Wnt Signaling Has Opposing Roles in the Developing and the Adult Brain That AreModulated by Hipk1
Cinzia Marinaro1, Maria Pannese2, Franziska Weinandy3, Alessandro Sessa4, Andrea Bergamaschi1, Makoto M. Taketo5,
Vania Broccoli4, Giancarlo Comi6, Magdalena Gotz3, Gianvito Martino1 and Luca Muzio1
1Neuroimmunology Unit, Institute of Experimental Neurology (INSPE), Division of Neuroscience and 2Genetic Expression Unit,
Department of Genetics and Cell Biology, San Raffaele Scientific Institute, Milan, Italy, 3Institute of Stem Cell Research, National
Research Center for Environment and Health Institute of Stem Cell Research, 85764 Neuherberg-Munich, Germany, 4Stem Cells and
Neurogenesis Unit, Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy, 5Department of Pharmacology,
Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan and 6Department of Neurology, Institute of Experimental
Neurology (INSPE), San Raffaele Scientific Institute, 20132 Milan, Italy.
Gianvito Martino and Luca Muzio have contributed equally to this work.
Address correspondence to Luca Muzio, Neuroimmunology Unit, Institute of Experimental Neurology (INSPE), Division of Neuroscience San Raffaele
Scientific Institute, 20132 Milan, Italy. Email: [email protected].
The canonical Wnt/Wingless pathway is implicated in regulatingcell proliferation and cell differentiation of neural stem/progenitorcells. Depending on the context, b-Catenin, a key mediator of theWnt signaling pathway, may regulate either cell proliferation ordifferentiation. Here, we show that b-Catenin signaling regulates thedifferentiation of neural stem/progenitor cells in the presence of theb-Catenin interactor Homeodomain interacting protein kinase-1 gene(Hipk1). On one hand, Hipk1 is expressed at low levels during theentire embryonic forebrain development, allowing b-Catenin to fosterproliferation and to inhibit differentiation of neural stem/progenitorcells. On the other hand, Hipk1 expression dramatically increases inneural stem/progenitor cells, residing within the subventricular zone(SVZ), at the time when the canonical Wnt signaling induces celldifferentiation. Analysis of mouse brains electroporated with Hipk1,and the active form of b-Catenin reveals that coexpression of bothgenes induces proliferating neural stem/progenitor cells to escapethe cell cycle. Moreover, in SVZ derive neurospheres cultures,the overexpression of both genes increases the expression of thecell-cycle inhibitor P16Ink4. Therefore, our data confirm that theb-Catenin signaling plays a dual role in controlling cell proliferation/differentiation in the brain and indicate that Hipk1 is the crucialinteractor able to revert the outcome of b-Catenin signaling in neuralstem/progenitor cells of adult germinal niches.
Keywords: b-Catenin, Hipk1, Stem cells, SVZ, Wnt
Introduction
During early forebrain development, cortical radial glia (RG)
preferentially undergo symmetric cell divisions (Haydar et al.
2003) to tangentially expand the cortical field (Chenn and
Walsh 2003). At later time points, RG switch to asymmetric cell
divisions, diminish their number into germinal niches, and
migrate as differentiated cells toward the cortical plate (Haydar
et al. 2003). Nevertheless, a subset of RG persist in germinal
neurogenic areas of the mature brain as adult neural stem/
precursor cells (aNPCs) (Gotz and Huttner 2005) where they
continue to generate new neurons fated to become olfactory
bulbs neurons. Several molecular machineries, acting in pro-
liferating RG of developing forebrain, are also operating in adult
germinal neurogenic areas (Doetsch and Alvarez-Buylla 1996;
Brill et al. 2009). Among them, the canonical Wnt signaling
exerts a critical role (Dickinson et al. 1994; Lee et al. 2000;
Backman et al. 2005; Subramanian et al. 2009). Wnts are
secreted glycoproteins (Galceran et al. 2000; Lee et al. 2000),
bind to specific cell-surface receptor belonging to the Frizzled
protein family, and activate the protein Disheveled (Dsh). This
latter protein, in turn, induces the reduction of Glycogen
Synthase Kinase (GSK-3b) activity (Cook et al. 1996). This
event leads to the accumulation of b-Catenin in the cytoplasm
as well as its nuclear migration and target gene regulation
(Willert and Nusse 1998). On the other hand, in the absence
of the Wnt intracellular signaling cascade, GSK-3b induces
b-Catenin degradation throughout its phosphorylation.
There is a large body of evidence indicating several different
roles exerted by Wnt signaling in both developing and aNPCs.
Wnts transiently induce RG to proliferate during early neuro-
genesis (Logan and Nusse 2004), induce midend gestational RG
to differentiate by promoting the expression of proneural genes,
such as Ngn1 (Hirabayashi et al. 2004; Israsena et al. 2004;
Guillemot 2007), force aNPCs of the adult dentate gyrus to exit
the cell cycle by modulating the expression of NeuroD1
(Kuwabara et al. 2009), and increase proliferating rates of
Mash1+progenitor cells belonging to the adult subventricular
zone (SVZ) (Adachi et al. 2007). The effects exerted by Wnt
signaling on RG versus aNPCs are largely depending on the
context in which such pathway operates and, above all, on the
presence of specific interactor proteins. For instance, secreted
Frizzled--related proteins (Sfrps) and Dickkopf (Dkk1) are
extracellular Wnt’s inhibitors (Semenov et al. 2001; Mao et al.
2002; Bovolenta et al. 2008), while Groucho proteins repress the
activation of Wnt/b-Catenin downstream genes by acting in the
nucleus on TCF/Lef-dependent targets (Daniels and Weis 2005).
Here, we show that the opposite role exerted by Wnts on
proliferating RG versus aNPCs is largely depending on the
presence of the Homeobox-interacting protein kinase1
(Hipk1) gene. Hipk1 is scarcely expressed in RG forced by
Wnts to proliferate while it raises in SVZ aNPCs forced by Wnts
to differentiate. We also found that Hipk1 operates within SVZ
aNPCs owing to the fact that it induces the expression of
p16INK4a by physically interacting with b-Catenin.
Materials and Methods
Mouse Strains and Husbandryb-Cateninflox/flox transgenic mouse line was provided by Jackson
laboratories (Brault et al. 2001), b-CateninEx3 mouse line contains
2 LoxP sequences upstream and downstream the b-Catenin third
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doi:10.1093/cercor/bhr320
Advance Access publication November 17, 2011
Cerebral Cortex October 2012;22:2415– 2427
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exon (Harada et al. 1999), BAT-Gal transgenic mouse line was provided
by Dr Piccolo (Maretto et al. 2003), and GlastCreERT2 transgenic mouse
line was provided by Dr Gotz (Mori et al. 2006). GfapCre mouse line
(Zhuo et al. 2001) was obtained from Jackson laboratory, and
NestinCreERT2 mouse line was provided by Dr Kageyama (Imayoshi
et al. 2008). Transgenic mice were repeatedly backcrossed onto
C57BL/6 (Charles River) mice and were genotyped by polymerase
chain reaction (PCR) as previously described (Harada et al. 1999; Brault
et al. 2001; Zhuo et al. 2001; Maretto et al. 2003; Mori et al. 2006).
