First published online 16 December 2004
doi: 10.1242/dev.01567
Development 132, 335-344 (2005)
Published by The Company of Biologists 2005
Sonic hedgehog controls stem cell behavior in the postnatal and adult brain
Verónica Palma1,*,
,
Daniel A. Lim2,*,
Nadia Dahmane1,*,
,
Pilar Sánchez1,3,*,
,
Thomas C. Brionne4,*,
Claudia D. Herzberg4,*,
Yorick Gitton1,¶,
Alan Carleton4,
Arturo Álvarez-Buylla2 and
Ariel Ruiz i Altaba1,3,**
1 The Skirball Institute, NYU School of Medicine, 540 First Avenue, New York, NY
10016, USA
2 Neurosurgery Research, UCSF, 10 Kirkham Street, San Francisco, CA 94143,
USA
3 University of Geneva Medical School, 8242 CMU, 1 rue Michel Servet, 1211
Geneva 4, Switzerland
4 Brain and Mind Institute, EPFL, Bat AAB, 1015 Lausanne, Switzerland
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Fig. 1. Gli1 and Shh gene expression in the SVZ. (A,C) Expression
of Shh mRNA in the lateral wall of the lateral ventricles (LV) of
adult mice. At high magnification, Shh expression is clearly detected
in SVZ cells (C). (B,D,F-H) Expression of Gli1 mRNA in the lateral
wall of the lateral ventricle of adult (B,D) and postnatal (P3; G,H) mice. (E)
Control section showing lack of hybridization of Shh antisense RNA
probes in the fourth ventricle (4V) of an adult mouse. Sense probe controls
gave no signal. (A-H) in situ hybridizations on cross sections. Arrows point
to sites of expression. Dorsal is to the top. Scale bar in F: 400 µm for
A,F; 200 µm for C-E; G, 150 µm; H, 30 µm.
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Fig. 2. Gene expression analyses in cell populations in the postnatal and adult
SVZ. (A) RT-PCR analyses of postnatal (P5) and adult sorted cells. Postnatal
whole SVZ is also shown as control. (B) RT-PCR analyses of Shh
expression in the SVZ and adjacent striatum (ST) from the same animal. Note
that Shh is expressed in the adult SVZ but it is not detected in
either B or E sorted cells (see text). All samples were tested with (+) or
without (-) reverse transcriptase to control for any possible signal resulting
from contaminating genomic DNA. (C) RT-PCR analyses of gene expression in P7
SVZ neurospheres (SVZ-NS). As positive control (+), RNA from a P7 brain was
used. As a negative control P7 SVZ RNA without reverse transcriptase was used
(-). As control for RT-PCR, all genes were found to be expressed in dissected
but non-cell-sorted SVZ pieces. As control for RNA recovery and amounts of
cDNA, the levels of the housekeeping gene Hprt were measured.
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Fig. 3. Gene expression in single SVZ cells. (A) Schematic representation of the
experimental procedure. Individual SVZ cells were randomly harvested with a
patch-clamp pipette from fresh living slices, and were used for single cell
multiplex RT-PCR. (B) Examples of RT-PCR assays showing gene expression in
single SVZ cells. (C) Summary graph showing the expression of GFAP,
Gli1 or both in single cells, shown as percentage of the total number of
collected cells (n=65).
