Persistent Sonic Hedgehog Signaling in Adult Brain Determines Neural Stem Cell Positional Identity

Neuron

Article

Persistent Sonic Hedgehog Signaling in Adult BrainDetermines Neural Stem Cell Positional IdentityRebecca A. Ihrie,1,2 Jugal K. Shah,1,2 Corey C. Harwell,1,3 Jacob H. Levine,1,2 Cristina D. Guinto,1,2 Melissa Lezameta,4

Arnold R. Kriegstein,1,3 and Arturo Alvarez-Buylla1,2,*1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research2Department of Neurosurgery3Department of Neurology

University of California, San Francisco, San Francisco, CA 94143, USA4Instituto Cavanilles de Biodiversitat i Biologia Evolutiva, 46980 Valencia, Spain

*Correspondence: [email protected] 10.1016/j.neuron.2011.05.018

SUMMARY

Neural stem cells (NSCs) persist in the subventricularzone (SVZ) of the adult brain. Location within thisgerminal region determines the type of neuronalprogeny NSCs generate, but the mechanism of adultNSC positional specification remains unknown. Weshow that sonic hedgehog (Shh) signaling, resultingin high gli1 levels, occurs in the ventral SVZ and isassociated with the genesis of specific neuronalprogeny. Shh is selectively produced by a smallgroup of ventral forebrain neurons. Ablation of Shhdecreases production of ventrally derived neurontypes, while ectopic activation of this pathway indorsal NSCs respecifies their progeny to deepgranule interneurons and calbindin-positive periglo-merular cells. These results show that Shh is neces-sary and sufficient for the specification of adultventral NSCs.

INTRODUCTION

Most of the neurons in the central nervous system are produced

in the embryo, and formany years it was thought that this was the

only source of neurons in the CNS. We now know that two

populations of neurons continue to be produced in the adult:

olfactory bulb interneurons and granule neurons in the dentate

gyrus of the hippocampus. Neuronal production is limited to

the subventricular zone (SVZ), along the walls of the lateral

ventricles, and the subgranular zone, within the dentate gyrus.

These two regions contain neural stem cells (NSCs) and continue

to generate new neurons throughout adult life (Zhao et al., 2008;

Ihrie and Alvarez-Buylla, 2011). Within the SVZ, glial fibrillary

acidic protein (GFAP)-expressing stem cells (type B cells) give

rise to rapidly dividing transit-amplifying progeny (type C cells),

which in turn generate immature neuroblasts (type A cells).

These neuroblasts migrate to the olfactory bulb (OB) within a

network of tangentially oriented chains that coalesce to form the

rostral migratory stream (RMS) (Luskin, 1993; Lois and Alvarez-

Buylla, 1994; Doetsch et al., 1999a, 1999b). Within the OB,

250 Neuron 71, 250–262, July 28, 2011 ª2011 Elsevier Inc.

young neurons migrate radially, complete their differentiation,

and integrate into the granular and periglomerular layers.

In the mouse, the SVZ covers an area greater than six square

millimeters along the rostrocaudal and dorsoventral axes

(Mirzadeh et al., 2008). Neuroblasts derived from the SVZ

traverse a significant distance to reach their final destination in

the OB. Why are neurons derived from such an extended prolif-

erative zone, and how does site of origin in the SVZ affect cell

fate? One clue comes from recent experiments using viral or

genetic lineage tracing to label specific subregions of the devel-

oping or adult SVZ (Kelsch et al., 2007; Kohwi et al., 2007;Merkle

et al., 2007; Ventura and Goldman, 2007; Young et al., 2007).

These results suggest that the SVZ is arranged as a mosaic; the

position of stem cells within the SVZ determines the types of

differentiated progeny generated. In particular, deep granule

interneurons and a subpopulation of periglomerular cells arise

from the ventral SVZ, while superficial granule interneurons and

distinct periglomerular cells are derived from the dorsal SVZ.

The molecular mechanisms responsible for this positional spec-

ification of primary progenitors in the adult SVZ are not known.

The Hedgehog (Hh) signaling pathway is central to the devel-

opment and patterning of the nervous system and other

organs (McMahon et al., 2003; Fuccillo et al., 2006). Inmammals,

this signaling pathway is initiated by one of three family

members—Indian hedgehog (Ihh), Desert hedgehog (Dhh), or

Sonic hedgehog (Shh). Secreted Hedgehog morphogen binds

Patched at the cell surface, relieving its inhibition of the trans-

membrane protein Smoothened (Rohatgi et al., 2007). Smooth-

ened triggers the activation of the Gli transcription factors. In

the absence of Hh signal, the Gli3 transcription factor acts as

a transcriptional repressor, while Gli2 functions primarily as

a transcriptional activator upon Hh stimulation and can initiate

transcription of Gli1, a constitutive transcriptional activator that

indicates high levels of pathway activity (Ahn and Joyner,

2005; Palma et al., 2005; Clement et al., 2007).

The responsiveness of a cell to a given level of Hh ligand is also

modulated by intrinsic expression of cell-surface proteins. The

transmembrane proteins Cdo, Boc, and Gas1 are thought to

positively regulate Hh pathway activation and allow cells at

a greater distance from a Hh source to respond to lower levels

within a Hh gradient (Tenzen et al., 2006; Allen et al., 2007;

McLellan et al., 2008). Conversely, Hedgehog interacting protein

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http://dx.doi.org/10.1016/j.neuron.2011.05.018

gli1 mRNA gli2 mRNA gli3 mRNA

dorsal

ventral

sep

str

sep

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sep

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sepstr sepstr

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ptc-lacZ gli1-nlacZ

dorsal

ventral sep

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2

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Nkx6.2 Nkx2.1 Gli1 Gli2 Gli3

vSVZ

septum

Relative transcript expression (compared to dorsal SVZ)

fold

cha

nge

(log2

)

K

*

**

*

Figure 1. Gli1 Expression Is Primarily

Ventral

(A–F) Dorsal and ventral areas shown in images

(A)–(F) are indicated in the cartoon at left. In situ

hybridization for the three Gli family members

reveals that gli1 mRNA is present in the ventral

SVZ (D), but not the dorsal SVZ (A). gli2 and gli3

mRNA are present at low levels in both the dorsal

and ventral SVZ (B, C, E, and F). Scale bars: 50

microns.

(G–J) LacZ staining in either ptc-lacZ (G and I) or

gli1-nlacZ (H and J) reporter strains is strongest in

the ventral SVZ (I and J) and is reduced or absent in

the dorsal SVZ (G and H). Red dashed lines indi-

cate the lateral ventricle.

(K) qRT-PCR detection of Gli family members in

microdissected ventral SVZ and septum recapit-

ulates the expression observed in in situ hybrid-

ization. Values shown are relative transcript

expression compared to microdissected dorsal

SVZ. Nkx2.1 and Nkx6.2, which are known to be

upregulated in ventral SVZ, are highly expressed in

both ventral SVZ samples (blue bars) and RNA

derived from the septum (red bars). gli1 is ex-

pressedmore highly in the ventral SVZ and septum

than in the dorsal SVZ. gli2 and gli3 transcript

expression did not differ significantly between the

dorsal and ventral SVZ, but both genes were ex-

pressed to higher levels in the septum. Asterisks =

p < 0.05.

