Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells.

Abstract

Neural stem cells that continue to produce neurons are retained in the adult hippocampal dentate gyrus. The mechanisms by which embryonic neural progenitors expand and transform into postnatal neural stem cells, an essential process for the continual production of neurons throughout life, remain unknown. We found that radial astrocytes, the postnatal progenitors in the dentate gyrus, failed to develop after embryonic ablation of ciliary genes or Smoothened (Smo), an essential component for Sonic hedgehog (Shh) signaling. Postnatal dentate neurogenesis failed in these mutant mice, and the dentate gyrus became severely hypotrophic. In contrast, expression of a constitutively active Smo (SmoM2-YFP) resulted in a marked expansion of the dentate gyrus. Double-mutant analyses suggested that both wild-type Smo and SmoM2-YFP function through the primary cilia. We conclude that Shh signaling, acting through the primary cilia, has a critical role in the expansion and establishment of postnatal hippocampal progenitors.

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Hedgehog signaling and primary cilia are required for
the formation of adult neural stem cells
Young-Goo Han1, Nathalie Spassky2, Miriam Romaguera-Ros3, Jose-Manuel Garcia-Verdugo3, Andrea Aguilar2,
Sylvie Schneider-Maunoury4& Arturo Alvarez-Buylla1
Neural stem cells that continue to produce neurons are retained in the adult hippocampal dentate gyrus. The mechanisms by
which embryonic neural progenitors expand and transform into postnatal neural stem cells, an essential process for the continual
production of neurons throughout life, remain unknown. We found that radial astrocytes, the postnatal progenitors in the dentate
gyrus, failed to develop after embryonic ablation of ciliary genes or Smoothened (Smo), an essential component for Sonic
hedgehog (Shh) signaling. Postnatal dentate neurogenesis failed in these mutant mice, and the dentate gyrus became severely
hypotrophic. In contrast, expression of a constitutively active Smo (SmoM2-YFP) resulted in a marked expansion of the dentate
gyrus. Double-mutant analyses suggested that both wild-type Smo and SmoM2-YFP function through the primary cilia. We
conclude that Shh signaling, acting through the primary cilia, has a critical role in the expansion and establishment of postnatal
hippocampal progenitors.
Granule neurons in the hippocampaldentate gyrus receive the primary
input from the cortex and have crucial roles in learning and memory.
The genesis of these neurons begins during mid-gestation and con-
tinues throughout life. Granule neuron precursors (GNPs) are initially
specified in the dentate neuroepithelium of the hippocampus, migrate
from this region into the developing dentate gyrus, expand in number
and assume final residence in the subgranular zone (SGZ) between the
condensing granular layer and the hilus1,2. Production of new granule
neurons continues postnatally, resulting in a substantial increase in the
size of the dentategyrus during the first 10d of lifeinmice.During this
period, GNPs acquire morphological features of astroglial cells, with
radial processes spanning the entire granular layer. These radial astro-
cytes, also known as type I progenitors, function as the SGZ stem cells
and continue to generate new granule neurons throughout life3–5.
Initial expansion of GNPs and their conversion into radial astrocytes
are essential steps for proper dentate gyrus development and the
maintenance of neurogenesis in the adult.
The primary cilium is a hair-like appendage extending from the
surface of a cell. This specialized structure, with a unique microtubular
cytoskeleton (axoneme) and a surrounding membrane, is assembled
andmaintained by the intraflagellar transport (IFT) machinery6. IFTis
a specialized intracellular trafficking between the cell body and the tip
of the cilia, in which kinesin-II and cytoplasmic dynein motors move
cargoesbound tomultiprotein complexescalledIFTparticlesalongthe
ciliary axoneme. IFT is a highly conserved process, as mutations in the
IFT motors or particles result in the loss of cilia from single-celled
organisms to mammals.
Recent work has shown that primary cilia concentrate receptors
and signal transduction components that have vital roles in develop-
ment7–9. In particular, evidence in the developing neural tubeand limb
bud has shown that genes encoding the IFTmotors (Kif3a, a subunit of
kinesin-II, and Dnchc2, a subunit of cytoplasmic dynein) and the IFT
particle subunits (Ift52, Ift88 and Ift172) are required for Shh signal-
ing10–14. Consistently, essential Shh signaling components, Patched
(Ptc), Smo, Suppressor of fused and Gli transcription factors, concen-
trate in primary cilia10,15,16.
Defective primary cilia have been associated with diverse human
diseases including Alstro ¨m, Bardet-Biedl, Joubert, Meckel-Gruber and
Oral-facial-digital type 1 syndromes17. The pathology of these pleio-
tropic disorders includes obesity, mental retardation and ataxia,
suggesting that primary cilia are required for the proper development
or function of the brain. A recent study showed that conditional
ablation of primary cilia in a subset of hypothalamic neurons caused
hyperphagia-induced obesity18. Neural stem cells in both embryonic
andadultbrainshaveaprimarycilium19,butitsfunctionisnotknown.
Using mutant mice that lack primary cilia in a subset of embryonic
neural stem cells, we show here that primarycilia and Shh signaling are
essential for the expansion and establishment of GNPs in the postnatal
dentate gyrus.
RESULTS
Loss of primary cilia in hGFAP::Cre; Kif3afl/flmice
Primary cilia are present in neural progenitor cells in the ventricular
zone of the embryo and in the subventricular zone of the adult
Received 19 December 2007; accepted 29 January 2008; published online 24 February 2008; doi:10.1038/nn2059
1Department of Neurological Surgery, Institute for Regeneration Medicine, University of California San Francisco, San Francisco, California 94143, USA.2Universite ´ Pierre
and Marie Curie-Paris 6, IFR des Neurosciences, Ho ˆpital Pitie ´-Salpe ˆtrie `re, INSERM, U711, Paris, F-75013 France.3Laboratorio de Morfologia Celular, Unidad asociada
Universidad de Valencia and Centro de Investigacion Principe Felipe, Valencia, 46100 Spain.4CNRS UMR7622, Universite ´ Pierre et Marie Curie, 9 Quai Saint Bernard,
75005 Paris, France. Correspondence should be addressed to A.A.-B. (abuylla@stemcell.ucsf.edu).
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mouse19, but have not beenshown to exist onneuralprogenitors inthe
developingdentategyrus.Wediscoveredthatprimaryciliawerepresent
in the developing dentate gyrus at postnatal day 0 (P0; Fig. 1a).