Postnatal mice were injected with bromodeoxyuridine (BrdU)
(100 mg/kg) for 9 h before the sacrifice. They were killed by anesthetic
overdose and transcardially perfused with 4% paraformaldehyde in
phosphate-buffered saline (PBS) pH 7.2. Brains were then fixed 4%
paraformaldehyde in PBS pH 7.2 for 12 h at +4 �C and cryoprotected for
24 h in 30% Sucrose (Sigma) in PBS at +4 �C. Brains were subsequently
sectioned at 10 lm. b-Galactosidase assay was performed on 200-lmthick slices adult BAT-Gal slices as previously described (Mallamaci
et al. 2000). Pregnant female was killed by cervical dislocation at
appropriate time points, and brains were collected in ice-cold PBS as
previously described (Muzio et al. 2002). Before the sacrifice, pregnant
dams received the injections of the S-phase tracer EdU (5-ethynyl-
2#-deoxyuridine; Invitrogen) at the concentration of 100 mg/kg. All
efforts were made to minimize animal suffering and to reduce the
number of mice used, in accordance with the European Communities
Council Directive of 24 November 1986 (86/609/EEC). All procedures
involving animals were performed according to the guidelines of the
Institutional Animal Care and Use Committee of the San Raffaele
Scientific Institute (IACUC number 429). GlastCreERT2 transgenic mice
were crossed with b-Cateninflox/flox and Rosa26YFP mice. Starting from
P30, mice received 2 Tam treatments (Tam, 2 mg/day, LASvendi) of
1 week each with a washing out period of 1 week in between. Long-
term effects of b-Catenin deprivation were analyzed 2 months after the
last injection. NestinCreERT2 mice were crossed with b-CateninEx3
mice, and starting from P30, double transgenic mice were injected with
Tam (5 mg/day, Sigma) for 5 consecutive days. A washing-out period of
time of 1 week was applied and then mice were injected with EdU for
9 h before the sacrifice.
Immunofluorescence and In Situ HybridizationImmunofluorescence was performed as previously described
(Centonze et al.). Sections were washed for 5 min 3 times in PBS and
then incubated in the blocking mix (PBS 13/FBS 10%/BSA 1 mg/mL/
Triton X 100 0.1%), for 1 h at room temperature. Antibodies were
diluted in blocking mix and incubated at + 4�C overnight as suggested
by manufacturer’s instructions. The following day, sections were
washed in PBS for 5 min, 3 times, and fluorescent secondary antibodies
diluted in blocking mix (concentration suggested by the manufac-
turer’s instructions) were applied. Slides were washed 3 times in PBS
for 5 min and incubated in Dapi solution for nuclei counterstaining.
When necessary, antigens were unmasked by boiling samples in 10 mM
sodium citrate (pH 6) for 5 min.
The following antibodies were used: a-mouse TuJ1 (Babco), rabbit
a-phospho H3 (Upstate), rabbit a-glial fibrillary acidic protein (GFAP;
Dako), mouse a-GFP (Abcam), chicken a-GFP (Chemicon), rabbit a-GFP(Molecular probes), rat a-Dcx (SantaCruz), rat a-BrdU (Abcam), mouse
a-BrdU (BD), EdU detection kit (Invitrogen), rabbit a-Tbr2 (Millipore),
mouse a-RC2 (Millipore), rabbit a-LacZ (Abcam), rabbit a-Olig2
(Millipore) and rabbit a-Id-1 (Bio Check Ink.), mouse a-Cre (Chemicon),
rabbit a-Iba-1 (Wako), mouse a-PSA-NCAM (Santa Cruz), and rabbit
a-NG2 (Chemicon). Appropriate fluorophore-conjugated secondary
antibodies (Alexa-fluor 488, 546, and 633, Molecular Probes) were used.
Nuclei were stained with 4#-6-Diamidino-2-phenylindole (DAPI; Roche).
Id-1 was detected by using the TSA System (Perkin Elmer). Light
(Olympus, BX51 with 34 and 320 objectives) and confocal (Leica, SP5
with340 objective)microscopywas performed to analyze tissue and cell
staining. Analysis was performed by using Leica LCS lite software and
Adobe Photoshop CS software.
In situ hybridization was performed as previously described (Muzio
et al. 2002; Centonze et al. 2009). Briefly, Ten-micrometer thick brain
sections were postfixed 15 min in 4% paraformaldehyde then washed
3 times in PBS. Slides were incubated in 0.5 mg/mL of Proteinase K in
100 mM Tris-HCl (pH 8) and 50 mM Ethylenediaminetetraacetic acid
(EDTA) for 10 min at 30 �C. This was followed by 15 min in 4%
paraformaldehyde. Slices were then washed 3 times in PBS then washed
in H2O. Sections were incubated in Triethanolamine 0.1 M (pH 8) for
5 min, then 400 lL of acetic anhydride was added 2 times for 5 min
each. Finally, sections were rinsed in H2O for 2 min and air-dried.
Hybridization was performed overnight at 60 �C with P33 riboprobes at
a concentration ranging from 106 to 107 counts per minute (cpm). The
following day, sections were rinsed in SSC 53 for 5 min then washed in
Formamide 50% SSC 2 3 for 30 min at 60 �C. Then slides were
incubated in Ribonuclease-A (Roche) 20 mg/mL in 0.5 M NaCl, 10 mM
Tris-HCl (pH 8), and 5 mM EDTA 30 min at 37 �C. Sections were
washed in Formamide 50% SSC 23 for 30 min at 60 �C then slides were
rinsed 2 times in SSC 23. Finally, slides were dried by using Ethanol
series. Lm1 (Amersham) emulsion was applied in dark room, according
to manufacturer instructions. After 1 week, sections were developed in
dark room, counterstained with Dapi, and mounted with DPX (BDH)
mounting solution. Mouse Hipk1 probe was generated by PCR on the
basis of the National Center for Biotechnology Information (NCBI)
reference (from nt735 to nt2160 of NCBI Reference Sequence
NM_010432.2), and LacZ riboprobes correspond to the entire
complementary deoxyribonucleic acid (cDNA) obtained from the
pcDNA 3.1-LacZ plasmid.
Tunel AssayTen-micrometer thick sections from E16.5 forebrains were postfixed
in 4% paraformaldehyde, then incubated in Proteinase K buffer for 5#at room temperature, and then exposed to 10 mg/mL Proteinase K
for 15# at room temperature. Reaction was stopped by washing samples
3 times in PBS 13. Slices were then incubated with terminal transferase
buffer for 15# before adding the following reagents: 10 lg/mL Biotin
16-dUTP, 1 mm CoCl2, and 10 U/mL of terminal transferase (Roche).
