COMMEMORATIVE LECTURE

Molecular and Cellular Mechanisms Contributing to
the Regulation, Proliferation and Differentiationof Neural Stem Cells in the Adult Dentate Gyrus
Fred H. Gage

Laboratory of Genetics, Salk Institute of Biological Studies, California, USA(Received for publication on January 16, 2010)

Adult neural stem cells (NSCs) are a potential endogenous source for neuronal cell replacement in the diseased adult central nervous system (CNS). However, the recruitment of adult
NSCs for repair is hampered by the current lack of knowledge about the cellular and molecularmechanisms that control their behavior in vivo. We have previously demonstrated that environment-derived signals control the fate choice of adult NSCs. More recently we have provided evidence that in the adult hippocampal dentate gyrus – one of the two neurogenic regions of
the adult CNS – specialized astrocytes provide signals that instruct NSCs to adopt a neuronalfate. In this review I will examine in vitro and in vivo the molecular mechanisms underlying theneuronal fate instruction of adult NSCs by the local astrocyte population in the adult hippocampus. In particular, I will focus on the Wnt family of proteins, which we have found to be
expressed in adult hippocampal astrocytes; our preliminary studies have also shown that these
proteins enhance the generation of neurons from adult NSCs in vitro. (Keio J Med 59 (3) : 79－
83, September 2010)

Keywords: neurogenesis, hippocampus, proliferation, differentiation, survival
Introduction

has not yet been elucidated.
NeuroD1 is a proneural bHLH transcription factor essential for the development of the central nervous system (CNS), in particular the generation of granule cells
in the hippocampus and cerebellum.1 0, 11 Environmental
signals regulate adult neurogenesis at least in partthrough the activation of NeuroD1.1 2, 1 3 In preliminary
studies, we showed that overexpression of NeuroD1 was
sufficient to promote neuronal differentiation in adulthippocampal neural progenitors,1 4 whereas deletion of
NeuroD1 resulted in decreased survival and maturationof newborn neurons.1 5 Thus, we hypothesized that astrocyte-derived Wnt signals might directly or indirectly
regulate the transcription of NeuroD1 to control the transition of NSCs to committed neuronal progenitors.
The HMG-box transcription factor Sox2 is expressedin embryonic stem cells and most uncommitted cells inthe developing CNS.1 6-1 8 Sox2, which can be detected

In the neurogenic niche of the adult mammalian brain,
self-renewing neural stem cells (NSCs) give rise to committed neuronal progenitors within the subgranular zone
(SGZ) of the dentate gyrus (DG).1 Astrocytes are an essential cell population that defines the SGZ niche, andastrocyte-derived factors have instructive effects that
promote adult neurogenesis.2, 3 Recently, we showed that
Wnt3 expression persists in the adult hippocampus andthat Wnt3 is released by astrocytes to regulate adult neurogenesis in vitro and in vivo.4 In the canonical Wnt/β-catenin pathway, TCF transcription factors transduce
Wnt/β-catenin signals to activate downstream target
genes.4 -9 However, the target gene(s) of Wnt/β-cateninsignaling responsible for promoting adult neurogenesis
have not been identified. Moreover, the regulatory mechanism underlying Wnt-mediated neuronal differentiation

Dr. Gage is the recipient of the 2008 Keio Medical Science Prize.
Presented at the Keio Medical Science Prize Commemorative Symposium, November 22, 2008.
Reprint requests to: Fred H. Gage, PhD, Laboratory of Genetics, Salk Institute of Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, CA 92037, USA,
E-mail: gage@salk.edu
79