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Sonic Hedgehog Regulates Neural Stem Cell Identity

(Hhip) binds and sequesters Hh ligand, and acts cooperatively

with Patched as a negative-feedback mechanism to regulate

pathway activation (Chuang and McMahon, 1999; Chuang

et al., 2003). Studies in the developing neural tube have demon-

strated that the varying levels of Hh ligand and interacting

proteins present over time along the dorsoventral axis of this

structure result in graded levels of activity of the Gli transcription

factors, allowing a morphogen gradient to be translated into

transcriptional control of neuronal identity (Jessell, 2000; Ribes

and Briscoe, 2009). Shh signaling also regulates cell proliferation

and fate in the developing forebrain and hindbrain (Machold

et al., 2003; Corrales et al., 2004; Blaess et al., 2006; Balordi

and Fishell, 2007a, 2007b; Xu et al., 2010). Shh is important in

the initial generation and proliferation of postnatal neural stem

cells in the SVZ and DG, but whether it has a role in the specifi-

cation of regional identity in the adult is not known (Lai et al.,

2003; Balordi and Fishell, 2007b; Han et al., 2008).

Here, we demonstrate that Shh signaling has a continuous,

critical role in the dorsoventral specification of adult SVZ neural

stem cells. We find that despite the ubiquitous expression of

Smoothened on stem cells throughout this region, Gli1 expres-

sion primarily occurs in the ventral SVZ and is associated with

particular neuronal fates. We show that Shh signaling is instruc-

tive in cell fate specification, and removal of Shh expression

affects the generation of specific neuronal subtypes. Further-

more, we identify a possible source of secreted Sonic hedgehog

(Shh) ligand close to the ventral SVZ: surprisingly, this source is

neuronal. These results are the first identification of a signaling

pathway that is sufficient to determine neuronal cell fate in adult

SVZ NSCs.

RESULTS

Gli1 Expression Occurs in the Ventral SVZShh pathwaymembers have been implicated in the development

and postnatal maintenance of SVZ neural stem cells (Machold

et al., 2003; Ahn and Joyner, 2005; Balordi and Fishell, 2007a,

2007b). Remarkably, in situ hybridization for gli1, gli2, and gli3

revealed that gli1 expression is higher in the ventral SVZ (Figures

1A and 1D), while gli2 and gli3 are present both ventrally and

dorsally (Figures 1B, 1C, 1E, and 1F). Likewise, staining of brain

sections from mice carrying gli1-nlacZ and ptc-lacZ reporter

alleles (Goodrich et al., 1997; Bai et al., 2002) showed high levels

of reporter expression in the ventral SVZ, in both the lateral and

medial walls (Figures 1I and 1J). We also microdissected these

regions from adult brains and performed qRT-PCR analysis. To

confirm that the correct areas were dissected, we measured

relative expression of the transcription factors Nkx2.1 and

Nkx6.2, which are expressed in the ventral forebrain during

development (Xu et al., 2008, 2010) and are present ventrally in

the adult SVZ (L. Fuentealba and A.A.-B., data not shown). Using

qRT-PCR, we observed elevated gli1 expression in the ventral

SVZ as well as the medial septum when compared to the dorsal

SVZ (Figure 1K).

Neuron 71, 250–262, July 28, 2011 ª2011 Elsevier Inc. 251

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We next stained adult SVZ for Smoothened (Smo), an obligate

component of the canonical Hh pathway. Smo protein was

present throughout the SVZ in a pattern reminiscent of GFAP,

which is expressed by type B cells in this region (see Figures

S1A–S1F; Doetsch et al., 1999a; Garcia et al., 2004; Tavazoie

et al., 2008). Confocal analysis of both dorsal and ventral SVZ

using two different antibodies demonstrated that Smo is ex-

pressed on a subset (�80%) of GFAP-positive cells in both

subregions. This staining was not observed when the antibody

was incubated with blocking peptide or when primary antibody

was omitted, and was almost entirely absent in the brains of

hGFAP::Cre; Smofl/fl mice, where Smoothened is lost in most

neural stem cells (Figures S1G and S1H; Han et al., 2008).

Smo did not colocalize with Dcx, CD24, or EGFR, which label

other cell types in the SVZ. To confirm that Smoothened is

primarily expressed on stem cells, we infused the antimitotic

cytosine-b-D-arabinofuranoside (Ara-C) into the brains of wild-

type mice for 6 days. This treatment eliminates fast-dividing

transit-amplifying (type C) cells and neuroblasts from the sub-

ventricular zone, while sparing slow-dividing stem cells (Doetsch

et al., 1999b; Long et al., 2001). While a 6 day Ara-C infusion was

sufficient to eliminate doublecortin-positive neuroblasts in wild-

type mice, Smo expression remained after Ara-C treatment

(Figures S1J–S1M).

We next examined pathway activation using Gli1-CreERT2;

R26YFP mice (Ahn and Joyner, 2004), in which cells responding

to high levels of Hedgehog ligand express a tamoxifen-inducible

Cre recombinase. By giving tamoxifen at postnatal day 60 (P60)

in adult mice, we identified SVZ stem cells in which the Hh

pathway was active and followed their YFP-labeled progeny

(Figure 2). We administered tamoxifen for 5 days and examined

YFP expression at 0 days, 5 days, or 30 days after the end of

treatment. In contrast to the widespread expression of Smooth-

ened, activation of Hh signaling was much more restricted.

Intriguingly, pathway activity occurred in a spatially specific

manner—the initial population of Hh-responsive cells was

distributed along the anterior-posterior axis of this region, but

largely confined to the ventral half of the SVZ (Figure 2D), an

area which primarily produces deep granule interneurons within

the OB (Merkle et al., 2007). This labeling was not due to limited

infiltration of tamoxifen in the dorsal SVZ, as equivalent treat-

ment of mice with ubiquitously expressed Cre-ER transgenes

caused recombination throughout the SVZ (Kuo et al., 2006;

data not shown).

The initial population of YFP-labeled ventral cells were largely

GFAP-positive (Figure 2D). YFP-positive migrating neuroblasts

appeared at 5 days after tamoxifen treatment in the dorsal and

ventral SVZ, RMS, and the core of the OB (Figures 2B, 2E, 2H,

2K, and S2). The presence of ventrally derived neuroblasts in

the dorsal wedge is consistent with the pattern of tangential

migration to the OB (Doetsch and Alvarez-Buylla, 1996). These

dorsal YFP-positive cells were GFAP negative and doublecortin

positive, confirming their identity as neuroblasts (Figures 2B and

S2). At 30 days after treatment, YFP-labeled cells were present

throughout the SVZ, in the RMS, and in the granular and periglo-

merular layers of the OB (Figures 2C, 2F, 2I, and 2L). We also

observed many YFP+ cells in the medial septum at 30 days after

tamoxifen treatment (Figure S3). These labeled septal cells were


primarily S100b+ astrocyte-like cells, similar to those observed

upon EGF infusion into the SVZ (Deloulme et al., 2004; Hachem

et al., 2005; Gonzalez-Perez et al., 2009). We next quantified the

localization of labeled progeny in the OBs of Gli1-CreERT2;

R26YFP animals at one month after tamoxifen. The labeled cells

were primarily interneurons located in the deep granule layer of

the OB, similar to the cells labeled by viral injection in the ventral

SVZ (Figures 2M–2O and S2M). The distribution of labeled cells

differed significantly from distributions generated by combining

dorsal and ventral injection data or from general BrdU labeling

and lineage tracing in the SVZ (shown in Figure 4), confirming

that Gli1 expression delineates a population of ventrally-derived

interneurons.