Reaction was incubated 1 h at 37 �C and stopped with H2O.
Endogenous peroxidase quenching was achieved by incubating slices
in 0.1% H2O2 for 15#. Slices were incubated in blocking buffer (10%
FBS, PBS 13) for 15# and then incubated in Streptavidin/Biotin
amplification kit (Vector) for 2 h. Reaction product was visualized
with 0.05% diaminobenzidine and 0.005% H2O2. Positive controls were
obtained by incubating slices in 3 U/mL DNase for 15# at room
temperature.
Neurospheres CulturesNeurospheres cultures were derived from the SVZ of P30 BAT-Gal
brains as previously described (Pluchino et al. 2008). Briefly, brains
were cut in coronal sections from anterior pole of the brain. The dorsal
SVZ were then microdissected from section approximately 2 mm far
from the anterior pole, and tissues were subsequently incubated 30 min
at 37 �C in Earle’s Balanced Salt solution supplemented with 1 mg/mL
of Papain (27 U/mg, Sigma), 0.2 mg/mL Cysteine (Sigma), and 0.2 mg/mL
EDTA (Sigma). Cells were then transferred in culture media Neurocult
NSCproliferationmedium(StemCell technology) supplementedwith 20
ng/mL Epidermal growth factor (Provitro), 10 ng/mL Basic fibroblast
growth factor (Provitro), and 2 lg/mLHeparin (Sigma). Cellswere plated
at the density of 8000/cm2 and maintained for more than 10 in vitro
passages. Differentiation of aNPCs (n = 3 independent cultures) was
obtained by using the Neurocult NSC differentiation medium (Stem Cell
technology). Briefly, single suspension of cells was plated on Matrigel
(BD) coated 10 mm dishes at the density of 30 000/cm2. Cells were
collected every 2 days starting from the day of plating for real-time (RT)
PCR assays. b-Cateninflox/flox neurospheres cultures were generated as
above and infected with lentiviruses (LVs) expressing the Cre
recombinase or the green fluorescent protein (GFP) (n = 3 independent
experiments) as previously described (Muzio et al. 2009). Vesicular
stomatitis virus-pseudotyped lentivirus generation: pRRLsin.PPT.hCMV
iCre-ires-GFP, Pre plasmid (Borello et al. 2006) was transfected with the
following plasmids: pMDLg/pRRE, pRSV-REV, and pMD2.VSVG into 293T
cells. Then, 14--16 h after DNA transfection, the mediumwas substituted
with fresh culture medium. Thirty-six hours later, cells supernatants
were collected and filtered through a 0.22-lm pore nitrocellulose filter.
To obtain high-titer vector stocks, the cells supernatants were
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concentrated by ultracentrifugation (55 kg, 140 min, 20 �C). Then,supernatantswerediscarded, and thepelletswere suspended in 100 lLof PBS 13, split in 20 lL aliquots, and stored at –80 �C. LV stocks
were titrated by infecting HeLa cells with serial dilution of the viral
stocks. Then, after 5 days, cells were harvested and titer was
calculated on the basis of GFP expression by flow cytometry.
b-Cateninflox/flox neurospheres were dissociated in single cells and
plated at the concentration of 50 000/well. Soon after plating cells
received iCre- or GFP-expressing lentiviruses, and cells were
collected at days 8 and 9 after lentivirus delivery for western blot
and RT analysis.
X-Gal staining on BAT-Gal derived aNPCs (n = 3 independent
neurospheres cultures) was performed as follow. Briefly, upon fixation
in 0.2% glutaraldehyde, 2% formaldehyde in 0.1 M phosphate buffer
pH 7.4, supplemented with 2 mM MgCl2, and 5 mM ethyleneglycol-bis
(2-aminoethylether)-N,N,N#,N#-tetra acetic acid for 5 min, cells were
washed 3 times in PBS 13 and incubated in the staining solution
(PBS 13, MgCl2 2 mM, Na-Deoxycholate 0.01%, Nonidet P.40 0.02%
supplemented with potassium ferricyanide 5 mM, potassium ferrocy-
anide 5 mM, and 1 mg/mL X-gal) overnight. Cells were then washed
3 times in PBS 13 and collected on cover slip for the analysis.
Neurospheres cultures were also nucleofected by using Amaxa
Mouse NSC Nucleofector kit. Briefly, 6 3 106 single cells derived from
established BAT-Gal neurospheres cultures (n = 3 independent
cultures) were combined with 1 lg of Nestin-D90-b-Catenin-GFPwith or without increasing amounts of CMV-fHIPK1 (1 or 2 lg) and
then transferred in a single cuvette for nucleofection (Program A-033)
accordingly with manufacturer’s instructions. After nucleofections,
cells were grown in standard culture medium for 24 h before total RNA
extraction.
Chromatin Immune PrecipitationNeurospheres cultures from the SVZ of P30 BAT-Gal mice2 Q3 were
obtained as above described and then used for chromatin immune
precipitation (ChiP) assay. Undifferentiated and differentiated neural
stem cells (2 3 106) were collected and fixed in 1% formaldehyde.
Extracts were incubated in cytoplasmic and nuclear lysis buffers.
Sonication was performed in ice (Branson sonicator 450, 8 3 10$), andextracts were subsequently precleaned in Protein-A sepharose (Sigma)
(20% of supernatants were collected as inputs). Each sample was then
probed overnight with following antibodies: rabbit a-b-Catenin (cell
signaling), rabbit a-GFP (molecular probes), or vehicle. Extracts were
then incubated with Protein-A sepharose (Sigma) for 3 h and washed
with low salt buffer, high salt buffer, LiCl buffer, and 10 mM Tris-HCl
pH8, 1 mM EDTA pH8.5 buffer. Elution of complexes was performed
by adding the elution buffer (1% Sodium dodecyl sulfate [SDS],
0.1%NaHCO3). Reverse cross-linking reaction was performed by
incubating extracts in RNase A (0.02 mg/mL), NaCl 0.3 M at 65 �Cfor 4 h. Samples were then extracted with Phenol/Chloroform
extraction buffer accordingly to standard laboratory procedures and
resuspended in 50 lL of H2O. One microliter from each sample was
used for the detection of p16INK4a promoter region by using PCR and
the following primers: p16INK4a ChiP F: GTTGCACTGGGGAGGAAG-
GAGAGA and p16INK4a ChiP R: CCTGCTACCCACGCTAACACC.
Amplicons were detected by standard agarose electrophoresis.
