmotic pump. At the end ofAraC treatment (day 0), PSA-NCAM immunoreactivity in whole-mounts of the lateral wall of the lateral ventricles (n ¼ 3) showed thatthe neuroblasts were almost completely depleted (Fig. 2b and data

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1Howard Hughes Medical Institute, Developmental Genetics Program, Skirball Institute of Biomolecular Medicine and Department of Cell Biology, and 2Department of Physiologyand Neuroscience, New York University School of Medicine, 540 First Avenue, New York, New York 10016, USA. †Present address: Unit on Developmental Neurogenetics,Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Building 6B,Bethesda, Maryland 20892, USA.

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not shown), confirming the efficacy of AraC treatment. Eliminationof fast-dividing precursors/neuroblasts by AraC treatment wasconfirmed on sections through the SVZ and DG, where only oneof 69 and one of 63 lacZþ cells (respectively) showed PSA-NCAMimmunoreactivity (Supplementary Table 1). In addition, Dlx2expression was abolished in the SVZ and was markedly diminishedin the RMS (Supplementary Fig. 2 and data not shown).

At the end of AraC treatment, 92.2 ^ 1.0% and 93.7 ^ 4.7% ofthe remaining labelled cells in the SVZ and SGZ, respectively, co-expressed GFAP (data not shown), indicating that quiescent neuralstem cells respond to Shh signalling in vivo. Strikingly, within oneweek of AraC removal (n ¼ 5), the number of labelled cells in theSVZ and DG of AraC-treated mice had increased more than in

control mice (n ¼ 4) (Fig. 2h–j and data not shown). No labelled cellswere observed in the OB at the end of AraC treatment (Fig. 2c), butlabelled cells were present in the granule cell layer of the OB one weekafter AraC removal (Fig. 2g). The extent of labelling in AraC-treatedmice, while continuing to expand, was comparable with controlmice after two months (n ¼ 3 each, data not shown), threemonths (n ¼ 4 each, data not shown) and six months (n ¼ 3 each,Fig. 2k–n). These results provide additional strong evidence thatShh-responding cells in the SVZ and SGZ that survive AraC treat-ment are rarely dividing neural stem cells that respond to this insultby increasing cell proliferation and the production of transit-amplifying cells.

Even though neural stem cells isolated from the SVZ and SGZ havecharacteristics of stem cells in vitro, including self-renewal andgeneration of multiple cell types10,16,17, it has not been demonstratedwhether quiescent neural stem cells in vivo self-renew or generate celltypes other than neurons in their respective target structures18,19.Previous lineage-tracing experiments have relied on the rareinfection of neural stem cells and transit-amplifying cells withreplication-defective retroviruses20,21 or BrdU labelling of progenitor

Figure 1 | Neural stem cells respond to Shh and expand over time in vivo.a, Left panel shows a schematic sagittal section of adult mouse brainwith theneurogenic regions and migratory paths indicated in red lines. Black areaindicates the lateral ventricle. The levels of coronal sections shown in panelsb–i are indicated by vertical lines. The two right panels show schematics ofhemi-coronal sections of adult forebrain at the level of the subventricularzone (SVZ) and the dentate gyrus (DG). b, c, Confocal images of singleoptical slices show expression of GFAP (green; neural stem cell) and nlacZ(red; Shh-responding cell) in the SVZ (b) and DG (c) of two-month-oldGli1-nlacZ mice. Arrowheads indicate double-labelled cells and the insetsshow the orthogonal analysis of a representative cell marked in a box.d–i, Genetic fate-mapping of Shh-responding cells over time usingGli1-CreERT2;R26R mice. Short-term fate mapping (1 week) shows nolabelled cells in the olfactory bulb (OB, d) and few cells in the SVZ (e) andDG (h). Six months after treatment with tamoxifen, many labelled cells arefound in the OB (f), SVZ (g) and DG (i). The red bracket in h indicates thesubgranular zone (SGZ) and the black bracket in i indicates the granularzone (GZ). Scale bars, 20 mm (b, c), 50mm (e–i).

Figure 2 | Quiescent neural stem cells respond to Shh signalling. a, Aschematic of the experimental time course. Tamoxifen (TM) wasadministered on two consecutive days (arrows) and AraC or saline wasinfused for one week. Forebrains of operated mice were analysed at theindicated survival time. b, Left panel shows a schematic sagittal section ofadult mouse forebrain. Vertical lines indicate the level of coronal sectionsshown in panels c–n. Yellow box indicates the area of the lateral wall of theSVZ analysed for PSA-NCAM (neuroblast marker) immunoreactivityshown in the right panels. One week of AraC treatment effectively abolishedthe proliferating neuroblasts from the lateral wall of the lateral ventriclecompared with control treatment. c–n, Fate-mapped cells were analysed atthe end of AraC treatment (day 0, c–e), and one week (g–i) and six months(k–m) after AraC treatment, and compared to saline-treated cells at thecorresponding time points (f, j, n). Shh-responding cells survived the anti-mitotic treatment with AraC and expanded over time in the SVZ and DG.Arrows indicate the few cells labelled after AraC treatment. Red bracketsindicate the subgranular zone (SGZ) and black brackets indicate thegranular zone (GZ) of the DG. Scale bars, 50mm.