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in cells of the mouse blastocyst, maintains precursor
cells in a multipotent state.1 9- 2 1 During CNS development, Sox2 prevents neurogenesis, and forced expression of Sox2 results in the loss of proneural cells. 2 1, 2 2Whereas overexpression of Sox2 in neural progenitor
cells derived from embryonic ventricular zone permitted
the differentiation of progenitors into astroglia, it inhibited neurogenesis.2 3 Although these analyses indicate thatSox2 is a transcriptional repressor of neuronal target
genes during development, the exact nature of Sox2 regulation during adult neurogenesis remains elusive.
Here I summarize recent evidence that the transcriptional activation of NeuroD1 is dependent on canonicalWnt/β-catenin activation and removal of Sox2 repression from the NeuroD1 promoter in a sequence-specificmanner. These studies provide evidence for possible
crosstalk between Sox2 and Wnt/β-catenin signaling,which represents an important mechanism underlying
adult granule cell neuronal differentiation through NeuroD1.
Characterization of the Effects of Astrocyte-derived
Wnt3 on the Neuronal Differentiation of
Adult Neural Stem Cells
To test the hypothesis that Wnt signaling is involved in
the regulation of neuronal differentiation of adult hippocampal progenitors (AHPs) induced by hippocampal astrocytes, GFP-expressing AHPs were co-cultured for 4days with hippocampal astrocytes in the presence or absence of secreted Frizzled-related proteins (sFRPs),
which are thought to inhibit Wnt signaling by competingwith Frizzled receptors for Wnt ligand and by forming
complexes with Frizzled. Differentiation into neurons
was evaluated every 24 hours by quantifying the percentage of AHPs expressing doublecortin, a microtubulebinding protein that is expressed by at least a subset ofproliferating neuroblasts and immature postmitotic neurons in the adult dentate gyrus. In addition, we determined the percentage of AHPs that expressed the matureneuronal marker MAP2ab after 4 days of co-culture. At
all time points, the percentage of AHPs that differentiated into neurons was significantly decreased in the presence of sFRP2/3, indicating that Wnts participate in thepreviously described induction of neuronal differentiation of AHPs by hippocampal astrocyte-derived factors.4
Furthermore, we analyzed astrocyte factors through gene
expression arrays of hippocampal astrocytes comparedto other cells and revealed several relevant changes in
expression of genes whose proteins could greatly affectneurogenesis.3

Gage FH: Adult Neurogenesis

Characterization of the Wnt-signaling Pathway
Regulating the Neuronal Differentiation of
Adult Neural Stem Cells
The interaction of Wnts with their receptors can trigger several signaling pathways, including the “canonical” β-catenin pathway and the “non-canonical” β-catenin-independent pathways. The β-catenin pathway
has been primarily linked to cell proliferation during development; more recently, however, it has also beenshown to influence fate determination and differentiationin several different organ systems during development.
To examine whether hippocampal astrocyte-derived
Wnts stimulated β-catenin signaling in AHPs, the Wnt/β-catenin signaling reporter Super8XTOPflash was electroporated into AHPs that were subsequently cultured in
the presence or absence of a hippocampal astrocyte feeder layer. Co-culture with hippocampal astrocytes resulted
in reporter activation, suggesting that hippocampal astrocytes stimulate Wnt signaling within AHPs. Next, AHPs
were electroporated with the Wnt/β-catenin signaling reporter TOPGAL and cultured on hippocampal astrocytes
for 4 days. Antibodies to β-galactosidase were used to
track the activation of Wnt/β-catenin signaling within individual cells. β-Galactosidase expression was never observed in the GFAP-positive astrocyte population, which
comprises not only the hippocampal astrocyte feeder
layer but also the small fraction of AHPs that differentiate into astrocytes. In contrast, we found expression of
the reporter almost exclusively in cells (>95% of the β-galactosidase-positive cells; n=300) that were positive
for the neuronal markers doublecortin or MAP2ab, indicating that Wnt/β-catenin signaling is associated with the
population of AHPs that differentiated into neurons.4
Gain and Loss of Function of Wnt Signaling
in the Hippocampal Neurogenic Niche
We tested whether Wnt signaling is involved in theregulation of neurogenesis in vivo. Adult BATGAL mice
were injected with a single dose of BrdU and sacrificed
24 hours later. Wnt/β-catenin pathway activity was observed in BrdU-positive cells in the SGZ and in doublecortin-expressing cells with morphological features ofproliferating neuroblasts. These in vivo results are consistent with our in vitro finding that Wnt/β-catenin signaling regulates neuronal fate commitment and neuroblast proliferation. To block Wnt signaling in vivo, weused a secreted mutant Wnt protein in which 71 aminoacids had been deleted from the carboxy-terminus ofmurine Wnt1 and had been shown to non-autonomously
block Wnt signaling in different in vivo assays. The
cDNA encoding for this dominant negative Wnt protein(dnWnt) was cloned into the recombinant lentivirus (LV)
vector CSC-PPT-CMV-IRES-GFP-WPRE, which contains an expression cassette under the control of theKeio J Med 2010; 59 (3): 79－83