Primary cilia in the developing dentate gyrus were 1.5–2 mm long
and present in cells containing numerous free ribosomes with little
rough endoplasmic reticulum, characteristic of dividing progenitor
cellsintheadult subventricular zone andSGZ5,20. To test whether these
primary cilia are involved in the development of the hippocampus,
we conditionally ablated primary cilia in a subset of neural progenitors
by crossing mice expressing Cre under the human glial fibrillary
acidic protein promoter (hGFAP::Cre) with mice carrying conditional
Kif3a alleles (Kif3afl/fl). Kif3a encodes a subunit of kinesin-II motor
that is essential for ciliogenesis21. GFAP was strongly expressed in a
subset of radial glia in the dentate neuroepithelium22(Supplemen-
tary Fig. 1 online). Consistently, the hGFAP promoter drove Cre
expression in the hippocampal ventricular zone, giving rise to recom-
bined cells in the hippocampus, including the dentate gyrus23
(Supplementary Fig. 1).
hGFAP::Cre; Kif3afl/flmice were born in Mendelian ratios. When
analyzedusingelectronmicroscopy,precursorcellsinthedentategyrus
of the P0 hGFAP::Cre; Kif3afl/flmice contained a basal body anchored
with transition fibers and a rootlet, but no primary cilium (Fig. 1b). At
the distal tip of this basal body, we observed a short expansion of
membrane, but this structure was much shorter than a primary cilium
and lacked an axoneme (Fig. 1b). Three-dimensional reconstructions
of serial ultra-thin sections in 15 dentate gyrus cells from hGFAP::Cre;
Kif3afl/flmice confirmed the absence of primary cilia (Supplementary
Fig. 2 online). In contrast, all 15 serially reconstructed cells from wild-
typemicehad a primarycilium.Thus, theconditionalremovalofKif3a
using hGFAP::Cre results in the loss of primary cilia in the developing
dentate gyrus.
Kif3a is required for postnatal dentate gyrus neurogenesis
At P21, the Ammon’s horn of the hippocampus in the hGFAP::Cre;
Kif3afl/flmice showed normal morphology (insets in Fig. 1c,d). How-
ever, the dentate gyrus in these mice was disorganized and was much
smaller than in wild-type mice (Fig. 1c,d). In wild-type mice, hema-
toxylin staining revealed numerous darkly stained cells corresponding
to type D cells5, also known as type II progenitors3,4, in the SGZ. These
cells are derived from radial astrocytes and correspond to polysialyated
neural cell adhesion molecule (PSA-NCAM)-positive neuroblasts and
young granule neurons. These cells were absent in the mutant dentate
gyrus (Fig. 1d). The small size of the dentate gyrus and the lack of
darkly stained cells suggested a defect in the postnatal generation of
granule neurons in hGFAP::Cre; Kif3afl/flmice. To test whether young
neurons are absent in the mutant dentate gyrus, we stained sections for
PSA-NCAM (Fig. 1e,f). Although many PSA-NCAM–positive cells
wereobservedinthewild-typeSGZ(Fig.1e),expressionofthismarker
in the mutant dentate gyrus was rare (Fig. 1f). This suggests that the
production of new neurons is defective. Consistently, proliferation was
severely reduced in the dentate gyrus of hGFAP::Cre; Kif3afl/flmice. On
average, eight cells were labeled per section in the wild-type dentate
gyrus (n ¼ 3) 1 h after bromodeoxyuridine (BrdU) injection (Fig. 1g).
However,inhGFAP::Cre;Kif3afl/flmice(n¼3),BrdU-labeledcellswere
rare (Fig. 1h); analysis of 41 sections yielded only four BrdU-labeled
cells. These observations suggest that germinal activity in the dentate
gyrus of hGFAP::Cre; Kif3afl/flmice is severely disrupted.
Staining for GFAP in hGFAP::Cre; Kif3afl/flmice revealed a marked
difference with wild-type mice. In wild-type mice, GFAP labeled
a regular palisade of radial astrocytes, the primary progenitors in
the SGZ3–5(Fig. 1i). These cells were absent in the mutant dentate
gyrus. Notably, this deficiency was specific to radial astrocytes, as
other GFAP-positive astrocytes in the dentate gyrus, hilus, molecular
layer or the rest of the hippocampus were present in the mutant
mice (Fig. 1j).
WT
Transmission
electron micrograph
Hematoxylin
PSA-NCAM
DAPI
BrdU
Hematoxylin
GFAP
DAPI
Kif3a mutant
a
b
c
d
e
f
gh
ij
Figure 1 Ablation of Kif3a removes primary cilia and disrupts germinal
activity in the adult dentate gyrus. (a,b) Transmission electron micrographs
showed that primary cilia present in the dentate gyrus (DG) of wild type (WT)
at P0 were absent in hGFAP::Cre; Kif3afl/flmutants. Magenta arrow indicates
the primary cilium and arrowheads indicate the basal body. The cilium
contains the 9+0 axoneme (inset). In the mutant, only a short extension of
membrane devoid of axoneme (green arrow) was associated with the basal
body with characteristic transition fibers (blue arrow) and rootlet (blue
arrowhead). Yellow arrow indicates a centriole attached to the basal body.