Real-Time PCRTotal RNA was extracted by using RNeasy Mini Kit (Qiagen) according
to manufacturer’s recommendations including DNase (Promega) di-
gestion. cDNA synthesis was performed by using ThermoScript RT-PCR
System (Invitrogen) and Random Hexamer (Invitrogen) according to
the manufacturer’s instructions in final a volume of 20 lL. The
LightCycler 480 System (Roche) and SYBR Green JumpStart Taq
ReadyMix for High Throughput Q-PCR (Sigma). Depending from
experiments, samples were normalized by using the following
housekeeping genes: Histone H3 and b-actin. b-actin F: GACTCC-
TATGTGGGTGACGAGG; b-actin R: CATGGCTGGGGTGTTGAAGGTC;
H3 F: GGTGAAGAAACCTCATCGTTACAGG CCTGGTAC; H3 R: CTGC-
AAAGCACCAATAGCTGCAC TCTGGAAGC. Gene specific primers:
p16INK4a F: CCTGGAACTTCGCGGCCAATCCC; p16INK4a R: GCTCC-
CTCCCTCTGCTCCCTCC; p19Arf F: CTGGGGGCGGCGCTTCTCACC;
p19Arf R: TCTAGCCTCAACAACATGTTCACG; Human p16INK4a
F: TTCCTGGACACGCTGGTGGTG; Human p16INK4a R: ATCGGG-
GATGTCTGAGGGACC; Hipk1 F: GCTAGCTGACTGGAGGAATGCC;
Hipk1 R: TGGTCTTGGACAGGAACTAGGG; Ki67 F: CCGAACAGACTT-
GCTCTGGCCTAC; Ki67 R: CTGGGCTGTGAGTGCCAAGAGAC; Lef1 F:
CCGTGGTGCAGCCCTCTCACGC; Lef1 R: ATTTCAGGAGCTGGAGGG-
TGTCTGG.
Luciferase AssayTOP-Flash luciferase reporter plasmid was a gift from R. T. Moon
(University of Washington, Seattle), while Fop-Flash plasmid was
purchased by Chemicon. Luciferase assays were performed by using
the dual-luciferase assay system (Promega, Madison, WI) accordingly
with manufacturer’ instructions. Hek293T cells (4.5 3 104) were
seeded in 12-well plates and transfected with TOP-Flash (0.5 lg), prlTK(0.1 lg), D90-bCatenin/GFP (0.5 lg), and increasing concentration of
fHipk1 (0.1, 0.5, 0.7, 1, and 2 lg). Control experiments were done by
transfecting cells with Fop-Flash plasmid (0.5 lg), prlTK (0.1 lg), D90-bCatenin/GFP (0.5 lg), and fHipk1 (2lg). Measurement of luciferase
activity was done by using the dual-luciferase system (Promega) on
a luminometer (GloMax 20/20 Luminometer; Promega). Relative
luciferase activity was reported as a ratio of firefly over Renilla
luciferase readouts.
Western Blot and Protein CoimmunoprecipitationWestern blot detection of antigens was performed as previously described
(Centonze et al. 2009). Briefly, 106 b-Cateninflox/flox cells were infected
with Cre-ires-GFP or GFP lentiviruses at Multiplicity Of Infection of 10.
Upon infection, cells were collected, respectively, at days 8 and 9. Total
cell extracts were obtained by incubating cells in lyses buffer (Sucrose 320
mM [Sigma], 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 1 mM pH
7.4 [Sigma], and MgCl2 1 mM (Sigma) supplemented with Protease
inhibitor cocktail, Sigma). Ten micrograms of each protein extract was run
on SDS--polyacrylamide gel electrophoresis (PAGE) electrophoresis and
subsequently blotted on nitrocellulose (Millipore). The following primary
antibodies were used: rabbit a-b-Catenin (Cell signaling), rabbit a-GFP(Chemicon), mouse a-Active Caspase3 (Cell Signaling), and mouse
a-b-Actin (Sigma). Secondary antibodies (Biorad) were incubated for 2 h
at RT, and signals were revealed by using Millipore ECL kit.
Coimmunoprecipitation was performed on HEK-293T cells trans-
fected with the following plasmids: TOP-Flash luciferase reporter, D90-b-Catenin/GFP, and pCMV-fHipk1. Briefly, 106 HEK-293T were trans-
fected with 2 lg of TOP-Flash plasmid, 4 lg of D90-b-Catenin/GFP with
or without 4 lg of pCMV-fHipk1. Forty-eight hours later, cells were
incubated in lyses buffer (Tris-HCl pH 7.5 20 mM, NaCl 100 mM, EDTA
5 mM, Triton 1%, Glycerol 10%, Na3VO4 1 mM, Na4P2O7 40 mM
supplemented with Protease inhibitor cocktail, Sigma). Then, 50 lL of
Protein-A (Sigma) sepharose beads were incubated with lysate for 1 h
at +4 �C. Lysates were then incubated with mouse a-Flag (2 lg/mL,
Sigma) for 2 h and then 100 lL of Protein A sepharose were added to
the lysates. Lysates were further incubated for 2 h with Protein A beads,
and subsequently, beads were separated by centrifugation. Protein A
beads were subsequently washed 6 times with lyses buffer, and finally,
elution was performed by adding SDS loading buffer directly to beads.
Co-IP samples, inputs, and washes were run on SDS--PAGE and then
blotted on nitrocellulose. Rabbit a-GFP (Abcam) primary antibody was
used to detect D90-b-Catenin-GFP protein. Western blot represents
1 over 3 independent experiments. Total extract from parallel
transfections was used for RT-PCR detection of human p16INK4a
expression as above described.
In Utero ElectroporationThe following plasmids were introduced into progenitor cells of E13.5
(CD1, Charles River) embryos: pCAG-GFP (0.1 lg/lL), Nestin-D90-b-Catenin-GFP (1 lg/lL), pCMV-fHipk1 (1 lg/lL). Plasmids were
mixed with 0.01% Fast green (Sigma), and 1--2 lL of each DNA mix was
injected into the ventricle through a fine glass capillary. Electrodes
(Tweezertrodes, BTX Harvard Apparatus) were placed flanking the
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ventricular region of each embryo covered by a drop of PBS and pulsed
4 times at 40 V for 50 ms separated by intervals of 950 ms with a square
wave electroporator (ECM 830, BTX Harvard Apparatus). Then, the
uterine horn was placed back into the abdominal cavity and filled with
warm PBS 13. Accordingly to the experimental plan, brains were
collected 3 days or 40 h after electroporations. BLOCK-it miR RNAi
selected oligos for Hipk1 interference (Mmi-51128; Mmi-51129; Mmi-
51130; Mmi-51131, efficient functioning was tested by transfecting
293T cells) were purchased by Invitrogen, cloned into pcDNA6.2-GW/
miR vector, and electroporated at the concentration of 250 ng/lL each
into E13.5 embryos. Controls were electroporated with pcDNA6.2-GW/
miR LacZ at the concentration of 1 lg/lL. Embryos were collected at
E16.5 and pulsed with EdU 2 h before the sacrifice. Electroporation was
also done at E16.5 (CD1, Charles River). Embryos received pCAG-GFP
(0.1 lg/lL) with or without the Nestin-D90-b-Catenin-GFP (1 lg/lL)plasmids. Electroporation was done as above described, and brains were
collected at P10. Six hours before the sacrifice mice were repeatedly
injected with EdU (100 mg/kg every 2 h). GFP-expressing brains were
fixed in 4% paraformaldehyde and processed for immune fluorescence.