CMV promoter and an IRES-GFP cassette to allow theidentification of infected cells. LV-dnWnt or control LV
encoding for GFP was stereotactically injected into the
DG of young adult rats (8-9 weeks; n=8). After a 3-weekperiod to allow transgene expression and to minimize
confounding factors caused by the surgery, animals were
injected with 200 mg/kg body weight BrdU daily for 7
days to label proliferating AHPs and their progeny. Animals were perfused 24 hours after the final BrdU injection. Compared to LV-GFP-injected animals, the LV-dnWnt-injected animals showed a dramatic reduction in thenumber (11170 ± 1122/mm3 and 2751 ± 281.4/mm3, respectively, p<0.0001, Student’s t-test) and in the percentage of BrdU-positive cells that differentiated into neurons (57.62 ± 4.65% and 31.03 ± 5.48%, respectively,
p<0.005, Student’s t-test), resulting in a total ~8-fold reduction in neurogenesis in LV-dnWnt-injected animals in
the infected areas (i.e., number of BrdU-positive cells
per DG volume multiplied by the percentage of BrdUpositive cells that differentiated into neurons; 6302 ±571.7 new doublecortin-positive neurons/mm3 vs. 813.6± 118.7 new doublecortin-positive neurons/mm 3).4 We
further showed that orphan nuclear receptor Tlx played
an important role in survival and differentiation of adultneurons in the DG, potentially acting through Wnt/
beta-catenin signaling.24, 25
Induction of Neurogenesis of Adult Neural Stem Cells
Through the Manipulation of Wnt Signaling In Vivo
We tested whether increased Wnt signaling is sufficient to enhance adult hippocampal neurogenesis. The
cDNA encoding for Wnt3 and for GFP were cloned into
the LV vector CCL-PPT-PGK-IRES-GFP-WPRE. The
expression cassette of this vector is under the control of
the PGK promoter, which allows higher levels of transgene expression than the CMV promoter. LV-Wnt3 or
LV-GFP was stereotactically injected into the DG of mature (15 weeks) adult rats (n=6). Within the infected areas, LV-Wnt3-injected animals showed a significantly
increased percentage of BrdU-positive cells expressing
doublecortin compared to LV-GFP-injected animals(53.93 ± 6.76% and 70.29 ± 4.29%, respectively, p<0.05,
Mann-Whitney rank sum test). These newborn immatureneurons were often found in large clusters of more than
8 DCX/BrdU double-labeled cells in LV-Wnt3-injectedanimals but not in control animals. 4 In addition we revealed that the Cdk5,2 6 NMDA receptor2 7, 2 8 and Mash
12 9 regulated and controlled the induction of neurogenesis of adult neural stem cells in vivo.
Functional Consequence of Loss of Wnt Signaling
in the Hippocampal Neurogenic Niche
Given the fundamental role of the hippocampus in processes underlying certain forms of learning and memory,81