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cells in their last division22,23 to determine the long-term fate ofneural stem cells. We took advantage of our efficient method formarking quiescent neural stem cells in the adult to determine theirlong-term fate after one year (Fig. 3). We first concentrated on theself-renewal capacity of the quiescent neural stem cells that respondto Shh signalling. The percentage of labelled cells co-expressing GFAPin the SVZ had increased significantly (P , 0.05) one year aftermarking (from 23.2 ^ 1.6% initially to 34.2 ^ 4.7% after one year,values mean ^ s.d.) and in the SGZ (from 18.8 ^ 3.3% initially to30.3 ^ 5.3% after one year). In addition, the absolute number oflabelled cells co-expressing GFAP in the SVZ and SGZ had increased,suggesting that Shh-responding neural stem cells had self-renewed togenerate more neural stem cells in both AraC-treated and controlmice (Fig. 3d, e and Supplementary Table 1). To further demonstratethe ability of Shh-responding neural stem cells to self-renew,Gli1-CreERT2;R26R mice (n ¼ 2) that were treated with AraC fol-lowing tamoxifen administration were allowed to recover for threemonths and then subjected to another round of AraC treatment.Even after a second round of eliminating the fast-dividing precursors,the remaining neural stem cells that were marked before the firstAraC treatment survived and generated more PSA-NCAMþ neuro-blasts (Supplementary Fig. 3). Together, these data show thatquiescent neural stem cells respond to Shh signalling and self-renew in vivo.

In addition to the GFAPþ cells that remain in the SVZ, many PSA-NCAM and lacZ co-expressing cells were observed in the dorsolateralSVZ (data not shown), where neuroblasts exit the SVZ to enter theRMS en route to the OB. In the anterior RMS, most of the lacZþ cellswere co-labelled with PSA-NCAM (Fig. 3c). Thus, quiescent neuralstem cells in the SVZ actively produce new neuroblasts even one yearafter marking. In the OB, the vast majority of labelled cells wereneurons as shown by their co-expression of lacZ and NeuN(92.3 ^ 4.5%, Fig. 3b) as well as their elaborate dendritic arboriza-tions. In the periglomerular region, there was extensive co-expression of lacZ and g-aminobutyric acid (GABA) or tyrosinehydroxylase, whereas labelled granular neurons typically expressedonly GABA (Supplementary Fig. 4). We also detected a few GFAP-expressing fate-mapped cells within the OB (3.5 ^ 0.5%, n ¼ 3)(Fig. 3a). This suggests that although most of the progeny generatedfrom the SVZ become interneurons of the OB, some also become

astrocytes in the OB. In addition, there were a few marked oligoden-drocytes in the corpus callosum (Fig. 3g, h). In the DG, the majorityof fate-mapped cells co-expressed NeuN (82.2 ^ 7.8%), indicatingthat they had become granule neurons in the deeper layers of thegranular zone (Fig. 3f). A few lacZ-expressing cells in the deeper layerof the granular zone also expressed GFAP (3.3 ^ 0.7%), suggestingthat the neural stem cells had also generated astrocytes in vivo (Fig. 3eand Supplementary Table 1). Our results therefore demonstrate thatquiescent neural stem cells have the potential to generate multiple celltypes in vivo.

Our genetic fate-mapping technique provides an opportunity toexplore the genetic history of neural stem cells and the formation ofneural stem cell niches. To determine whether adult neural stem cellsin the SVZ and DG are derived from cells that had previouslyresponded to Shh in the embryo, we analysed the long-term fate ofShh-responding cells marked at various embryonic stages. No

Figure 3 | Shh-responding neural stem cells generate multiple cell typesin vivo. Long-term fate-mapped cells (one year after tamoxifen treatment)were analysed for co-expression of lacZ (red) and the indicated molecularmarkers (green). a–f, The vast majority of labelled cells are interneurons(NeuNþ) in the olfactory bulb (b) and the dentate gyrus (f), as well as afew astrocytes (GFAPþ, arrowheads in a, e). Fate-mapped neuroblasts

(PSA-NCAMþ) are found in the rostral migratory stream (RMS, c).Quiescent neural stem cells (GFAPþ) are found in the SVZ (d) and SGZ(e, arrows). g, h, Few fate-mapped cells in the corpus callosum areoligodendrocytes (arrowheads). CC1 and CNPA are oligodendrocytemarkers. Scale bars, 10mm (a–e, g, h), 20mm (f).