(c–j) Analysis of P21 mice. (c,d) Hematoxylin staining showed that the DG
of hGFAP::Cre; Kif3afl/flmice was small and disorganized compared with WT.
Insets, only the DG was severely affected. Arrows indicate numerous darkly
stained type D cells in the WT SGZ. The mutant DG lacked a well-defined
SGZ and darkly stained cells. (e,f) WT mice had many PSA-NCAM–positive
young neurons (arrows) in the SGZ, whereas only rare cells were positive
for PSA-NCAM in the mutant. (g,h) Analysis of proliferation by BrdU
incorporation for 1 h. Several BrdU-positive cells (arrows) were present in the
WT SGZ, whereas no cells were positive for BrdU in the mutant. (i,j) Antibody
to GFAP stained the palisade of radial astrocytes (arrow) that function as
primary progenitors in the WT SGZ. The mutant DG lacked these radial
astrocytes. Notably, GFAP-positive astrocytes were still present in
non-neurogenic locations (arrowheads) of the mutant DG. Scale bars,
0.5 mm (a,b), 0.1 mm (inset in a) and 100 mm (c–j).
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Kif3a is required to expand GNP and form radial astrocytes
The radial organization of the dentate gyrus becomes apparent by P10
and coincides with the appearance of radial astrocytes at the border
between the condensing granular layer and the hilus2. To test if the
initialorganizationoftheradiallyorientedGNPpopulationisdefective
in the mutant dentate gyrus, we labeled dividing GNPs at P10, after
GNP translocation is completed (Fig. 2). A 1-h pulse of BrdU resulted
in many labeled cells in the SGZ of wild-type mice (Fig. 2a). In
contrast, the dentate gyrus of hGFAP::Cre; Kif3afl/flmice had only one
third of the number of BrdU-labeled cells that we observed in controls
(Fig.2k),andthefewlabeledcellsinthemutantdentategyruswerenot
in the SGZ, but were mostly in the granular layer (Fig. 2b). Mash1 is
expressed by GNPs throughout development and by adult GNPs in the
SGZ24. At P10, the number of Mash1-expressing cells in the dentate
gyrus of hGFAP::Cre; Kif3afl/flmice was greatly reduced compared with
controls (Fig. 2c,d). The few Mash1-positive cells in the mutant mice
were not in the SGZ, but were again distributed throughout the
granular layer. Consistently, radial astrocytes in the mutant dentate
gyrus were rare, in contrast with their abundance in the wild-type
dentate gyrus (Fig. 2e,f). These observations suggest that, in
hGFAP::Cre;Kif3afl/flmice,thenumberofproliferatingGNPsisseverely
reduced by P10 and very few cells become radial astrocytes.
We next examined the dentate gyrus of hGFAP::Cre; Kif3afl/flmice at
ages younger than P10 to determine when the defect in proliferation
occurs.Byembryonicday18.5(E18.5),GNPproliferationinthemutant
dentate gyrus was already significantly decreased compared with wild
type (P o 0.000001; Fig. 2g,h,l). However, proliferation in the dentate
neuroepithelium and the migratory stream to the developing dentate
gyrus was not affected in the mutant mice, suggesting that the earlier
development of GNPs was not affected. Consistently, at E16.5, prolife-
ration in the developing dentate gyrus was indistinguishable between
wild-type and hGFAP::Cre; Kif3afl/flmice (Fig. 2i,j,m). The number of
apoptotic cells that were positive for cleaved caspase-3 in the dentate
gyrusatE18.5wasindistinguishablebetweenwild-typeandhGFAP::Cre;
WT
P10
c
P10
e
P10
g
E18.5
i
E16.5
k
BrdU
Hematoxylin
Mash1
Hematoxylin
GFAP
Hematoxylin
BrdU
DAPI
BrdU
DAPI
Kif3a mutant
Kif3a mutant
25
20
15
10
5
0
P10
E18.5
E16.5
120
100
80
60
40
20
*
*
200
150
100
50
0
0
BrdU+ cells per section
BrdU+ cells per section
BrdU+ cells per section
DG
WT
DG MSDEDG+MS
DE
a
b
d
f
lm
h
j
Figure 2 hGFAP::Cre; Kif3afl/flmice fail to expand GNPs and to establish
adult stem cells in the dentate gyrus. (a,b) Analysis of proliferation by BrdU
incorporation for 1 h at P10. Many BrdU-positive cells (arrows) were present
in the SGZ of WT. In the mutant DG, BrdU-positive cells were rare and were
not in the SGZ, but were mostly in the granular layer. (c,d) GNPs in the WT
SGZ expressed Mash1 (arrows). In the mutant DG, Mash1-positive cells were
rare and were not confined to the SGZ. (e,f) Many GFAP-positive radial
astrocytes were established in the WT SGZ at P10 (arrow), but these cells
were rare in the mutant SGZ. (g–j) Analysis of proliferation by BrdU
incorporation for 1 h at E18.5 (g,h) and E16.5 (i,j). Refer to Figure 5i for a
schematic illustration identifying the dentate neuroepithelium (DE), migratory
stream (MS) and DG. Dashed lines indicate the outline of the DG. The
number of BrdU-positive cells (arrows) was significantly decreased in the
mutant DG (h) compared with WTat E18.5 (g), but not at E16.5 (i,j).
Proliferation in the DE and MS were not significantly different between the
mutants and WT. (k–m) Quantification of BrdU incorporation at P10 (k),
E18.5 (l) and E16.5 (m). Data are mean ± s.e.m. * indicates P o 0.000001
from Student’s t-test. Scale bar, 100 mm.