Sonic Hedgehog Ligand Is Produced in the VentralForebrainWe next investigated possible sources of Hh ligand in the adult

brain. As previously reported (Echelard et al., 1993), we did not

observe expression of indian hedgehog or desert hedgehog in

the adult brain by in situ hybridization (data not shown). We did

not observe expression of shh in the dorsal SVZ. However, shh

mRNA was present in the medial septum, ventral forebrain,

and in infrequent cells close to the ventral SVZ (Figures S4A–

S4C). This was confirmed by qRT-PCR on dissected ventral

SVZ and septum (Figure S4D) and is consistent with previous

reports of in situ hybridization in the rat brain (Traiffort et al.,

1999). We next used a genetic approach to label the cells

producing Shh and visualize cell morphology. We first examined

ShhCre; R26YFP animals (Harfe et al., 2004), in which cells

expressing Shh at any point in development recombine the

R26YFP reporter (Figure S4E). In addition to labeling in the cere-

bellum and cortex, we also observed an accumulation of YFP+

cells in the ventral forebrain.

By administering tamoxifen to ShhCreER; R26YFP animals,

we induced YFP expression in cells producing Shh in the adult

(Figure 3; Harfe et al., 2004). YFP expression identified cells in

the ventral forebrain, extending along the rostrocaudal axis

adjacent and ventral to the SVZ. These cells primarily localized

to the medial and ventral septum, the preoptic nuclei near the

hypothalamus, and the bed nuclei of the stria terminalis. We

also observed rare labeled cells in cortex (Garcia et al., 2010).

Within the septum, YFP+ cells localized to both the horizontal

and vertical limbs of the diagonal band, approximately 0.25–

1 mm ventromedial to the SVZ (Figures 3A and 3B). A number

of YFP+ cells in the bed nuclei of the stria terminalis were in close

proximity to the ventral tip of the lateral ventricle (boxed area in

Figures 3A and 3D). We did not observe YFP-labeled cells near

the dorsal SVZ, the RMS, OB, or in the choroid plexus—other

sites which have been suggested to produce this ligand (Balordi

and Fishell, 2007a; Angot et al., 2008). Labeled cells had the

morphology of neurons, and all were colabeled by the neuronal

marker protein NeuN (Figure 3H). Most YFP-labeled cells were

GABAergic (GABA-positive; Figure 3I), with a small number of

cholinergic (ChAT-positive), YFP-positive cells (Figure 3J). We

did not observe YFP-positive dopaminergic (TH-positive) cells

(data not shown). Labeled cells within the bed nucleus of the stria

terminalis were of particular interest because of their close prox-

imity to the ventral SVZ, and we examined these cells in greater

YFP / GFAP

0 d post tamoxifen 5 d post tamoxifen 30 d post tamoxifen

dorsalSVZ

ventralSVZ

RMS

olfactorybulb

YFP / GFAP YFP / GFAP YFP / GFAP

YFP / GFAP YFP / GFAP YFP / GFAP

YFP / Dcx YFP / Dcx YFP / Dcx

YFP / Dcx YFP / Dcx YFP / Dcx

A

D

G

J K L

IH

E F

B C

YFP / DcxN YFP / DcxO

tamoxifen(5 days)

Gli1CreER; R26YFP YFP labeling in SVZ, RMS, OB

M 1

0.8

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0.4

0.2

00 to 0.33

deep0.33 to 0.67intermediate

0.67 to 1superficial

Dorsal viral injection (n = 3)

Ventral viral injection (n = 3)

Gli1CreER; R26YFP (n = 3)

Frac

tion

of to

tal l

abel

ed c

ells

Normalized distance from core

Figure 2. High Hedgehog Pathway Activity Is Limited to the Ventral SVZ

(A–L) Lineage tracing of Gli1-expressing cells in the SVZ. Schematic at top indicates the experimental timeline. Immediately after 5 days of tamoxifen admin-

istration inGli1CreERT2; R26YFP animals, a small number of YFP/GFAP double-positive cells are present in the ventral SVZ (arrowhead and inset, D). YFP-labeled

cells are absent from the dorsal SVZ, RMS, and OB (A, G, and J). At 5 days after tamoxifen administration, YFP-labeled cells are present in the ventral SVZ, and

some YFP labeling coincides with GFAP labeling (arrowheads and inset, E). YFP-labeled cells are also present in the dorsal SVZ (B), medial septum (right side in

E), RMS (H), and the core of the OB, where doublecortin (Dcx)-expressing migrating neuroblasts enter this structure (K). One month after tamoxifen adminis-

tration, there is widespread YFP labeling in the SVZ (C and F), RMS (I), and OB (L). Scale bars: 50 microns.

(M–O) Quantification of the localization of labeled granular layer neurons inGli1CreERT2; R26YFP animals at 1 month after tamoxifen administration (red bars, M).

The neuronal progeny of labeled cells tend to localize in the deep OB granular layer close to the core, a phenotype that is consistent with an origin in the ventral

SVZ. The distributions of cells resulting from dorsal (dark blue) or ventral (light blue) viral injection are shown for comparison. Data shown are average ± SEM

(n in figure is number of animals). This localization can be observed consistently in multiple samples (N and O). The OB core (dashed line) is located at the bottom

of both images.

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detail. Computerized tracings of YFP-labeled cells highlighted

thin processes that were located close to the basal side of ventral

SVZ cells (Figures 3E and 3F). In order to better characterize

these cells, we used whole-mount preparations of ShhCreER;

R26YFP animals, allowing en face visualization of the lateral

and medial walls of the ventricles. ShhCreER; R26YFP-labeled

cells had thin processes that interdigitated with the basal side

of cell bodies within the SVZ (Figure 3G).

To test whether these cells are actively producing Shh and

might contact the subventricular zone, we pursued two strate-

gies. First, we injected Ad:CreStopLight (Ad:CSL) virus in the

ventral SVZ of Shh-Cre animals. This virus contains a CMV


αYFP αYFP αYFP

Shh-CreER; R26YFP - 4 days post tamoxifen administration

B C D

YFP / DAPIE

YFP / NeuN /DAPI

YFP / GABA /DAPI

H I YFP / ChAT /DAPI

J

YFP / DAPIGYFP / DAPIF

A

YFP Fluorogold YFP / FG / DAPI

K L M

tamoxifen(2 days)

(2 days)

ShhCreER; R26YFP Inject Fluorogold in ventricle YFP and Fluorogold

Shh/ NeuN /DAPI

Shh / DAPI Shh / RFP /DAPI

Q R

N O P

Shh / DAPIShh / DAPI

Figure 3. Sonic Hedgehog-Producing Cells Localize to the Ventral Forebrain and Can Be Labeled via the SVZ or Lateral Ventricles

(A–D) Colorimetric staining for YFP expression in coronal sections of ShhCreER; R26YFP animals 4 days after tamoxifen treatment. Clusters of labeled cells are

visible in themedial septal nucleus (red box in left section in A, enlarged in B), the preoptic nuclei (middle section in A), and the bed nuclei of the stria terminalis (red

boxes in middle section of A, enlarged in D). A small number of labeled cells approached the ventral regions of the lateral ventricles (D). We did not observe YFP-

labeled processes or cell bodies in the dorsal SVZ (red box in middle section of A, enlarged in C). Scale bars throughout figure: 50 microns.