Only embryos showing comparable electroporated patches were
included in our analysis.
StatisticsBar graphs represent the mean value ± standard error of the mean or
the mean value ± standard deviation. Data were analyzed as appropriate
by Student’s t-test or by one-way analysis of variance (ANOVA) using
Graph-Pad Prism version 4. A Significance was accepted when P < 0.05.
Results
b-Catenin Stimulates Cell Proliferation and Inhibits theExpression of Tbr2 in Progenitors of the DevelopingForebrain
We generated embryos expressing the constitutively active
form of b-Catenin by crossing the b-CateninEx3 transgenic
mouse line, which contains 2 loxP sequences upstream and
downstream the third exon of b-Catenin (Harada et al. 1999)
with GfapCre mice. In this new transgenic mouse line,
recombined b-Catenin becomes stabilized and constitutively
active in the nucleus in virtually all RG of E13.5 cerebral cortex
and the hippocampus (Fig. 1A) (Zhuo et al. 2001). GfapCre/
b-CateninEx3 brains collected at P21 and P30 displayed
a significant expansion of the tangential surface of the brain
(Fig. 1B and not shown) and a severe reduction of the cortical
thickness (Fig. 1C and not shown). We further characterized
double transgenic mice at E14.5, that is, 1 day after the activation
of the constitutively active form of b-Catenin; they showed
a significant increase of the cerebral cortex surface (Fig. 1D--F).
At this stage, we also analyzed the effects of b-Cateninstabilization on RG by staining sections for the RG marker RC2
(Hartfuss et al. 2001). In wild-type (WT) mice, RG showed the
classical palisade organization, while they were severely disor-
ganized in GfapCre/b-CateninEx3 brain, displaying distortion of
cytoplasmic bundles at the level of the ventricular zone (VZ)/
SVZ (Fig. 1G,I). We next measured the thickness of the neural
layer (NL) by staining E14.5 sections for TuJ1. NL layer
was significantly reduced in mice overexpressing stabilized
b-Catenin (Fig. 1K,L). In contrast, the thickness of the germinal
layer did not show any alteration (Fig. 1M). The distribution of
pH3+proliferating cells in the germinal layer of double trans-
genic mice was also severely altered. In WT embryos, pH3+
proliferating cells were placed within the ventricular lining
and the SVZ (Carney et al. 2007), while in GfapCre/b-CateninEx3
mice, they were scattered throughout the entire germinal layer
(Fig. 1N--P). These scattered proliferating cells may represent
abventricular mitoses of Tbr2+basal progenitor (BP) cells (Sessa
et al. 2008). However, in GfapCre/b-CateninEx3 mice, the total
number of Tbr2+cells was significantly reduced (Fig. 1Q--S),
confirming previous results that showed a delay of maturation
of BP cells in mice overexpressing the Wnt signaling (Wrobel
et al. 2007;Mutch et al.). To studyproliferatingRGandTbr2+cells
and the contribution of BP cells to the total proliferating pools of
the forebrain, embryos were pulsed with the s-phase tracer EdU.
Proliferating Tbr2+cells accounted for a small fraction of the
brain total proliferating pool (Fig. 1T). Nevertheless, the fraction
of Tbr2+cells, incorporating the EdU tracer, over the total
number Tbr2-expressing cells was similar in both genotypes
(Fig. 1T), suggesting that their limited number derives from
a derangement of their genesis rather than from alterations of
their progression along the cell cycle. Thus, the persistent
activation of the canonical Wnt signaling forces RG to maintain
their cell identity and to reenter the cell cycle, inducing
a tangential expansion of the cortical field. Similar results have
been reported in previously published works (Wrobel et al.
2007; Mutch et al.), however, very few data are available on the
role of b-Catenin at later time points of forebrain maturation.
In order to fill this gap, we overexpressed the stabilized form of
b-Catenin (Nes-D90-b-Catenin-GFP) (Chenn and Walsh 2002,
2003) at the end of neurogenesis by in utero electroporation.
E16.5 embryos received Nes-D90-b-Catenin-GFP along with
the GFP-expressing plasmid, while control embryos were
injected with the GFP-expressing plasmid alone. Brains were
then collected at P10, and GFP+cells were scored in the SVZ
(Supplementary Fig. 1A,B) and in the cortical plate (Supple-
mentary Fig. 1D,E). The active form of b-Catenin significantly
increased the number of GFP+cells that were retained in the
SVZ (Supplementary Fig. 1C). Since mice were pulsed with the
EdU at the day of the sacrifice, we also established the fraction
of proliferating cells in the SVZ. The number of GFP/EdU
double positive was significantly increased in mice over-
expressing Nes-D90-b-Catenin-GFP plasmids (Supplementary
Fig. 1F,G). Thus, unlike previous published results (Hirabayashi
et al. 2004), our data suggest that the activation of Wnt/
b-Catenin signaling induces RG to proliferate also at late
gestational time points similarly to earlier embryonic stages
(Chenn and Walsh 2002; Wrobel et al. 2007; Mutch et al.).
Confined Expression of the Canonical Wnt Signaling inthe Postnatal SVZ
Given the pivotal role of b-Catenin in regulating cell pro-
liferation during forebrain development (Chenn and Walsh
2002; Wrobel et al. 2007; Mutch et al.), we turned our attention
to the postnatal SVZ. We used the reporter BAT-Gal mice
(Maretto et al. 2003) to trace cells in which the canonical Wnt/
b-Catenin signaling is still active. These mice have multiple
TCF/Lef-binding sites coupled to a minimal promoter that
drives the expression of the LacZ reporter (Maretto et al.
2003). The analysis of P30 BAT-Gal brains showed the presence
of many X-Gal+cells that were placed in the dorsal but not in
the ventrolateral SVZ (Fig. 2A and inset) (Doetsch et al. 1999;
Temple and Alvarez-Buylla 1999). The presence of LacZ
transcripts in the dorsal SVZ was confirmed by in situ
hybridization (Fig. 2B). A subset of LacZ-expressing cells
incorporated the BrdU tracer, suggesting that they belong to
a proliferating SVZ aNPCs (Fig. 2C). In addition, many LacZ+
cells coexpressed Olig2, which labels parenchymal
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oligodendrocyte precursors and type-C aNPCs of the SVZ (Fig.