*
200
Ki67
DAPI
150
Ki67+ cells per section
100
50
WT
lft88orpk/orpk
lft88orpk/orpk
0
WT
a
b
cdef
Figure 3 Ift88orpk/orpkmice show decreased proliferation of GNPs in the
dentate gyrus. (a–c) Analysis of proliferation in the DG by staining a
proliferation marker Ki67 at P1. Dashed lines indicate the outline of the DG.
Proliferation was significantly decreased in the Ift88orpk/orpkDG (b) compared
with WTat P1 (a). (c) Quantification of Ki67-positive cells. (d–f) Transmission
electron micrographs of primary cilia present in the developing DG of
Ift88orpk/orpkat P1; three representative pictures (mid-cilia sections) from
30 cilia serially reconstructed. Arrows indicate the primary cilium and
arrowheads indicate the basal body. One third of reconstructed cilia were
severely disrupted (d), similar to those in the DG of hGFAP::Cre; Kif3afl/fl
mice (Fig. 1b). The other two thirds (e,f) were shorter than those in wild-type
(Fig. 1a), but maintained relatively normal structures. Data are mean ± s.e.m.
* indicates P ¼ 0.027 from Student’s t-test. Scale bar, 100 mm (a,b) and
1 mm (d–f).
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Kif3afl/flmice (Supplementary Fig. 3 online). Therefore, differences in
cell death do not appear to explain the reduction of the numbers of
GNPs. Instead, the loss of Kif3a in GNPs resulted in a marked decrease
in GNP proliferation sometime between E16.5 and E18.5.
In hGFAP::Cre; Kif3afl/flmice, expansion of dentate gyrus progeni-
tors is specifically affected; the hippocampus and other telencephalic
brain regions appear to develop normally. This suggested that a lack of
proper kinesin-II function does not result in generalized defects in
intracellular trafficking or cytokinesis, as has been suggested by
experiments in cultured cells using overexpression of dominant-
negative subunits of kinesin-II25–27. We tested whether intracellular
trafficking and cytokinesis were affected in dentate gyrus progenitors
from hGFAP::Cre; Kif3afl/flmutant dentate gyrus and found no
differences from controls (Supplementary Fig. 4 online). Taken
together, these observations suggest that the loss of progenitor cells
and hypoplasia of the dentate gyrus in hGFAP::Cre; Kif3afl/flmice are
results of the loss of primary cilia and not of defective intracellular
trafficking or cytokinesis.
Decreased GNP proliferation in Ift88 and Ftm mutants
To verify that the phenotype in the dentate gyrus of hGFAP::Cre;
Kif3afl/flmice is a result of the loss of cilia, we examined proliferation
of GNPs in mice that were defective for Ift88, a gene that encodes an
essential subunit of IFT particles. Mice with null alleles of Ift88 die
around E1028, which is much earlier than the development of the
dentate gyrus. We therefore analyzed a hypomorphic insertional
mutant allele, Ift88orpk/orpk, which dies postnatally29, allowing us to
study the early stages of dentate gyrus development. The prolifera-
tion of GNPs in the dentate gyrus of Ift88orpk/orpkmice decreased
significantly by 38% compared with wild type at P1 (P ¼ 0.027;
Fig. 3a–c). This decline was smaller compared with the 64% (at
E18.5) or 72% (at P10) decreases that we observed in hGFAP::Cre;
Kif3afl/flmice. This is likely due to the hypomorphic nature of the
Ift88orpk/orpkmutation. Three-dimensional reconstructions of serial
ultra-thin sections revealed that one third of primary cilia (10 out of
30 reconstructions) in the dentate gyrus of Ift88orpk/orpkmice were
severely disrupted (Fig. 3d), as shown above for hGFAP::Cre; Kif3afl/fl
mice. The other 20 cilia reconstructed in the dentate gyrus of
Ift88orpk/orpkmice were shorter than controls, but maintained rela-
tively normal structures (Fig. 3e,f).
Fantom (Ftm; also known as Rpgrip1l, or retinitis pigmentosa GTPase
regulator interacting protein 1 like) encodes a basal body protein, and
mutations in this gene are found in ciliary disorders including Joubert
and Meckel syndromes30. Consistently, Ftm mutant mice (Ftm–/–) have
malformed cilia and reduced numbers of cilia31. The Ftm–/–phenotype
shows variable penetrance, with death beginning early during embryo-
nic development in the severely affected mice and occurring at birth in
less severe ones. We analyzed proliferation in the dentate gyrus in
Gli1
Gapdh
WT
WT
Mutant
RT-PCR
Gli1 in situ
β-gal activity
from PtclacZ/+
Kif3a mutant
a
b
c
d
e
Figure 4 hGFAP::Cre; Kif3afl//flmice show defective Shh signaling in the
dentate gyrus. (a–c) Gli1 expression in the WTand mutant DG. Gli1
expression was detected by RT-PCR (a) and in situ hybridization (b) from
the WT DG, but was barely detectable in the mutant DG (a,c). Each lane
represents amplification from an individual mouse. Primers were designed
to exclude amplification of genomic DNA. Gapdh was used for an internal
control. (d,e) b-galactosidase activity from Ptclacz/+locus revealed active and
defective Shh signaling in the WT (d) and mutant DG (e), respectively.
Arrows indicate the DG. Scale bar, 100 mm.
Hematoxylin
BrdU
DAPI
BrdU
DAPI
GFAP, PSA-NCAM
DAPI
WT
Smo mutant
140
120
100
80
60
40
20
DE
MS
DG
GNP
E18.5
0
BrdU+ cells per section
DG MSDE
WT
Smo mutant
*
*
P21
c
P21
e
P21
g
E18.5
i
a
b
d
f
h
j
Figure 5 hGFAP::Cre; Smofl/flmice show defects in dentate gyrus
development that resemble hGFAP::Cre; Kif3afl/flmice. (a,b) Hematoxylin
staining showed that hGFAP::Cre; Smofl/flmutant mice have small and
disorganized DG (b) similar to hGFAP::Cre; Kif3afl/flmice (Fig. 1d). Insets
show a low-magnification view of the hippocampus. Arrows indicate darkly
stained type D cells in the WT SGZ. (c,d) BrdU incorporation for 1 h revealed
a lack of dividing cells in the mutant SGZ at P21. Arrow indicates a BrdU-
positive cell in WT. (e,f) The mutant DG had few PSA-NCAM–positive young
neurons (yellow arrow) and no GFAP-positive radial astrocytes (white arrow).