(E–G) Labeled cells in ShhCreER; R26YFP animals extend processes toward the ventral SVZ. Frontal sections of the ventral tip of the SVZ (delineated by red

dashed lines) are shown in (F) and (G) with accompanying computerized tracing of processes shown in white. En-face visualization of a whole-mount specimen is

shown with tracing of processes in (G).

(H–J) Labeled cells in ShhCreER; R26YFP animals express the neuronal marker NeuN (H). A subset of these neurons express the neurotransmitter GABA (I) or the

cholinergic cell marker choline acetyltransferase (ChAT, shown in J). No colabeling of YFP and the neuronal subtype marker tyrosine hydroxylase (TH) was

observed (not shown).

(K–M) Colabeling of Shh-producing cells in the septum with Fluorogold retrograde tracer. At 4 days after tamoxifen treatment and 2 days after injection of

Fluorogold into the lateral ventricle of ShhCreER; R26YFPmice, many Fluorogold-labeled cells (L) can be observed in the medial septum. YFP-positive cells are

present in this region (K), and these cells are also labeled with Fluorogold (arrow, M).

(N–R) Shh protein is present in the septum and SVZ. (N) Antibody against Shh protein robustly labels the Purkinje cells of P5 cerebellum, a known source of Shh

ligand. In tamoxifen-treatedShhCreER; tdTomatomice, Shh protein is found around the ventral SVZ and is found in the cell body of an RFP-labeled neuron (arrow,

O). Within the septum, Shh is present in the neuropil and in cell bodies associated with NeuN-positive nuclei (arrows, P). In the dorsal SVZ, minimal staining is

observed, although infrequent punctuate staining is present (Q). In contrast, higher levels of Shh protein can be observed in and around the ventral SVZ (red

dashed line, R).

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promoter driving the expression of the gene encoding red

fluorescent protein (and a STOP sequence) flanked by loxP

sequences, followed by the gene encoding green fluorescent

protein (Yang and Hughes, 2001). After injecting Ad:CSL in the

ventral region of the SVZ, we observed many RFP-positive cells

at the injection site (Figure S4F). We also observed a subset of

GFP-positive cells located near the ventral SVZ in the bed

nucleus of the stria terminalis, similar to the labeled cells identi-

fied in ShhCreER; R26YFP animals (Figures S4G and S4H). We

did not observe GFP-labeled cells after an equivalent injection

in the dorsal SVZ (Figures S4I and S4J). These ventral cells


were therefore actively producing Cre recombinase under the

control of the Shh promoter.

Previous studies in rodent brain have suggested that Shh may

be transported anterogradely along axons and secreted distally

at axon terminals (Traiffort et al., 2001). To determine if more

distant Shh-producing cells might also contact the SVZ, we

used the retrograde tracer Fluorogold (hydroxystilbamidine).

After treating P90 ShhCreER; R26YFP animals with tamoxifen,

we waited two days to allow accumulation of YFP label in the

absence of injury and injected Fluorogold into the lateral

ventricle. At 2 days after tracer injection, we found significant

Shh orfl/+

ShhCreER; Shh fl

tamoxifengli1 mRNAin SVZ

olfactorybulb BrdU distribution

BrdU(1 week)(2 weeks) (3 weeks)

sep

str

sep

str

gli1 mRNA (SVZ)

Shh protein (septum and SVZ)

Shhfl/+ ShhCreER; Shhfl

Shhfl/+ ShhCreER; ShhflBrdU (olfactory bulb)

BrdU / CalB (olfactory bulb)Shhfl/+

ShhCreER; Shhfl

E F

GFr

actio

n of

tota

l Brd

U+Shh / DAPI Shh / DAPIA B

DC Shh / DAPI Shh / DAPI 0

0.1

0.2

0.3

0.4

0.5

BrdU / CalB DAPI

BrdU / CalB DAPI

0 to 0.33deep

0.33 to 0.67intermediate


Shhfl/+ (n = 3)ShhCreER; Shh fl

(n = 5)

I

J

**


H

Per

cent

Cal

B-B

rdU

/ to

tal B

rdU

+

genotype

Shh (n = 3)fl/+

ShhCreER; Shh fl(n=6)

0

1

2

3*

Figure 4. Loss of Shh Alters the Production

of Olfactory Interneurons

(A–F) Treatment of ShhCreER/Shhfl animals with

tamoxifen results in the loss of Shh protein and gli1

expression. Schematic at top shows the timeline of

tamoxifen and BrdU administration. At six weeks

after tamoxifen administration, Shh protein is

present in the septum and ventral SVZ of control

animals (A and C). The SVZ is outlined by a red

dashed line. In situ hybridization for gli1 shows the

expected pattern of ventral expression in Shhfl/+

animals (arrowhead, E). Shh protein is largely

absent in ShhCreER/Shhfl animals (B and D).

gli1 expression is also substantially reduced or

absent in ShhCreER/Shhfl animals, indicating that

pathway activity is compromised (arrowhead, F).

White scale bars throughout figure: 50 microns.

Black scale bars: 500 microns.

(G) The distribution of newly born granule neurons

in the OB is altered in the absence of Shh. At

3 weeks after a 1 week pulse of BrdU in Shhfl/+

animals, labeled cells are distributed throughout

the granular layers of the OB, with a greater

percentage of the total present in the deep granule

layers close to the core (blue bars, G). However, in

ShhCreER/Shhfl animals, BrdU-labeled cells were

less frequent in the deep granule layers and more

prevalent in the superficial layers of the OB.

Quantification of the distribution of BrdU-labeled

cells by genotype shows a statistically significant

decrease in the number of deep granule cells and

concomitant increase in the number of superficial

granule cells (asterisks = p = 0.01, unpaired t test).

Data shown are average ± SEM. n indicates

number of biological replicates counted, with at

least 100 OB granular layer cells counted per

animal.

(H–J) The production of calbindin-positive peri-

glomerular cells is decreased in the absence of

Shh. At 3 weeks after a 1 week pulse of BrdU,

a subpopulation of BrdU-labeled cells in the peri-

glomerular layer of Shhfl/+ OBs are calbindin-

positive (colabeling shown in I). This population is

substantially reduced in ShhCreER/Shhfl animals

(p = 0.0033, unpaired t test).

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Fluorogold labeling around the ventricles and in the bed nuclei

of the stria terminalis, as well as many labeled cells in the

medial septum and preoptic nuclei. A small number of Fluoro-

gold-labeled cells in the septum also expressed YFP (Figures

3K–3M), suggesting that Shh produced in the septum could

also reach the SVZ by transport along axonal extensions. We

did not observe more distant Fluorogold-labeled cells in the

midbrain or hindbrain.

Finally, we performed immunohistochemical staining for Shh

protein (rabbit mAb 95.9, kind gift from Genentech). We found

that Shh protein was present in the septum in cell bodies asso-

ciated with NeuN-positive nuclei and at low levels in the associ-

ated neuropil (Figure 3P). In the ventral SVZ, we also found Shh

protein in the neuropil and in association with the apical and

basal surfaces of SVZ cells (Figures 3O, 3R, and 4C). Infrequent,

punctate staining for Shh protein was also observed in the dorsal

SVZ (Figure 3Q), suggesting that a low level of ligand may be

present in this region despite the absence of Shh-producing cells

or their processes.