2D). Some LacZ+cells coexpressed Dcx, which is a marker for
type-A aNPCs (Lois et al. 1996; Hack et al. 2005) (Fig. 2E), while
a relatively high number of them coexpressed GFAP, a marker
of astrocytes in the CNS parenchyma and type-B aNPCs in the
SVZ (Fig. 2F) (Doetsch et al. 1999; Ahn and Joyner 2005;
Tavazoie et al. 2008). LacZ+cells of the most dorsal SVZ were
positive for Id-1 (Fig. 2G), a marker of SVZ-restricted slow
dividing aNPCs (Nam and Benezra 2009). The SVZ, however,
contains also microglia and oligodendrocytes (Menn et al.
2006). Thus, we tested whether some of LacZ+cells might
belong to these lineages by probing sections for Iba-1 or NG2.
However, neither LacZ/Iba-1 nor LacZ/NG2 double positive
cells were detected in the SVZ of BAT-Gal mice (Fig. 2H,I).
Taken together, these results indicate, for the first time, that
the canonical Wnt signaling is confined in adult brains to
a subset of aNPCs of the dorsal SVZ.
The Activation of b-Catenin Signaling in the Adult SVZinhibits Cell Proliferation and Activates the Expression ofp16INK4a
In order to define the contribution of the canonical Wnt
signaling to adult neurogenesis, we took advantage an inducible
Cre allele. NestinCreERT2 transgenic mouse line expresses the
Tamoxifen-dependent Cre recombinase in aNPCs of the SVZ
(Imayoshi et al. 2008). Starting from P30, NestinCreERT2/
b-CateninEx3 mice and their control litters—that is, mice
containing only the NestinCreERT2 allele—were treated with
Tamoxifen for 5 consecutive days. Brains were collected 1 week
after the last Tamoxifen injection. At the day of the sacrifice, we
labeled proliferating cells using EdU. Tamoxifen treatments did
not alter gross morphology but significantly inhibited aNPC
proliferation in the SVZ (Fig. 3A--C). On the other hand, the
number of PSA-NCAM+cells was significantly increased in the
SVZ of NestinCreERT2/b-CateninEx3 mice (Fig. 3D--F), thus
suggesting that the stabilization of b-Catenin in the adult SVZ
inhibits cell proliferation, favoring cell differentiation. Accord-
ingly, also the number of Id-1+slow dividing progenitor cells
was greatly diminished in double transgenic mice (Fig. 3G,H).
A large body of experimental evidence suggests that
b-Catenin signaling, in certain circumstances, might positively
or negatively regulate of the cell cycle--dependent kinase
inhibitor (Cdki) p16INK4a (Saegusa et al. 2006; Delmas et al.
2007). For example, in tumor cells, the b-Catenin--mediated
modulation of p16INK4a forces these cells to differentiate
(Saegusa et al. 2006). To determine whether or not b-Catenin
Figure 1. Wnt/b-Catenin signaling promotes the tangential expansion of the cerebral cortex. Panel (A) provides double staining for Cre and YFP in E13.5 GfapCre/Rosa26YFPforebrain coronal section. Double positive cells were detected in the VZ/SVZ, while YFPþ recombined cells are evident in the outer cortical wall (n 5 3). Comparison between P21cerebral hemispheres of GfapCre/b-CateninEx3 (right in panel B) and WT littermates (left in B, n 5 5 for each group). A significant enlargement of the forebrain is evident in doubletransgenic mice. Panel (C) shows hematoxylin staining of coronal sections from P21 GfapCre/b-CateninEx3 and WT brains (n 5 4 for each group). One coronal section every 300lm of E14.5 GfapCre/b-CateninEx3 and WT brains was used to measure the extension of cortical fields (D--F, n 5 3 for each group). Linear extensions of cortical fields,encompassing the corticostriatal and cortical hem notches, were measured at 3 different levels along the anterior--posterior axes and plotted in panel (F) (arbitrary units ± SD).RG cell cytoarchitecture derangement is evident in E14.5 GfapCre/b-CateninEx3 (H, J) brains when compared with WT (G, I). Arrows in the high magnification of the panel (J)indicate RC2þ cells (n 5 3 for each group). Coronal sections from E14.5 WT (K) and GfapCre/b-CateninEx3 (L) mice were stained for TuJ1 to measure the thickness of the neurallayer (NL) and the germinal niche (VZ/SVZ). One section every 300 lm along the anterior--posterior axes was used for cell counting (n 5 3 for each group). Mean values (arbitraryunits ± standard deviation [SD]) are plotted in panel (M). E14.5 WT (N) and GfapCre/b-CateninEx3 (O) sections were probed for pH3 detection. PH3þ cells were counted in areasof 0.07 mm2 belonging to the VZ/SVZ (see boxed areas). The mean numbers (±SD) of apical and abventricular (arrows in N and O) mitoses are plotted in panel (P). E14.5 WT (Q)and GfapCre/b-CateninEx3 (R) brains received the EdU tracer for 1 h before the sacrifice and were subsequently labeled for Tbr2 and EdU. Cell counts were done withina rectangular areas of 0.07 mm2 belonging to the VZ/SVZ (see boxed area). The mean numbers (±SD) of EdUþ and Tbr2þ cells per section (n 5 3 for each group) are plotted inpanel (S). Fractions of EdU/Tbr2 double positive cells above the total number of EdU or the total number of Tbr2-expressing cells (mean ± SD) are plotted in panel (T). Scale bar100 lm. *P \ 0.05; ***P \ 0.001, t-test.
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can modulate p16INK4a in aNPCs of the adult SVZ, we
microdissected SVZs from NestinCreERT2/b-CateninEx3 mice
administered with Tamoxifen as above (Fig. 3I). The stabilization
of b-Catenin induced the expression of the target gene Lef1
(Fig. 3J) (Hovanes et al. 2001; Filali et al. 2002) as well as of
p16INK4a (Fig. 3K), suggesting that the activation of ectopic
b-Catenin is operating, and such manipulation induced the
expression of p16INK4a, that in turn orchestrate the opposite
role of b-Catenin functioning in adult germinal niche. In order to
confirm these results, we raised neurospheres cultures (Rey-
nolds and Weiss 1992) from the dorsal SVZ of P30 BAT-Gal mice
(Maretto et al. 2003) to establish if the canonical Wnt signaling is
still working in vitro. X-Gal staining of neurospheres revealed
the presence of LacZ+cells in approximately 40% of them
(Supplementary Fig. 2A). However, upon serial in vitro passages
(ivp), this number greatly diminished, possibly because the
prolonged exposure to high levels of growth factors may
hamper their capacity to express Wnt genes (not shown). Early
generated neurospheres cultures were used for chromatin
immunoprecipitation (ChIP) of b-Catenin targets. By using
a specific antibody for b-Catenin, we demonstrated that the
promoter region of p16INK4a is a direct target of b-Catenin(Supplementary Fig. 2B). In addition, we also demonstrated that
p16INK4a messenger RNA (mRNA) levels were significantly
increased when aNPCs were induced to differentiate by absence
of growth factors (Supplementary Fig. 2C). We also raised
neurospheres cultures form P30 b-Cateninflox/flox SVZs (a
b-Catenin conditional knockout allele) (Brault et al. 2001).