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Fig. 4. Cyclopamine inhibits SVZ cell proliferation in vivo. (A,B,E) Cross section
through the forebrain SVZ of an adult mouse showing normal BrdU incorporation
(arrows in A,B) or the expression of GFAP and Nestin (E) following one week's
injection of HBC carrier (cyclodextrin) alone. Animals were perfused
 12-24 hours after the last injection. (C,D,F) Decrease of
BrdU+ cells in adult mice treated with cyclopamine for one week
(C,D) does not lead to the loss of Nestin+ or GFAP+
cells (F). (G) Quantification of the number of BrdU+ cells in the
SVZ of control and cyclopamine-treated adult mice. Counts are averaged and
shown per section. Error bars=s.e.m., n=13 for control HBC-injected
mice and n=18 for cyclopamine-injected mice in four independent
experiments pooled together. Out of 18 cyclopamine-injected mice, three
animals did not respond, five animals decreased the number of BrdU+
cells by 50%, and ten animals reduced incorporation by 100%. No
reduction was observed in the HBC-injected mice. (H) RT-PCR of fresh SVZ
tissue from adult control or cyclopamine-treated mice, dissected 4 hours after
the last injection. Hprt levels are used as loading controls.
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Fig. 5. Reduced number of newborn interneurons in the adult olfactory bulb after
cyclopamine treatment. (A) Experimental procedure. BrdU injections are done
during cyclopamine or vehicle treatment. One month post-injection, the number
of newborn neurons is quantified after BrdU staining. (B,C,D,E) Photographs of
the BrdU staining in the olfactory bulb of vehicle- (B,C) or cyclopamine-
(D,E) treated mice. (F) The number of BrdU+ cells in the olfactory
bulb of cyclopamine-treated mice (open circle, n=5) is significantly
reduced in comparison to vehicle-treated mice (filled circle, n=5)
along the antero-posterior axis. Error bars=s.e.m.
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Fig. 6. Shh signaling regulates SVZ proliferation and neurogenesis. (A)
Quantification of the effects of Shh on the proliferation of dissociated P5
SVZ cells plated on a quiescent astrocytic monolayer. BrdU incorporation was
quantified by immunofluorescence. Under these conditions, SVZ precursors
proliferate and generate new neurons, as they normally do in vivo. (B)
Quantification of the effects of blocking anti-Shh monoclonal antibody (5E1)
on the proliferation of P5 SVZ cells after dissociation and reaggregation.
Cell proliferation was measured by radioactive thymidine incorporation. (C)
Quantification of the effect of Shh on neurogenesis in dissociated adult SVZ
cells plated on an astrocytic monolayer. Generation of new neurons was
measured by co-labeling with Tuj1, identifying neurons, and anti-BrdU
antibodies, identifying cells that replicated after BrdU addition. (D)
Quantification of the effects of Shh on isolated type A SVZ neuroblasts. Type
A cells were purified from P5 mice and cultured with or without Shh. At 3 and
7 days, the number of Tuj1+ cells in Shh-treated cultures were compared to
control cultures. Error bars=s.e.m. (E) Immunocytochemistry of a 7-day SVZ
cell culture on an astrocytic monolayer showing the labeling of neurons with
Tuj1 (red) and recently divided cells with anti-BrdU (green) antibodies. Note
the large number of doubly labeled (yellow) cells representing newly born
neurons. (F) Nomarski optics image of the same panel shown in E.
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Fig. 7. Shh regulates proliferation and neurosphere formation in cooperation with
EGF. (A) Image of neurospheres from adult SVZ cultures. (B) Quantification of
the number of primary neurospheres formed from cultures of SVZ cells
previously grown on astrocytic monolayers with or without exogenous Shh (5
nM). Shh was not added to the neurosphere cultures. (C) Synergism of Shh and
EGF on neurosphere proliferation. The assay was done with a constant dose of
EGF at 1 ng/ml, and varying doses of Shh at 5 or 0.5 nM (left) or with a
constant does of Shh at 5 nM and varying doses of EGF at 5 and 0.5 ng/ml
(right). Treatments were for 48 hours. (D,E) Quantification of proliferation
as measured by the percentage of BrdU+ cells (D) and the number of
clones obtained in cloning assays (E) in adult SVZ neurospheres treated with
cyclopamine (5 µM) or treated with an equal dose of ethanol used as carrier
for in vitro work. In all cases, error bars indicate s.e.m. of triplicate
cultures.
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© The Company of Biologists Ltd 2005