Removal of Shh Reduces the Production of VentrallyDerived Olfactory NeuronsAlthough Shh ligand production and pathway activity both

occur in the ventral forebrain, this did not necessarily indicate

a requirement for this signaling in the generation of particular

olfactory interneurons. We examined the requirement for Shh

ligand in the adult brain by conditionally deleting Shh in mice

carrying one copy of a knockin allele of ShhCreER and a floxed

Shh allele in the second locus (ShhCreER/Shhfl). Adult (P60)

ShhCreER/Shhflmice and littermateShhfl/+ controls were treated

with tamoxifen for 5 days to induce deletion of the functional Shh

allele. After a 2 week period to ensure loss of Shh protein expres-

sion and tamoxifen clearance, mice were given a 1 week pulse of

BrdU, followed by a 3 week chase, to label newly produced


saline cyclopamine αShh (5E1) SAG

dorsalSVZ

ventralSVZ

tamoxifen

Gli1CreER; R26YFP YFP labeling in SVZ

(5 days)

YFP/GFAP YFP/GFAP YFP/GFAP YFP/GFAP

YFP/GFAPYFP/GFAPYFP/GFAPYFP/GFAP

A B C D

E F G H

Figure 5. Dorsal SVZ Cells Are Resistant to Pathway Activation

(A–H) Infusion of Hedgehog pathway agonists in the SVZ. Schematic at top illustrates pump cannula placement, allowing infusion of solution into the SVZ and

CSF. Infusion of vehicle (sterile saline) does not alter the predominantly ventral distribution of cells labeled by theGli1CreERT2; R26YFP reporter (A and E). Infusion

of either the small-molecule antagonist cyclopamine (B and F) or an anti-Shh antibody (C and G) results in a reduction in YFP-positive cells. Administration of the

small-molecule agonist SAG causes a robust increase in YFP labeling in the ventral SVZ (H), but does not cause a similar upregulation of YFP expression in the

dorsal SVZ (D). Scale bars: 50 microns.

Neuron


interneurons in the OB. At the end of this time course, we

observed a decrease in Shh protein in both the septum and

ventral SVZ of ShhCreER/Shhfl mice (Figures 4A–4D). Impor-

tantly, we also observed a loss of gli1 mRNA expression in

tamoxifen-treated ShhCreER/Shhfl mice but not treated Shhfl/+

controls, indicating that Shh pathway activity was significantly

decreased in ventral SVZ (Figures 4E and 4F). As expected,

the OBs of control animals had BrdU–labeled cells distributed

throughout the granular layer, with 36% of cells observed in

the deep granule layer (Figures 4G and S5). Similar numbers of

BrdU-labeled interneurons were present in all genotypes, and

label-retaining cells as well as proliferating cells were present

in the SVZ for both genotypes, suggesting that stem cell self-

renewal and progenitor proliferation were not grossly affected

(data not shown). However, in ShhCreER/Shhfl animals, there

was a shift in the distribution of labeled cells, with 15% fewer

deep granule cells present and a 25% increase in superficial

granule cells in the labeled population when compared to

controls (Figure 4G; p = 0.02, unpaired t test). This suggests

that a subset of deep granule interneurons is lost when Shh

ligand is removed from the adult brain.

We also examined the effects of Shh loss on the population

of calbindin (CalB)-positive periglomerular cells normally pro-

duced by the ventral SVZ (Merkle et al., 2007). In ShhCreER/

Shhfl animals, production of new CalB+ periglomerular cells

(BrdU/CalB double-positive cells) was decreased by almost

90% compared to controls (Figures 4H–4J; p = 0.0033, unpaired

t test). This reduction in CalB-positive cells was more pro-


nounced than the reduction in deep granule cells. Shh signaling

may be required for the generation of specific subgroups of

deep granule cells, but not others, resulting in a smaller decrease

in the total population of deep granule cells. However, in both

populations of cells, we observed a meaningful change in the

cell types generated, indicating that Shh production plays

a role in the production of different types of neurons destined

for the OB by ventral NSCs.

Dorsal SVZ Cells Are Intrinsically Resistantto Smo AgonistTo test whether dorsal and ventral SVZ cells are equivalently

responsive to Shh pathway activation, we administered Smooth-

ened agonist (SAG) in the cerebrospinal fluid via an intracranial

osmotic pump into the lateral ventricles. SAG is a small molecule

that efficiently activates the Hh pathway (Chen et al., 2002).

Vehicle (saline), SAG, or the pathway antagonists cyclopamine

or anti-Shh antibody (5E1) were infused for 5 days inGli1CreERT2;

R26YFPmice. During infusion, mice were treated with tamoxifen

to acutely label cells in which the Hh pathway was active. YFP

labeling in vehicle-infused mice was predominantly ventral,

demonstrating that pump installation alone did not alter the

pattern of Hh signaling (Figures 5A and 5E). We observed

increased GFAP labeling after all pump implantations, likely

due to increased numbers of reactive astrocytes. Administration

of either cyclopamine or 5E1 antibody reduced the number of

YFP-positive cells in the ventral SVZ, confirming that YFP

labeling was dependent on pathway activation (Figures 5B, 5C,

Neuron


5F, and 5G). SAG infusion resulted in a dramatic increase in YFP-

positive cells, both GFAP-positive and –negative, in the ventral

SVZ, but did not significantly alter the pattern of YFP labeling

in the dorsal SVZ (Figures 5D and 5H).

Infusion of cyclopamine, 5E1, or SAG may also affect SVZ

cell survival or proliferation, as suggested by previous experi-

ments in which Smoothened was ablated in the SVZ (Balordi

and Fishell, 2007b). Staining for the proliferation marker Ki67

indicated that large changes in proliferation did not occur during

the time frame of this experiment. In both controls and SAG-

infused animals, we observed small populations of YFP-positive

cells in the dorsal SVZ. Most of these cells were GFAP negative

and Dcx positive, suggesting that they correspond to young

migrating neurons (Figure 5 and data not shown). We cannot

exclude that a small subpopulation of Gli1-expressing type B

or C cells are present in dorsal regions. The regional difference

in Gli1 distribution remains after agonist infusion, suggesting

that additional cell-intrinsic factorsmay affect the ability of dorsal

cells to activate the Hh pathway.

Ectopic Pathway Activation ReprogramsNeuronal ProgenyWhile high Hedgehog pathway activity was required for the

production of particular types of neuronal progeny, this observa-

tion did not necessarily indicate that Hedgehog signaling had an

instructive role in cell fate. To investigate this possibility, we per-

formed targeted injections of Ad:GFAPpCre virus in SmoM2-

YFP; R26R mice (Mao et al., 2006). The Ad:GFAPp-Cre virus,

in which Cre recombinase expression is driven by the murine

GFAP promoter, results in recombination in primary progenitors

(type B cells) within this region (Merkle et al., 2007). In these

animals, Cre-mediated recombination causes the expression of

SmoM2, a constitutively active mutant of Smo, and activation

of the Hh pathway. By injecting Ad:GFAPpCre in the dorsal

SVZ of SmoM2-YFP; R26R animals, we activated the Hh

pathway to high levels in a ligand-independent, cell-intrinsic

fashion while simultaneously labeling these cells with b-galacto-

sidase. This allowed us to follow the labeled progeny of these in-

fected stem cells. Remarkably, while expression of the SmoM2

protein is sufficient to drive rapid tumorigenesis in other contexts

(Schuller et al., 2008), we did not observe abnormalities within

the SVZ of injected SmoM2-YFP; R26R animals. One month

after injection, lacZ-labeled cells were found at the site of injec-

tion in control and SmoM2-YFP; R26R animals (Figure 6). gli1

expression was absent in the dorsal SVZ of injected R26R

mice. SmoM2-YFP; R26R animals showed a marked upregula-

tion of gli1, but not Shh, mRNA at the site of virus injection

(Figures S6A–S6I), confirming that Hh signaling was active in

infected cells.