These cultures were infected with lentivirus expressing either
the Cre-IRES-GFP cassette (Borello et al. 2006) or the GFP gene.
Starting from day 8, after lentivirus delivery, the Cre-mediated
recombination of the b-Cateninflox/flox locus abolished the
expression of b-Catenin (Supplementary Fig. 2D). Because
b-Catenin is a component of adherens junctions (Aberle et al.
1996), its depletion deranged cell morphology of targeted aNPCs
and affected their long-term survival. Indeed, 9 days after lentivirus
delivery, we detected a slight activation of Caspase-3 (Supple-
mentary Fig. 2D), and starting from 10 days after viruses, delivery
targeted cells did not adhere to each other (not shown)
(Holowacz et al.). At day 8, a time when infected cells have
already lost b-Catenin expression but the activation of Caspase-3
isundetectable (SupplementaryFig. 2D),weobserveda significant
reduction of p16INK4a expression (Supplementary Fig. 2E).
Because the INK4a locus contains 2 distinct gene variants, the
p16INK4a and the p19Arf, in addition to the short p16INK4a
variant,wemeasured also the expressionof the longp19ArfmRNA
levels that did not show any alteration (Supplementary Fig. 2E).
Thus, we demonstrated that b-Catenin can directly interact with
the promoter region of p16INK4a in vivo and in aNPCs derived
from the adult SVZ.
Figure 2. The canonical Wnt signaling is still active in progenitor cells of the adult dorsal SVZ. X-Gal staining of a 200-lm thick P30 BAT-Gal coronal section (panel A, n 5 4)shows the presence of many cells expressing LacZ at levels in the dorsal, but not in the ventral, SVZ (inset represents lateral SVZ). Radioactive in situ hybridization of P30 BAT-Galcoronal sections confirmed the presence of LacZ-expressing cells (panel B, n 5 3). P30 BAT-Gal received BrdU injections for 9 h at the day of the sacrifice. LacZ/BrdU doublepositive cells are indicated by arrows in panel C (n 5 3). LacZþ cells coexpressing Olig2, are shown in panel (D), while LacZþ cells coexpressing the type-A marker DCX areshown in panel E (n 5 3 for each group). Several LacZþ cells at the ventricular lining are positive for the type-B marker GFAP (arrowheads in panel F, n 5 3) and Id-1(arrowheads in G indicate double positive cells in dorsal SVZ). We did not find any LacZþ cells expressing either the microglial/macrophages marker Iba-1 (panel H, n 5 3) or theoligodendrocytes marker NG2 (panel I, n 5 3). Scale bar 50 lm.
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Dynamic Expression of Hipk1 during ForebrainNeurogenesis
The context-dependent role of b-Catenin pathway in pro-
moting cell self-renewal or neuronal differentiation may rely on
the presence of specific molecular interactors (Cavallo et al.
1998). Some of these molecules dictate the outcomes, in term
of cell proliferation and cell differentiation, for the canonical
Wnt/b-Catenin signaling (Sierra et al. 2006; Li and Wang 2008).
Among them, Hipk genes have been recently indicated
as modulators of b-Catenin signaling in the neural plate cells
of Xenopus laevis embryos (Louie et al. 2009) and in stem cells
of the skin (Wei et al. 2007). Hipk1 is expressed in germinal
niches of E9.5, E14.5, E18.5, and P30 brains, as we showed by in
situ hybridization assay (Fig. 4A--E) (Isono et al. 2006).
However, starting from E14.5, also cells of the presumptive
hippocampal plate and cells of the choroid plexus exhibited
detectable levels of Hipk1 (Fig. 4B,C). Thus, the pattern of
expression of Hipk1 overlaps the pattern of expression of the
Wnt signaling (Maretto et al. 2003; Muzio et al. 2005). Hipk1
expression levels are very low during forebrain development
but dramatically raise in germinal niches of the adult brain (Fig.
4D). Indeed, by coupling in situ hybridization for Hipk1 with
immunofluorescence-mediated detection of GFAP, we were
able to detect, in the adult SVZ, a large number of
periventricular GFAP+cells coexpressing at high levels Hipk1
mRNA (Fig. 4E). The expression of Hipk1 levels was also
evaluated by RT-PCR on cortical microdissections of develop-
ing forebrains (E9.5, E11.5, E14.5, E18.5, and P4) and postnatal
SVZs (P10 and P30). Hipk1 mRNA levels were normalized on
the expression of the housekeeping gene b-Actin, and data
from each time point were compared with the normalized
expression of Hipk1 detected at E9.5. During forebrain
development, Hipk1 expression levels were pretty low, if
compared with levels measured in P30 SVZs (Fig. 4F). We also
established that Hipk1 was also expressed in neurospheres
cultures (Fig. 5F), and its expression increased during their
differentiation (Fig. 4G).
CMV-fHipk1 and Nes-D90-b-Catenin-GFPCoelectroporation Reduces RG Cells Proliferation
This temporal gradient of Hipk1 prompted us to investi-
gate whether Hipk1 can regulate b-Catenin functioning in
Figure 3. The stabilization of b-Catenin in adult SVZ progenitors affects cell proliferation and induces p16INK4a expression. P30 NestinCreERT2 (panels A, D, and G) andNestinCreERT2/b�CateninEx3 (panels B, E, H, and I) mice were injected with Tam for 5 days and sacrificed 1 week after the last Tam injection (n 5 3 for each group). Micereceived multiple EdU injections at the day of sacrifice (9 h of cumulative labeling). Cell proliferation was evaluated in the SVZ by staining sections for GFAP and EdU (panels A andD). Cell counts were done in one sections every 300 lm along the anterior--posterior axis, and mean values (±standard deviation [SD]) are plotted in panel (C). Parallel sectionswere probed for PSA-NCAM (panels D and E, arrows indicate the thickness of neuroblast streams), cell counts were done as above, and percentages (±SD) of PSA-CAMþ cellsare plotted in panel (F). Panels (G and H) show adjacent sections stained for Id-1 detection. Coronal sections from P30 NestinCreERT2 and NestinCreERT2/b�CateninEx3 brainstreated with Tam as above were taken in a region spanning from 2 mm from the anterior pole of the brain to 3 mm posterior to the previous cut. SVZs were furthermicrodissected and used for RT-PCR analysis (arrow in panel (I) shows the region of interest in a representative sample, n 5 3 for each group). Expression levels (mean ± SD) ofLef1 and p16INK4a are shown in panels (J and K), respectively. Scale bar 50 and 200 lm for the panel (G). ***P \ 0.001; **P \ 0.01. Scale bar 50 lm.