As in hGFAP::Cre; Kif3afl/flmice, GFAP-positive astrocytes in non-neurogenic
locations (arrowheads) were still present in hGFAP::Cre; Smofl/flmice. (g–j)
Analysis of proliferation by BrdU incorporation for 1 h at E18.5 (g,h) and an
illustration identifying DE, MS and DG (i). Dashed lines indicate the outline
of the DG. Proliferation was significantly decreased in the mutant DG and MS
compared with WT (j). Arrows indicate BrdU-positive cells. Data are mean
± s.e.m. * indicates P o 0.000005 from Student’s t-test. Scale bar, 100 mm.
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Ftm–/–mice that survived to E18, when we detected defects in the
hGFAP::Cre; Kif3afl/flmice.InFtm–/–mice,the proliferationof GNPsin
the dentate gyrus was significantly reduced by 48% compared with
wild-type littermates (P ¼ 0.01, data not shown). The reduced
proliferation observed in the dentate gyrus of Ift88orpk/orpkand Ftm–/–
mice is consistent with our results from hGFAP::Cre; Kif3afl/flmice,
indicatingthatprimaryciliaareimportantintheperinatalproliferation
of GNPs in the dentate gyrus. Notably, both Ift88 and Ftm mutations
result in defective Shh signaling12,13,30,31.
Kif3a is required for Shh signaling in the dentate gyrus
Recent evidence in the developing neural tube, cerebellum and limb
bud has shown that primary cilia are required for Shh signaling10–14,32.
Notably, the defects described above in the expansion of GNPs in the
dentate gyrus of hGFAP::Cre; Kif3afl/flmice coincided with the
time when Shh-responding cells normally appear in the developing
dentate gyrus33. We therefore tested whether the loss of primary
cilia results in defective Shh signaling in the dentate gyrus of
hGFAP::Cre; Kif3afl/flmice. A marked decrease in the expression of a
Shh target gene, Gli1, in the hGFAP::Cre; Kif3afl/flmutant dentate
gyrus was observed using RT-PCR and in situ hybridization at P1
and E18.5 (Fig. 4a–c). We confirmed the decrease of Shh signaling
by examining the activity of a lacZ gene knocked into the locus
of another Shh target gene, Ptc (PtclacZ/+)34(Fig. 4d,e). At P0,
b-galactosidase activity was detected in the wild-type dentate gyrus,
but not in the mutant. Thus, Shh signaling in the developing dentate
gyrus is abolished or significantly decreased when Kif3a is mutated
in GNPs.
Smo also required for postnatal dentate gyrus neurogenesis
The above observations indicate that primary cilia are essential for the
proliferation of transitory progenitor cells and generation of the
postnatal GNP population. The alterations in Shh target gene expres-
sionthatwefoundsuggestedthatShhsignaling,viatheprimarycilium,
is central to this process. To test whether defects in Shh signaling
recapitulate the defects observed in Kif3a conditional mutants, we
examined the dentate gyrus of conditional animals for Smo (Smofl/fl)
crossed with the hGFAP::Cre mice. These hGFAP::Cre; Smofl/flmice had
a very small dentate gyrus with no darkly stained cells (Fig. 5a,b),
markedly reduced proliferation (Fig. 5c,d) and few PSA-NCAM–
positive young neurons (Fig. 5e,f). A similar reduction in the size of
the dentate is observed in Nestin::Cre; Smofl/–mice at P1535. The above
phenotype suggests a defect in neurogenesis. We stained the hippo-
campal sections of hGFAP::Cre; Smofl/flwith GFAP antibodies to label
astrocytes. Astrocytes were observedthroughoutthe hippocampus, but
radial astrocytes in the dentate gyrus were missing at P21 (Fig. 5e,f).
These defects were similar to those observed in the hGFAP::Cre;
Kif3afl/flmice, but were more severe. The number of BrdU-labeled
GNPsinhGFAP::Cre;Smofl/flatE18.5(Fig.5g–j)wasmarkedlyreduced
not only in the dentate gyrus, but also in the migratory stream.
Both Smo and SmoM2 require Kif3a for function
Persistent Shh signaling in extra-ciliary regions could explain the
additional proliferation defects that we observed in hGFAP::Cre;
Smofl/flmice compared with hGFAP::Cre; Kif3afl/flmutants. We there-
fore examined the proliferation of GNPs in mutant mice defective
for both primary cilia and Smo (hGFAP::Cre; Kif3afl/fl; Smofl/flmice)
(Fig. 6). These double mutants showed the less severe phenotype that
was similar to that of Kif3a conditional mutant animals (Fig. 6a,c).
Residual Shh signaling outside the primary cilia is, therefore, not the
cause of the more severe phenotype that we observed in hGFAP::Cre;
Smofl/flmice. Previous studies have shown that the formation of Gli3
repressor,which isconstitutive inthe absenceofShh signaling,requires
primarycilia10,11,13,14.Thus, the loss ofboth Gli repressorandactivator
functions could explain the less severe phenotype that we observed in
both hGFAP::Cre; Kif3afl/fland hGFAP::Cre; Kif3afl/fl; Smofl/flmice.