it has been speculated that newborn granule cells contribute to cognition. However, previous strategies aiming
to causally link newborn neurons with hippocampal
function used ablation strategies that were not exclusive
to the hippocampus or that were associated with substantial side effects, such as inflammation. We used a lentiviral approach to specifically block neurogenesis in the
DG of adult male rats by inhibiting Wnt signaling, whichis critically involved in the generation of newborn neurons, using a dominant-negative WNT (dnWNT). We
found a level-dependent effect of adult neurogenesis on
the long-term retention of spatial memory in the water
maze task: rats with substantially reduced levels of newborn neurons showed less preference for the target zone
in probe trials > 2 weeks after acquisition compared tocontrol rats. Furthermore, animals with strongly reduced
levels of neurogenesis were impaired in a hippocampusdependent object recognition task. Social transmission offood preference, a behavioral test that also depends on
hippocampal function, was not affected by knockdownof neurogenesis. We identified a role for newborn neurons in distinct aspects of hippocampal function that will
lay the groundwork to further elucidate, using experimental and computational strategies, the mechanism bywhich newborn neurons contribute to behavior.30
Transition from Sox2-expressing NSCs to
NeuroD1-positive Cells in Adult Dentate Gyrus
To visualize the transcription factors determining thetransition from the undifferentiated state of NSC to neuronal differentiation, we stained adult hippocampus sections using several antibodies. We used the Sox2 promoter-driven EGFP transgenic mouse1 and compared the
EGFP expression pattern with that of NeuroD1.
NeuroD1-expressing cells were clearly detected in the
DG area. Importantly, Sox2- and NeuroD1-positive cells
were mutually exclusive, and neither Sox2 nor NeuroD1
was immunoreactive for astrocytic S100β. Cells expressing NeuroD1 were detected only in the inner layer of the
SGZ of the DG, where adult neurogenesis continuously
occurs.1 2, 3 1 Also, Sox2-positive cells were negative forthe mature neuron marker NeuN, and NeuroD1-positive
cells never co-localized with NeuN-positive cells.
To characterize the properties of NeuroD1-positive
cells, we performed an immunohistochemical analysis
with specific proneural markers. NeuroD1-positive cells
co-localized with nestin, calretinin and doublecortin.
Sox2-GFP cells colocalized with the stem cell marker
nestin and only a few and weakly expressing GFP-positive cells colocalized with the weaker DCX.1 A major
proportion of the NeuroD1-positive cells co-localizedwith DCX, and a few of them were nestin-positive, suggesting that NeuroD1-positive cells had recently transitioned from Sox2-positive NSCs toward neurogenesis.
NeuroD1-positive cells co-localized with Ki67, indicat-