Figure 4 | Shh-responding neural stem cells are established during lateembryogenesis inmice. Tamoxifen was administered on E15.5 and E17.5 tomark cells responding to Shh at E16–17 and E18–19, respectively. One-month-old mouse forebrains show labelled cells in the OB (a, d) and ventralSVZ (b, e) when marked at both time points. Labelled subgranular zone(SGZ; red bracket and arrowhead) cells of the DG were seen by marking atE18–19 (f) but not at E16–17 (c). Scale bar, 50 mm. GZ, granular zone.

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labelled cells were found in the adult SVZ and SGZ when tamoxifenwas administered between embryonic days (E)7.5 and E11.5 (datanot shown). The appearance of marked cells was first observed in theSVZ when tamoxifen was given at E15.5, and labelled cells in the SGZdid not appear until tamoxifen was given at E17.5 (Fig. 4). This showsthat Shh does not act on forebrain neural stem cells until lateembryogenesis, and further suggests that the two stem cell nichesin the forebrain are established sequentially in mice. In addition, wefound that the Shh-responding cells in the DG marked at E17.5showed a different behaviour from cells marked in the adult. Cellsmarked as a consequence of tamoxifen treatment on E17.5 populateddeeper layers of the granular zone of the DG than cells marked in theadult (compare Figs 1i, 2n and 4f, black brackets), suggesting atemporal restriction in the extent of integration of newly generatedcells as the brain matures.

Our genetic fate-mapping studies demonstrate that Shh first actson stem cell niches in the SVZ and SGZ at late embryonic stages.Quiescent neural stem cells are then set aside and are regulated byShh signalling. We present in vivo evidence that the neural stem cellscan self-renew for at least a year and generate multiple celltypes over time. Hedgehog signalling has recently been implicatedin the stem cell biology associated with tissue repair and theprogression of tumours in many non-neural tissues24–26. Our in vivogenetic fate-mapping approach provides a unique opportunity todetermine whether in fact stem cells regulated by Hedgehog signal-ling have a role in cell replenishment of various organs, and toelucidate the mechanism by which they contribute to tissue repairand cancer.

METHODSFate mapping and drug treatment. Two- to three-month-old Gli1-CreERT2/þ;R26R/R26R adult mice were administered 10 mg tamoxifen (Sigma) in cornoil by oral gavage on two consecutive days using feeding needles (FineScience Tools). For AraC treatment (two days after tamoxifen treatment), amini-osmotic pump (model 1007D, Duret) was implanted to infuse either salineor 2% AraC (Sigma) for one week as described15. Brains of mice killed at the endof AraC or control treatment were designated as day 0 samples. For long-termsurvival experiments, the pump was removed one week after implantation andmice were later killed at the indicated survival time. The second application ofAraC was performed in the same way as the first AraC treatment.Immunostaining and analysis of brain tissues. Mice were perfused with 4%paraformaldehyde (PFA), processed for frozen sections, and stained with X-galas described on http://saturn.med.nyu.edu/research/dg/joynerlab/. For immuno-fluorescent staining, dissected brains were fixed in 4% PFA overnight at 4 8C and50-mm vibratome (Leica) sections were obtained. Free-floating sections werestained overnight at 4 8C with goat anti-b-galactosidase (1:500, Biogenesis) andone of the following antibodies: mouse anti-GFAP (1:500), anti-PSA-NCAM(1:300), anti-NeuN (1:200, Chemicon), anti-CC1 (1:20, Calbiochem), anti-CNPase (1:150), anti-MBP (1:200, SMI), rabbit anti-GABA (1:500, Sigma)and anti-Olig2 (1:20,000; ref. 27). Secondary antibodies for double labellingwere donkey anti-species conjugated with Alexa 488, Alexa 555 (1:500, Molecu-lar Probes) or FITC (1:200, Jackson ImmunoResearch). Fluorescent images werecaptured in 1.5-mm optical sections using a confocal laser-scanning microscope(LSM 510, Zeiss) and processed using Adobe Photoshop. Orthogonal analysiswas performed to confirm co-expression of two markers. Cells co-expressinglacZ and one of the various markers were counted and divided by total numberof lacZ-expressing cells in the indicated region. At least three sections eachthrough the SVZ and DG were analysed from one mouse. Data from at least threemice were pooled together to determine the average and standard deviation(s.d.). Student’s t-tests were performed to calculate P values and to determinewhether the results between two time points were significantly different.

Received 4 April; accepted 29 June 2005.

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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We would like to thank J. Wolf, K. Harper, D. Stephens,O. Aristizabal and R. Turnbull for technical assistance; M. Fuccillo forexperimental advice; P. Soriano for R26R mice; D. Rowitch and C. Stiles for theanti-Olig2 antibody; G. Fishell for his insightful input throughout the project, andM. Zervas and S. Blaess for critical reading of the manuscript. S.A. is supportedby a NIH National Research Service Award postdoctoral fellowship and A.L.J. isa Howard Hughes Medical Institute investigator.

Author Information Reprints and permissions information is available atnpg.nature.com/reprintsandpermissions. The authors declare no competingfinancial interests. Correspondence and requests for materials should beaddressed to A.L.J. (joyner@saturn.med.nyu.edu).

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