Next we tested whether constitutively active Shh signaling could
rescue the defects that we observed in hGFAP::Cre; Kif3afl/flmice. We
first tested whether Shh signaling is sufficientto expand GNPs by using
a conditionally expressed, constitutively active Smo (SmoM2) fused to
YFP (SmoM2-YFP)36. The expression of SmoM2-YFP in hGFAP::Cre;
SmoM2-YFPfl/+mice resulted in a marked hyperplasia of the dentate
gyrus (Fig. 6b, middle). Prox1 is a homeobox transcription factor
specifically expressed by dentate granule neurons37. Prox1-positive
granule neurons were greatly expanded, resulting in ectopic granule
100
80
60
40
20
0
DG
BrdU+ cells per section
MS DE
*
Kif3a mutant
Smo mutant
Double mutant
BrdU
E18.5 Kif3a
BrdU
E18.5 Kif3a Smo
c
Prox1
P2 Kif3a
Prox1
P2 SmoM2
Prox1
P2 Kif3a SmoM2
YFP γ-Tub
P2 Kif3a SmoM2
YFP γ-Tub
P2 SmoM2
BrdU
E18.5 Smo
Hematoxylin
P2 Kif3a
Hematoxylin
P2 SmoM2
Hematoxylin
P2 Kif3a SmoM2
a
b
d
e
Figure 6 Primary cilia are required for the function of both wild-type and
constitutively active Smo. (a,c) Analysis of proliferation by BrdU incorporation
for 1 h at E18.5. hGFAP::Cre; Smofl/flmice showed decreased proliferation in
MS, which was recovered in hGFAP::Cre; Kif3afl/fl; Smofl/flmice. Data are
mean ± s.e.m. * indicates P o 0.0005 from Student’s t-test.
(b) Hematoxylin staining of P2 DG sections. hGFAP::Cre; SmoM2-YFPfl/+
mice had hypertrophic DG with elongated lower blade (arrowheads) and
ectopic cells in the DE (arrow), whereas hGFAP::Cre; SmoM2-YFPfl/+; Kif3afl/fl
mice showed atrophic DG similar to hGFAP::Cre; Kif3afl/flmice. (d) Prox1-
positive granule neurons in P2 DG. Prox1-positive ectopic granule neurons
(arrows) and elongated lower blade of DG in hGFAP::Cre; SmoM2-YFPfl/+
mice were reversed in hGFAP::Cre; SmoM2-YFPfl/+; Kif3afl/flmice.
(e) SmoM2-YFP localized to the primary cilia (arrow) associated with the
basal body (arrowhead). SmoM2-YFP distributed to the cell body on loss of
primary cilia. Note the high level of SmoM2-YFP in the cell body (magenta
arrow). Scale bars, 100 mm (a,b,d) and 5 mm (e).
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neuronsinventral and lateral hippocampus (Fig.6d, middle). Further-
more, morphogenesis of the dentate gyrus was abnormal, as granule
neurons expanded laterally and did not fold into the characteristic
V-shaped granular layer. To our surprise, removal of Kif3a in cells
expressing SmoM2-YFP (hGFAP::Cre; Kif3afl/fl; SmoM2-YFPfl/+)
reversed the hypertrophic defects in histogenesis, resulting in an
atrophic dentate gyrus similar to that observed in Kif3a conditional
mutants (Fig. 6b,d). Thus, constitutively active Smo was not able to
rescue the defects in hGFAP::Cre; Kif3afl/flmice and required Kif3a for
its action. A previous study showed that Smo localizes to primary
cilia on Shh treatment, but SmoM2 localizes to primary cilia indepen-
dent ofShhinculturedMDCKcells15.WefoundthatSmoM2-YFPalso
localized to primary cilia in hGFAP::Cre; SmoM2-YFPfl/+mice in vivo,
but distributed to the cell body on removal of primary cilia in
hGFAP::Cre; Kif3afl/fl; SmoM2-YFPfl/+mice (Fig. 6e).
DISCUSSION
Shh has been shown to be important for the proliferation and
maintenance of adult neural stem cells in the SGZ35,38. In the present
study, we have shown that Shh signaling is essential for expanding
GNPs during perinatal development to establish the adult stem cell
population. These effects of Shh signaling required Kif3a, an essential
motor for assembling primary cilia. The conditional removal of Kif3a
in a subset of hippocampal progenitors resulted in defective Shh
signaling in GNPs, leading to defective proliferation. In these mice,
the earliest populations of GNPs were specified, but during later stages
of perinataldevelopment, GNPs failedtoexpandand togenerate radial
astrocytes that function as the SGZ stem cells. Notably, other types of
astrocytes in the dentate gyrus were produced. Thus, Kif3a is required
for the production or maintenance of radial astrocytes in the SGZ, but
not for the production or differentiation of other astrocytes in the
hippocampus. This suggests a fundamental difference between non-
germinalastrocytesandastrocytesthatfunctionasprimary progenitors
in the adult brain.
The removal of Smo in the same GNP population resulted in a
similar, but more severe, phenotype, and this severe phenotype was
ameliorated on removal of both Kif3a and Smo, confirming that Shh
acts through primary cilia to expand GNPs and to convert them to
adultprogenitors. Proliferationinthe othermajoradult germinal layer,
the subventricular zone, also showed a reduction in the hGFAP::Cre;
Kif3afl/flmice (data not shown), but this reduction was not as large as
that observed in the SGZ of the hippocampus. Shh signaling and
primary cilia may be particularly important for the expansion and
establishment of germinal centers away from the ventricular zone, as is
the case in the SGZ. Consistently, in hGFAP::Cre; Kif3afl/fland
hGFAP::Cre;Smofl/flmice,wedidnotobservemajordefectsinneuronal
layers that are derived directly from proliferating ventricular zone cells,
such as the cortex and Ammon’s horn of the hippocampus, but we and
others32observed a major deficit in the expansion of cerebellar granule
neuron precursors, which also occurs away from the ventricular zone
(N.S., Y.-G.H., A.A., L. Strehl, M.R.-R., J.-M.G.-V. and A.A.-B.,
unpublished data).