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Gage FH: Adult Neurogenesis

ing that they are in the proliferating population in the
DG. To examine the proliferative status of NeuroD1-expressing cells, BrdU (100 mg/kg) was injected once a
day for 1 week into Fisher344 rats (7- to 8-week-old animals). Within the subgranular layer of the DG,
NeuroD1-positive cells apparently incorporated BrdU.
Taken together with an immunohistochemical analysis, it
was confirmed that NeuroD1-expressing cells appearedin the mitotically active progenitor stage first, and they
were mainly in immature neurons within the granule cell
layer of the DG.
Based on the expression of NeuroD1 during adult neurogenesis and the essential role of NeuroD1 in newborn
neurons in the adult hippocampus, we focused on themechanism by which the transcription of the NeuroD1
gene is regulated to better understand neurogenesis in
adult NSCs. To gain insight into the possible transcription regulation of NeuroD during the transition from
NSCs to neuronal cells, we analyzed the DNA binding
sequences of Sox2 and TCF/LEF transcription factors on
the NeuroD1 and NeuroD2 promoters. The binding sites
for the TCF/LEF and Sox2 transcription factors were
found within 3 kb upstream of the promoter region ofboth the NeuroD1 and NeuroD2 promoters. Interestingly, some sequences overlapped. These overlapping DNA
regulatory elements were conserved among the human,
rat and murine NeuroD1 promoters. This observation led
to the subsequent analysis of the functional significanceof these overlapping sequences and their reciprocal control over neurogenesis in the adult DG.32
Does Wnt Signal through β-catenin to
Activate TCF/LEF to Regulate Neurogenesis?
We now have evidence that NeuroD1 is up-regulated
in early committed neuronal cells. To evaluate this effect in vivo, LV-β-catenin shRNA or control lentivirus(LV) expressing only GFP (LV-GFP) was stereotactically injected into the DG of young adult mice (i.e., 6 to 8
weeks old; n = 12/group). Two weeks after the injection,
BrdU (100 mg/kg) was injected once a day for 1 week.
Three weeks after the original injection, the animals
were analyzed. Our results revealed that control LV-GFP
injections resulted in many GFP-positive cells in the
granule cell layer of the DG. Some of those GFP-positive cells were Sox2-positive and BrdU-positive, indicating that the lentivirus preferentially infected not only
mature neurons but also proliferating cells, including undifferentiated NSCs.32 This finding is consistent with ourprevious report in which LV-based tracing analysis of
Sox2-positive cells within the DG was described.1
LV-β-catenin shRNA-infected cells were observed in
the inner layer of the DG, and the GFP-positive cells expressing β-catenin shRNA (shRNA-GFP) also co-localized with Sox2. The shRNA-GFP cells co-localizing
with Sox2 were labeled by BrdU, indicating that thecells in which the β-catenin gene was down-regulated
survived and remained in an undifferentiated state. 3 2Thus, compared to dnWnt, LV-β-catenin shRNA results
in a partial loss of function; therefore, it is possible that
small changes in Wnt signaling may regulate proliferation vs. differentiation, whereas complete loss of Wnt
signaling would be more detrimental to the neurogenic
niche. Thus, as with other molecules involved in these
dynamic processes, there are gradients of activity, andthe magnitude of signaling within a given signaling
pathway may have qualitative as well as quantitative effects.
Discussion and Summary
Adult NSCs are a potential endogenous source forneuronal cell replacement in the diseased adult CNS.
However, the recruitment of adult NSCs for repair ishampered by the current lack of knowledge about thecellular and molecular mechanisms that control their behavior in vivo. We have previously demonstrated that environment-derived signals control the fate choice ofadult NSCs. More recently we have provided evidence
that in the adult hippocampal DG – one of the two neurogenic regions of the adult CNS – specialized astrocytes provide signals that instruct NSCs to adopt a neuronal fate. Here I summarized the in vitro and in vivo
molecular mechanisms underlying the neuronal fate instruction of adult NSCs by the local astrocyte populationin the adult hippocampus. In particular, I focused on theWnt family of proteins, which we have found to be expressed in adult hippocampal astrocytes; our studies
have also shown that these proteins enhance the generation of neurons from adult NSCs in vitro.
Adult neurogenesis represents an extreme form of neural plasticity that has the potential to have significant impact on memory capabilities throughout life. It is clear
from many reports that neurogenesis is regulated bymany normal life conditions (e.g., stress, age and environmental enrichment), is impaired in a variety of neurological and psychiatric diseases, and can be actively regulated by both behavioral and pharmaceutical interventions (e.g., exercise, SSRIs and chemotherapy), suggesting that the birth of new neurons may be a mechanism totarget when addressing neurological and psychiatric diseases. If neurogenesis is going to be used as a therapeutic target in addressing age- and stress-related disorders,for instance, it is important to understand the molecularand cellular mechanisms that control and regulate theprocess. We have uncovered a potential interaction between three separate molecular pathways that work together to sustain NSCs in the brain and regulate the generation of new neurons in the brain. Understanding theway these pathways coordinate to generate neurons will
provide new targets to protect or enhance adult neurogenesis. A better understanding of how adult neurogene-Keio J Med 2010; 59 (3): 79－83

sis affects and contributes to the function of the hippocampus will help to address why the dysregulation of
adult neurogenesis may relate to certain natural and neurological conditions.
Acknowledgments
I am particularly indebted to those scientists who collaborated with me on the projects reported in the review,
including Tomoko Kuwabara, Sebastian Jessberger, Chichung Lie, Hong Jun Song, Hoonkyo Suh, Xinyu Zhao,
Kinichi Nakashima, Ayumu Tashiro and Ahmet Denli.
The work was supported by grants from NINDS/NIH
and the McDonnell Foundation.
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