At 1 month after dorsal injection of Ad:GFAPpCre in control

R26R animals, lacZ labeling marked a population of cells

located in the superficial granular layer of the OB, consistent

with previous work (Figure 6B). Remarkably, dorsal injections

in SmoM2-YFP; R26R animals generated a population of labeled

cells that localized to the deep granule layer of the OB (Figures

6E, 6M, and S6J), similar to the progeny resulting from injections

in the ventral SVZ of R26R or SmoM2-YFP/R26R animals (Fig-

ures 6H and 6K). These labeled cells expressed NeuN (Figures

6C, 6F, 6I, and 6L), suggesting that SmoM2 expression in

infected neural stem cells did not block maturation but did alter

the type of progeny generated.

We next injected Ad:GFAPpCre in SmoM2-YFP; CAG animals

andCAG littermates to generate progeny expressingGFP, which

fills the cell and allows visualization of cell morphology (Figure 7).

Ad:GFAPpCre infection of dorsal SVZ cells in SmoM2-YFP; CAG

animals caused a shift in the localization of GFP-expressing

progeny in the OB like that observed with the R26R reporter

(Figures 7A and 7D). Within the SmoM2-YFP; CAG SVZ, we

observed an almost 4-fold increase (p = 0.0025) in the dorsal

expression of the transcription factor Pbx3a, which is normally

limited to the ventral SVZ (Figures 7J, 7L, and 7R). We also

observed a decrease in expression of Pax6, which is normally

present in the dorsal SVZ, in YFP-positive cells in SmoM2/CAG

animals (Figures 7N and 7P). Within the OB, SmoM2/GFP-

expressing cells were positive for NeuN and the neurotransmitter

GABA (Figures 7B, 7C, 7E, and 7F), confirming that the relocali-

zation of progeny does not block their maturation into inter-

neurons. The projection patterns of deep and superficial inter-

neurons differ (Merkle et al., 2007; Whitman and Greer, 2009),

so in addition to soma location, we traced the arborizations of

GFP-labeled cells. The progeny of dorsally injectedCAG animals

were primarily superficial interneurons with dendrites that

reached past the midline of the external plexiform layer of the

OB (Figure 7G). After dorsal injections in SmoM2/CAG animals,

labeled olfactory interneurons tended to have dendrites that

contacted the inner half of the external plexiform layer, a feature

that is typical of deep granule interneurons (Figure 7H). In addi-

tion to the deep granule cells that arise from the ventral SVZ,

calbindin-expressing periglomerular cells are also derived from

this region. Within the periglomerular cell population derived

from dorsal injections, we saw a three-fold enrichment of CalB/

GFP-double positive cells in SmoM2-YFP; CAG animals when

compared to controls (Figures 7O, 7Q, and 7S). We conclude

that ectopic activation of the Hh pathway is sufficient to respecify

dorsal SVZ neuronal progeny.

DISCUSSION

We demonstrate that the Hedgehog pathway has a critical func-

tion in the regional specification of stem cells of the adult brain.

We found that Smo is expressed by stem cells throughout the

SVZ. However, high Hh pathway activation is more prevalent in

the ventral SVZ and is sufficient to respecify dorsal SVZ stem

cells when ectopically induced. In the forebrain, we found that

Shh is the only Hh isoform expressed. Interestingly, Shh produc-

tion occurs in neurons that are located very close to the ventral

subventricular zone. These results indicate that specification of

subpopulations of neural stem cells and their progeny in the adult

brain is actively regulated by Hh signaling.

It has previously been suggested that the Hh pathway func-

tions in multiple cell types within the SVZ. Genetic and pharma-

cologic studies have argued that this pathway is important in

stem cell self-renewal, the generation of transit-amplifying

progeny, and neuroblast migration to the OB (Machold et al.,

2003; Palma et al., 2005; Balordi and Fishell, 2007a, 2007b).

Our results indicate that astrocytes of the SVZ are the major


SVZ (injection site) olfactory bulbβ-galactosidase

R26Ronly -dorsal

injection

SmoM2/R26R -dorsal

injection

R26Ronly -

ventralinjection

SmoM2/R26R -ventral

injection

β-gal / NeuN / DAPI

A

D

B C

M

FE

G

J

H I

LK

0

0.2

0.4

0.6

0.8

1 R26R dorsal (n = 3)

SmoM2 ventral (n = 3)

R26R ventral (n = 3)

SmoM2 dorsal (n = 5)

0 to 0.33deep

0.33 to 0.67intermediate


Frac

tion

of to

tal l

acZ+

gra

nule

cel

ls


*

* *

Figure 6. High Pathway Activity Causes Relocali-

zation of Neuronal Progeny

(A–L) Ad:GFAPpCre was injected into the dorsal or ventral

SVZ of R26R and SmoM2-YFP/R26R animals and

brains were collected one month later to analyze labeled

progeny. LacZ labeling within the SVZ (A, D, G, and J)

confirmed the site of virus injection. Dorsal injections in

control animals primarily generated superficial labeled

cells (B) that were distant from the core of the OB (indi-

cated by a dashed line). Injections in the dorsal SVZ of

SmoM2 animals gave rise to deep cells located close to

the core of the OB (E), similar to those derived from ventral

viral injections in either genotype (H and K). b-galactosi-

dase-expressing progeny in both control and SmoM2

animals coexpressed the NeuN protein, a marker of

mature neurons (arrows in C, F, I, and L). Black scale bars:

50 microns. White scale bars: 14 microns.

(M) Quantification of the spatial distribution of labeled

progeny in R26R and SmoM2-YFP; R26R animals after

dorsal or ventral Ad:GFAPpCre injection. Ventral virus

injection in either genotype (pale red and pale blue bars)

results in cells that are mostly located close to the core of

the OB. Dorsal virus injection in R26R animals (bright blue

bars) results in many cells that are distant from the core.

However, dorsal virus injection in SmoM2; R26R animals

results in cells that are located close to the core (bright red

bars), with a distribution similar to that from ventral injec-

tions. Data shown are average ± SEM, with total number of

animals indicated on graph. Asterisks = p < 0.01.

Neuron


Hh-responsive population within the SVZ: expression of Smo

protein, which is required for a cell to respond to Hh ligand, is

restricted to the GFAP-positive population (including type B1

cells) in this region. Intriguingly, Smo expression labels a subset

of GFAP-expressing SVZ cells, suggesting either that Smo is


only expressed in a subset of stem cells, or

that it is a more specific marker of stem cells

than GFAP, which is also expressed by some

astrocytes in non-germinal regions (Garcia

et al., 2010).