Analyses of double mutants for both Kif3a and Smo demonstrate
thatKif3aactsdownstreamofSmoandthatKif3aisrequiredforallShh
signaling in the developing dentate gyrus. Consistent with our genetic
data, mutant Smo protein that fails to localize to cilia is unable to
activate Shh signaling15. Notably, our results show that even constitu-
tively active Smo (SmoM2) requires primary cilia for its function. Why
SmoM2 is constitutively active in the absence of Shh is unknown. Our
data indicate that, similar to wild-type Smo, SmoM2 signals through
the primary cilia. Recent work has shown that the Shh receptor, Ptc,
localizes to primary cilia, where it binds to Shh16. On binding to Shh,
Ptc exits the primary cilia and Smo accumulates there, resulting in
signaling. The mutation in SmoM2 may target this protein to the
cilium independent of Ptc or Shh, and this localization may be
sufficient to promote constitutive signaling.
Whydovertebratecellsrequire thisintricatelocalization of receptors
and other downstream components in a tiny projection from the cell
surface? The cilia’s small volume relative to the surface area could
allow high sensitivity for detection of low levels of extracellular signals.
The primary cilium, although continuous from the cell body, is a
distinct subcellular compartment, in which trafficking is restricted
by IFT. This restriction may allow the coordinated spread or move-
ment of secondary messengers or effecter molecules. In addition, the
juxtaposition of the basal body and the Golgi complex at the base
of the cilia may facilitate rapid trafficking of molecules such as Smo
into the cilia after receiving extracellular signals and could help
coordinate the cell cycle.
We used a conditional mutant for Kif3a to remove primary cilia.
Kinesin-II has been also suggested to participate in intracellular
trafficking and mitosis in cultured cells25–27. In cells dissociated from
the dentate gyrus of hGFAP::Cre; Kif3afl/flmice, we did not observe
increased incidence of intracellular trafficking or mitotic defects.
Moreover, our genetic evidence indicates that kinesin-II function is
essential for Shh signaling; loss of Kif3a partially rescued the severe
defects that we observed when Smo was removed or when SmoM2 was
expressed. This rescue cannot be explained if Kif3a had a general
function in mitosis or intracellular trafficking. Consistently, condi-
tional removal of either Kif3a or Ift88 results in identical phenotypes in
three different organs, including brain18,32,39. In vivo, Kif3a appears to
be essential for the formation and function of primary cilia, but is
dispensable for mitosis and intracellular trafficking. The discrepancy
between these and previous in vitro studies could be the result of
differences in the cell types used or of differences in the experimental
approaches used to interfere with kinesin-II function. We and
others18,32,39analyzed loss-of-function mutant cells, whereas previous
in vitro studies used overexpression of dominant-negative subunits of
kinesin-II,which,asdemonstratedinChlamydomonas,couldhaveoff-
target effects. In this flagellated unicellular organism, point mutations
in kinesin-II can cause defective mitosis, but null mutations cause
defects only in flagellar assembly. Notably, mitotic defects in
these mutants are dominant, whereas flagellar assembly defects are
recessive40,41. This demonstrates that mitotic defects arise from
acquired aberrant or toxic functions of the mutant kinesin-II protein
in Chlamydomonas.
If kinesin-II had essential roles in mitosis or intracellular trafficking,
generalizeddefectsinour Kif3amutantswould beexpected, but we did
not observe increased apoptosis or general hippocampal defects.
Defects in the dentate gyrus were first detected 5 d after the onset of
Creactivity(atE13)andwerespecifictoGNPsinthedentategyrusand
notinthedentateneuroepitheliumorthemigratorystream,suggesting
that the loss of primary cilia specifically affects GNP expansion in the
dentate gyrus. This proliferation deficit coincides with the time when
Shh signaling starts to occur in the developing dentate gyrus33. Note
also that mutations in Ftm and Ofd1, genes that are not linked to IFT,
but are required for ciliogenesis, also cause defects in Shh signal-
ing30,31,42. Lastly, we observed a substantial reduction in GNP prolife-
ration in other ciliary mutants (Ift88orpk/orpkand Ftm–/–). Altogether,
the results listed here indicate that the phenotype that we observed in
hGFAP::Cre; Kif3afl/flmice is not the result of defects in mitosis or
intracellular transport, but of deficits in Shh signaling mediated by
the cilia.
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Our work does not eliminate the possibility that Kif3a, Ift88 or Ftm,
could also have functions outside of the primary cilia. Kinesin-II
has been shown to localize to axons and synapses in neurons43,44.
Therefore, disruption of other cellular functions of kinesin-II could
have contributed to the phenotypes observed in hGFAP::Cre; Kif3afl/fl
mice.The resultsfrom Kif3a and Smodouble mutants indicate that any
possible nonciliary function forkinesin-II must also beinvolvedin Shh
signaling. Mutations in different ciliogenic genes (Kif3a, Dnchc2, Ift52,
Ift88, Ift172, Ftm and Ofd1)10–14,30,31,42all result in Shh signaling
defects. Although we cannot totally exclude that Kif3a, Ift88 or Ftm
could have some role outside of the primary cilia, cumulative evidence
points to this organelle as an essential component of Shh signaling.
Mental retardation is a prominent feature of several systemic
disorders associated with dysfunctional cilia17. Our findings showing
that primary cilia are essential for hippocampal development could
help to elucidate the molecular etiology of mental retardation found in
these diseases. The present results show that dentate gyrus progenitors
require primary cilia to mediate of Shh signaling at a critical stage of
their development, when they are transitioning and expanding from
embryonic to postnatal neural stem cells.
METHODS
Mice. All animal procedures were approved by the University of California, San
Francisco Institutional Animal Care and Use Committee. The genetically
modified mice used in the study, hGFAP::Cre23, Kif3afl/fl(ref. 45), PtclacZ
(ref. 34), Smofl/fl(ref. 46), SmoM2-YFPfl/fl(ref. 36), Ift88orpk/orpk(ref. 29) and
Ftm–/–(ref. 30), have been described previously.
Tissue preparation and staining. Mice were injected with BrdU (50 mg
per kg of body weight) and perfused 1 h later with 4% paraformaldehyde
(w/v). The brains were postfixed overnight at 4 1C. Embryonic brains were
fixed in the same fixative overnight at 4 1C. For frozen sections, brains were
cryoprotected in 30% sucrose, embedded in Tissue-Tek Optimal Cutting
Temperature compound (Sakura) and cut into 12-mm sections. For poly-
ethylene glycol (PEG) sections, brains were dehydrated in an ethanol
series, embedded in a 2:1 mixture of PEG 1,000 and PEG 1,500, and cut into
10-mm sections. For immunostaining, sections were incubated with primary
antibodies overnight at 4 1C, followed by incubation with secondary antibodies
at 20–23.5 1C for 2 h. For X-gal staining and electron microscopy, brains were
perfused with 2% paraformaldehyde and 2.5% glutaraldehyde and postfixed in
2.5% glutaraldehyde overnight at 4 1C. Coronal 300-mm sections were cut on a
vibratome. The sections were postfixed in 2% osmium for 2 h, dehydrated and
embedded in Araldite (Durcupan, Fluka Biochemica). Semi-thin (1.5 mm) and
ultra-thin (0.06 mm) sections were cut with a diamond knife and stained with
1% toluidine blue and lead citrate, respectively.
Images were taken with an Olympus AX70 light microscope, a Leica
SP2 confocal microscope or a Jeol electron microscope, and processed using
Adobe Photoshop.
Antibodies and reagents. The following primary antibodies were used: rabbit
antibody to GFAP (1:300, DAKO), mouse antibody to Cre (1:500, Abcam), rat
antibody to BrdU (1:10, Oxford Biotech), rabbit antibody to Ki67 (1:1,000,
Novo Castra Labs), mouse antibody to PSA-NCAM (1:400, AbCys), mouse
antibody to Mash1 (1:200, Pharmingen), mouse antibody to acetylated tubulin
(1:1,000, Sigma), rabbit antibody to g-tubulin (1:1,000, Sigma), rabbit antibody
to Prox1 (1:2,500, gift from S. Pleasure, University of California San Francisco),
rabbit antibody to cleaved caspase-3 (1:50, Cell Signaling Technology), chicken
antibody to GFP (1:500, Aves Labs) and mouse antibody to Lamp1 (1:100,
Stressgen). Antibody to GFP was used to visualize the localization of SmoM2-
YFP. For fluorescence staining, sections were incubated with Alexa Fluor–
conjugated secondary antibodies (1:400, Molecular Probes) and counter stained
with DAPI (100 ng ml–1). For enzymatic staining, biotin-conjugated secondary
antibodies (1:200, Jackson Lab) and ABC reaction (Vector Laboratories) were
used. Standard hematoxylin staining was used for counterstaining. FITC-
conjugated HP lectin was used to stain Golgi (0.1 mg ml–1, Sigma).
Cell counting. The immunostained cells were counted in at least three
sections from each mouse at comparable rostrocaudal levels. Data from at
least three mice per each group were pooled for statistical analysis with
Student’s t-test.
RT-PCR. Total RNA was isolated from the dentate gyrus of individual P1 mice
using Qiagen RNeasy Mini Kit and was reverse transcribed by Superscript II
reverse transcriptase (Invitrogen). Both Gli1 and Gapdh cDNAs were amplified
from individual mice by PCR in a single reaction.
Cell culture. Cells were dissociated from the dentate gyrus of P1 mice using
papain (0.6 mg ml–1, Worthington), followed by trituration, resuspended in
DMEM and F12 (50:50) medium supplemented with N2 (Gibco) and B27
(Gibco), and plated in chamber slides coated with poly-D-lysine and laminin.
In situ hybridization. Standard in situ protocol and antisense riboprobe from
Gli1 cDNA (gift from A. Ruiz i Altaba, University of Geneva Medical School)
were used.
Note: Supplementary information is available on the Nature Neuroscience website.
ACKNOWLEDGMENTS
We are grateful to L.S. Goldstein for providing us with the Kif3a conditional
mutant mice, N. Murcia for Ift88orpkmice, and J. Reiter and D. Rowitch for Smo
and SmoM2 conditional mutant mice and PtclacZ/+mice. We thank R. Ihrie,
D. Lim, S. Pleasure, J. Reiter and C. Yaschine on the manuscript. The work was
supported by a Mark Linder/American Brain Tumor Association Fellowship to
Y.-G.H. and by grants (NS28478 and HD32116) from the US National Institutes
of Health and a grant from the Goldhirsh foundation to A.A.-B. N.S. was
supported by the Human Frontier Science Program and the Agence Nationale de
la Recherche. Confocal microscopy at Diabetes Endocrinology Research Center
Microscopy and Imaging Core was supported by an US National Institute of
Health grant (P30 DK063720).
AUTHOR CONTRIBUTIONS
Y.-G.H. designed and performed most of the experiments. M.R.-R. and
J.-M.G.-V. carried out the electron microscopic analyses. N.S. assisted with the
initial analysis of hGFAP::Cre; Kif3afl/flmice. N.S. and A.A. analyzed the Ftm–/–
mice. S.S.-M. provided the Ftm–/–mice. A.A.-B. supervised the project.
Y.-G.H. and A.A.-B. wrote the manuscript.
Published online at http://www.nature.com/natureneuroscience
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions
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