We searched for components of the Hh

signaling pathway that might be differentially

expressed in the subregions of the SVZ. We

did not observe significant differences in the

expression of Smo, Gli2, or Gli3 between the

ventral and dorsal SVZ, but found that Gli1

expression is much higher in the ventral SVZ.

Lineage tracing of these cells using Gli1-

CreERT2 mice indicates that progenitors re-

sponding to high levels of Shh are located in

the ventral SVZ and primarily generate deep

granule interneurons. Moreover, Hh signaling

appears to be instructive in cell fate deci-

sions: ablation of Shh expression reduced the

production of ventrally-derived progeny, and

ectopic activation of the pathway resulted in

the respecification of dorsally-derived progeny

to a ventral fate. These results are surprising,

as previous studies using viral targeting and

cell culture indicated that cell identitywas largely

encoded using a cell-intrinsic program: transplantation of dorsal

cells to a ventral location, even after intervening time in culture,

was not sufficient to respecify their progeny (Merkle et al., 2007).

The differences in cell fate between previous transplantation

studies and the results shown here may be due to several

olfactory bulb

CAGonly

SmoM2/CAG

dorsal SVZ(injection site) ventral SVZ

sep

str

sep

str

str

str

GFP / Pbx3a / DAPI GFP / CalB / DAPIGFP / Pbx3a / DAPI

dorsal SVZ(injection site)

GFP / Pax6 / DAPI

I

L M Q

R S

OJ

str

str

P

N

0

4

8

12

16SmoM2/

CAG

CAG only

genotype

Pbx

3a-G

FP /

tota

l GFP

+

0

2

4

6

8

10SmoM2/

CAG

CAG only

genotype

Cal

B-G

FP /

tota

l GFP

+

olfactory bulb

CAGonly

SmoM2/CAG

αGFP / hematoxylin GFP / NeuN / DAPI GFP / GABA / DAPI

A

D E F

CB

CAG dorsal SmoM2 / CAG dorsalG H

exte

rnal

ple

xifo

rmla

yer

Figure 7. SmoM2 Expression Results in

Relocalization, Altered Dendritic Projec-

tions, and Ventral Marker Expression

(A–F) Dorsal injection of Ad:GFAPpCre in SmoM2-

YFP; CAG animals (D–F) also resulted in relocali-

zation of labeled progeny close to the core of

the OB (dashed line) when compared to CAG

controls (A–C). GFP-expressing progeny of both

genotypes expressed NeuN (arrows, B and E) and

the neurotransmitter GABA (arrows, C and F),

consistent with their status as differentiated

inhibitory interneurons. Scale bars: 50 microns.

(G and H). Camera lucida tracings of individual

GFP-labeled cells in CAG and SmoM2; CAG

animals show the projections of these cells into the

external plexiform layer of the OB (delineated by

gray shading). Dashed lines indicate the mitral cell

layer (lower line) and periglomerular layer (upper

line) of the OB. While dorsal SVZ-derived cells in

CAG animals typically project to the outer half of

the EPL (G), dorsal SVZ-derived cells from

SmoM2; CAG animals project to the inner half of

the EPL, a feature associated with a deep granule

interneuron identity (H).

(I, J, L–N, P, andR) At 1month after dorsal injection

of Ad:GFAPpCre in CAG animals, many GFP-

labeled cells are present at the site of injection in

the dorsal SVZ (I). However, the transcription

factor Pbx3a is more prevalent in the ventral SVZ

(J). After a similar injection in SmoM2-YFP; CAG

animals, Pbx3a is present at similar frequency in

the uninjected cells of the ventral SVZ (M), but is

also enriched in dorsal GFP-positive cells (L). str =

striatum, sep = septum. This enrichment is quan-

tified in (R)—graph shows average ± SEM for three

independent injections per genotype (p = 0.0025,

unpaired t test). In concert with the increased

expression of the ventral marker Pbx3a, we also

observed a loss of the dorsal marker Pax6—while

many dorsal GFP-positive cells normally express

Pax6 (arrows, N), this expression is absent in

SmoM2/CAG animals (P).

(O, Q, and S) Within the OBs of CAG animals at

one month after dorsal Ad:GFAPpCre injection,

few GFP-positive periglomerular cells (O and S)

expressed the marker calbindin (shown in red).

However, we did observe an increase in the

number of CalB/GFP double-positive cells in

periglomerular cells derived from injections in

SmoM2-YFP; CAG animals (Q and S). Graph

shows average ± SEM for three independent

injections per genotype.

Neuron



Neuron


possiblemechanisms: dorsal SVZ cellsmay be intrinsically resis-

tant to the Hh signal, resulting in lower pathway activation even

upon exposure to equivalent levels of ligand, or a second devel-

opmental pathway may act to oppose Hh action in the dorsal

region, as is the case in the developing neural tube (Liem et al.,

2000). It has also recently been recognized that SVZ stem cells

have a specialized apical-basal orientation within the SVZ niche

(Mirzadeh et al., 2008; Shen et al., 2008; Tavazoie et al., 2008).

Transplantation experiments may not allow the grafted popula-

tion to integrate and adopt proper apical-basal positioning within

the niche. Hh ligand may be delivered to ventral SVZ cells via

specialized local contacts which are not recapitulated after

transplantation. Importantly, the present results indicate that

strong activation of the Shh pathway can override the intrinsic

programming of dorsal neural progenitors, suggesting that

reprogramming of neural stem cells for therapeutic purposes

may depend on the identification of the relevant molecular signal

for a desired cell type. This study also provides the first in vivo

respecification of adult neural cell fate by modulation of the Hh

pathway.

We identify clusters of Shh-producing neurons in the ventral

forebrain, in locations that are consistent with previous studies

at the RNA level. A subset of these cells, in the bed nucleus of

the stria terminalis, have processes that are immediately adja-

cent to the ventral SVZ. In addition, some Shh-producing cells

in the ventral and medial septum are able to take up retrograde

tracer molecules that are injected into the lateral ventricle, sug-

gesting that Shh ligand may also reach the ventral SVZ by

anterograde transport from the septum (Traiffort et al., 2001).

The localized activation of ventral SVZ stem cells, and expres-

sion of Gli1 in these cells, might be part of an adult brain regula-

tory mechanism to locally modulate production of specific

neuronal subtypes destined for different OB circuits.

Shh is produced by Purkinje neurons in the developing

cerebellum and by cells of the floor plate in the neural tube

(Ho and Scott, 2002; Fuccillo et al., 2006). In these instances,

Shh signaling directs significant large-scale remodeling and

patterning of developing tissue. Our results demonstrate that in

the adult brain, this pathway remains active and directs the

production of specific subtypes of neurons. The finding that

mature neurons in the adult brain are a likely source of Shh ligand

suggests that neural network activity may regulate generation of

certain types of neurons within the SVZ. It remains unclear if Shh

reaches ventral stem cells via diffusion or whether more special-

ized contacts exist between Shh-producing neurons and stem

cells. It will be of interest to study the mechanism of Hh ligand

delivery to the SVZ, and to identify what circuits in the brainmight

activate the release of Shh ligand.

The SVZ is the largest germinal zone in the adult brain, and

is capable of generating thousands of immature neurons each

day. Importantly, it is organized as a mosaic, with stem cells in

different locations producing different types of neurons (Hack

et al., 2005; Kohwi et al., 2005; Kelsch et al., 2007; Merkle et al.,

2007; Ventura and Goldman, 2007; Young et al., 2007; Alvarez-

Buylla et al., 2008). These groups of olfactory interneurons

differ in location and are also thought to differ functionally

(Gheusi et al., 2000; Cecchi et al., 2001; Kosaka and Kosaka,

2007). Here, we elucidate a molecular mechanism for the spec-


ification of a subpopulation of neural stem cells within this exten-

sive adult germinal layer. We show that manipulation of this

pathway allows stem cells to be redirected to a different fate.

These studies demonstrate that adult neural stem cells, which

normally produce a restricted repertoire of progeny, may be

reprogrammed if the relevant specification signals are identified.

EXPERIMENTAL PROCEDURES

Tamoxifen Administration

All animal procedures were carried out in accordancewith institutional (IACUC)

and NIH guidelines. Tamoxifen was prepared at 20 mg/ml in corn oil and

administered via oral gavage. Adult mice (P60–P90) were treated with 1 mg

(Gli1-CreER) or 5 mg (Shh-CreER)/day tamoxifen for 5 consecutive days and

sacrificed at the specified times.

Antimitotic Treatment

Adult mice were treated with a solution of 2% cytosine-b-arabinofuranoside

(Sigma) or sterile saline alone as a control. Solutions were infused for 6 days

at the pial surface using a miniosmotic pump (Alzet 1007D).

Injections and Adenovirus Preparation

Ad:GFAPpCre into was injected into dorsal and ventral adult SVZ as described

(Merkle et al., 2007) using 50 nl of virus. Injections of Ad:CSL were carried out

using 100 nl of virus, and Fluorogold injections were carried out using 250 nl of

tracer. Injections used the following coordinates: dorsal SVZ —0.5 anterior,

3.2 lateral, 1.8 deep, needle at 45� angle; ventral SVZ—0.5 anterior, 4.6 lateral,

3.3 deep, needle at 45� angle; lateral ventricle—0.38 posterior, 1.0 lateral,

2.25 deep, with needle vertical. x and y coordinates were zeroed at bregma,

and the z coordinate was zeroed at the pial surface.

Miniosmotic Pump Implantation

Miniosmotic pumps (Alzet 1007D) were assembled under sterile conditions,

filled with vehicle (0.9% saline), 0.5 mMSmoothened agonist (EMDChemicals),

5 mM cyclopamine (Sigma), or 5 mg/ml 5E1 antibody (DSHB), and allowed to

equilibrate overnight at 37�C. Pump installation was performed on a stereo-

taxic rig as previously described.

Immunostaining

Immunostainings were carried out on methanol-fixed 10 micron frozen

sections (Figure S1) or paraformaldehyde-fixed 50 micron free floating Vibra-

tome sections (all other stains) according to standard procedures. Primary

antibodies used were goat anti-Smo (Santa Cruz, C-17), rabbit anti-Smo

(MBL 2668, kind gift of J. Reiter), mouse anti-GFAP (Chemicon), rabbit anti-

doublecortin (Cell Signaling), mouse anti-PSA-NCAM (Chemicon), chicken

anti-GFP (Aves Labs), rabbit anti-RFP (MBL), mouse anti-b-galactosidase

(Promega), anti-NG2, rabbit anti-Olig2 (kind gift of D. Rowitch), rabbit anti-

S100b, mouse anti-GABA (Sigma), goat anti-ChAT (Chemicon), anti-NeuN

(Chemicon), rabbit anti-tyrosine hydroxylase (Pel-Freez Biologicals), rabbit

95.9 anti-Shh (kind gift from S. Scales, Genentech), mouse anti-Pbx3a (Santa

Cruz), and rabbi anti-calbindin D28k (Chemicon). The secondary antibodies

used were conjugated to AlexaFluor dyes (Invitrogen/Molecular Probes). For

preblocking of Smoothened antibody (Santa Cruz, C-17), the antibody was

incubated with the accompanying blocking peptide (sc-6367P, Santa Cruz).

LacZ (X-gal) staining was carried out after brief fixation in paraformaldehyde

according to standard procedures.

Microscopic Analysis, Tracing, and Quantification

Fluorescent staining was visualized using a Leica SP5 confocal microscope

and analyzed using NIH ImageJ. Tracings of neuronal processes from fluores-

cent staining were completed using the Filament module in Imaris analysis

software (Bitplane). Colorimetric staining was visualized using an Olympus

AX70 microscope, Retiga 2000R camera and LabVelocity software. Measure-

ments of olfactory interneuron localization were carried out using the Measure

and Label plugin in NIH ImageJ, with normalization to granular layer width

carried out as described (Merkle et al., 2007). Data were quantified and

Neuron


analyzed using GraphPad Prism 5. Tracing of colorimetric anti-GFP staining

was completed using a Nikon microscope with camera lucida adaptor. Hand

tracings were scanned and imported into Adobe Illustrator using the LiveTrace

and LivePaint modules.

qRT-PCR

For qRT-PCR, dorsal SVZ, ventral SVZ, striatum, and septum were microdis-

sected from 2 mm slices of unfixed brain. Dissected tissue was immediately

placed in RNAlater solution (Ambion) and stored at �20�C until all brains

(14 total) were dissected. RNA isolation was done with the RNEasy Mini kit

(QIAGEN). cDNA was synthesized using SuperScript III RT (Invitrogen), and

qRT-PCR was completed using SYBR Green PCR Master Mix (Applied

BioSystems) on an ABI7900HT. Data analysis and statistical tests were carried

out using the REST algorithm (Pfaffl et al., 2002).

Whole-Mount Dissection

Whole-mount dissection of Shh-CreER; R26YFP brains was performed as

described (Mirzadeh et al., 2008). Full methods including video are available

online (Mirzadeh et al., 2010).

In Situ Hybridization

In situ hybridization was performed using standard protocols (Han et al., 2008).

Antisense and sense RNA probes were labeled with digoxigenin and visualized

with alkaline phosphatase-NBT/BCIP reaction (Roche).

SUPPLEMENTAL INFORMATION

Supplemental Information includes six figures and can be found with this

article online at doi:10.1016/j.neuron.2011.05.018.

ACKNOWLEDGMENTS

The authors thank Alexandra Joyner for sharing the Gli1CreERT2 mouse line

prior to publication, David Rowitch and the members of the Rowitch and

Alvarez-Buylla labs for many useful conversations, and T. Nguyen for

assistance with qRT-PCR preparations. R.A.I. was supported by postdoctoral

fellowships from the Damon Runyon Cancer Research Foundation (DRG1935-

07) and the AACR/NBTS. C.C.H. was supported by a Ford Foundation Post-

doctoral Fellowship and a UNCF Merck Postdoctoral Science Research

Fellowship. This work was supported by grants from the US National Institutes

of Health (NS28478 and HD32116), the John G. Bowes Research Fund, and

a grant from the Goldhirsh Foundation to A.A.-B. A.A.-B. is the Heather and

Melanie Muss Endowed Chair of Neurological Surgery at UCSF.

Accepted: May 16, 2011

Published: July 27, 2011

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Persistent Sonic Hedgehog Signaling in Adult Brain Determines Neural Stem Cell Positional Identity

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