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proliferating SVZ aNPCs. To address this issue, we overex-
pressedHipk1 along with the activated form of b-Catenin in the
developing forebrain, at the time when the Hipk1 physiological
expression levels are low. In utero electroporation of E13.5
forebrains was done by injecting mice with Nes-D90-b-Catenin/GFP, flag-Hipk1 (fHipk1), and a mix of both plasmids. Following
electroporation, embryos were collected at E16.5, and only
forebrains displaying electroporated cells in the lateral cerebral
cortex were included in our analysis. Sections were stained for
GFP and RC2. The number of double positive cells was counted
in the VZs of embryos receiving Nes-D90-b-Catenin-GFP(Fig. 5A), fHipk1 (Fig. 5B), or both plasmids (Fig. 5C). The
overexpression of both Nes-D90-b-Catenin-GFP and fHipk1
significantly reduced the number of GFP cells displaying RG
features (Fig. 5D). Parallel sections were stained for GFP and
TuJ1 to count the number of targeted cells persisting in the
VZ/SVZ and not expressing the neuronal marker TuJ1
(Supplementary Fig. 3A--C). fHipk1, per se, slightly dimin-
ished this number, but only embryos receiving Nes-D90-b-Catenin-GFP and fHipk1 plasmids showed a significant
reduction of these cells (Supplementary Fig. 3D). We next
assessed the role of Hipk1 and b-Catenin on proliferating RG
of the brain. In utero electroporation was done as above, but
brains were collected at E15.5. Double staining for TuJ1 and
GFP allowed the estimation of GFP+cells exiting the cell
cycle—that is, the Q fraction (Takahashi et al. 1996).
Accordingly to previously published report, the fraction of
GFP/TuJ1+
cells was 0.54 ± 0.07 in brains receiving the
pCAG-GFP plasmids (Fig. 5E,I) (Takahashi et al. 1996). This
number was reduced in brains receiving the Nes-D90-b-Catenin-GFP plasmids (Fig. 5F,I), and 1.3-fold increased in
mice receiving both fHipk1 and Nes-D90-b-Catenin-GFPplasmids (Fig. 5H,I). Electroporated embryos were also pulsed
with EdU for 2 h to estimate the proliferating fractions in
each experimental group. The percentage of GFP+
cells
incorporating the EdU tracer was 25.3 ± 0.8% in embryos
receiving pCAG-GFP (Fig. 5J,N). This percentage increased in
mice electroporated with Nes-D90-b-Catenin-GFP (Fig. 5K,N)
butwas significantly reduced in embryos receiving bothplasmids
(Fig. 5M,N). Brains containing large numbers of GFP+cells
(Supplementary Fig. 4A--C) were scored for the presence of
Tunel+cells. However, we did not find any discernable differ-
ences in Tunel staining between each group (Supplementary
Fig. 4A--C). Although the Hipk1 physiological expression levels
are generally low during forebrain development, we further
investigated the effects of Hipk1 inhibition on neurogenesis by
performing its knockdown. E13.5 embryos were electroporated
with short interfering RNAs plasmids targeting Hipk1. The
efficiency of these Hipk1-RNAi vectors was examined by
transfecting 293T cells (Supplementary Fig. 5). E16.5 brains
receiving Hipk1-RNAi, or control plasmids, were injected for 2 h
with the EdU tracer and then sacrificed. The effects of Hipk1-
RNAi on proliferating cells were investigated by probing sections
for GFP and EdU. The fraction of GFP+cells incorporating the
S-phase tracer was 10.5 ± 2 in mice receiving unrelated RNAi
vectors but was doubled in mice receiving Hipk1-RNAi plasmids
(Fig. 5O--Q). Thus, the knockin and the knockdown analyses
suggest that Hipk1 has a repressor activity on proliferating cells
of the forebrain.
Hipk1 Can Directly Interact with b-Catenin
To examine whether Hipk1 might directly interact with
b-Catenin, we performed a coimmunoprecipitation assay
(co-IP). Hek-293T cells were cotransfected with Nes-D90-b-Catenin-GFP, the Top-flash reporter plasmid (Veeman et al.
2003), with or without fHipk1. Two days after transfection,
cells were collected and used for fHipk1 immunoprecipitation
using a specific a-Flag antibody. Immune-precipitated com-
plexes were run on standard SDS--PAGE electroporation and
then probed for D90-b-Catenin-GFP detection. The immune
Figure 4. Hipk1 is confined in proliferating niches of both developing and postnatal brains. Panels (A--D) show radioactive in situ hybridization for Hipk1 at E9.5, E14.5, E18.5, andP30 (n 5 3 for each group). Hipk1 expressing cells are detectable into the VZ of E9.5 forebrain as shown in panel (A). Cells of the VZ/SVZ of both E14.5 and E18.5 brains expressHipk1 as shown in panels (B and C). However, at both time points, neurons of the medial cortical wall and cells of the choroid plexus express Hipk1 at detectable levels(arrowheads in B and C). Cells confined in the P30 SVZ express Hipk1 at robust levels, as shown in panel D (high magnification of the dorsal SVZ is shown in the inset).Immunomediated detection of GFAP coupled with Dig-in situ hybridization for Hipk1 is provided in panel (E) and arrowheads indicate double positive cells in the SVZ. Total mRNAswere extracted from the E9.5 forebrain, E11.5, E14.5, E18.5, and P4 cortices; P10 and P30 S6VZs; and neurospheres cultures. Histogram of panel (F) shows relative expressionlevels of Hipk1. Each data are normalized on the total amount of the housekeeping gene b-Actin, and fold changes (±standard deviation) are calculated respect the meanexpression levels of Hipk1 that is detectable at E9.5. The expression of Hipk1 mRNA was also evaluated in resting and differentiating neurospheres cultures (n 5 3), as shown inhistogram of panel (I). ***P \ 0.001, t-test. Scale bar 100 lm.
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precipitation of D90-b-Catenin-GFP was obtained only when
fHipk1 plasmids were included in the plasmids mixes (Fig. 6A,
right panel), while no detectable signals were found when
we omitted fHipk1 (Fig. 6A, left panel). We next performed
a standard Luciferase assay on Hek-293T cells that were
transfected with Nes-D90-b-Catenin-GFP, the Top-flash re-
porter, with or without fHipk1. The cotransfection of Nes-
D90-b-Catenin-GFP and the Top-flash reporter produced high
luciferase levels (Fig. 6B), while the inclusion of fHipk1
plasmids induced a significant reduction of the luciferase
activity (Fig. 6B). Control transfections were done by using the
Top-flash plasmids (Fig. 6B).
Beside the effects as corepressors (Choi et al. 1999), the
Hipk family have been identified among genes that can also
positively regulate the gene transcription. These 2 opposite
effects are largely dependent on promoters of target genes and
on the context in which Hipk genes operate (Rinaldo et al.
2007). We next assessed whether Hipk1 along with b-Catenincan modulate the expression of p16INK4a in SVZ-derived
neurosphere cultures. We nucleofected primary neurosphere
cultures with Nes-D90-b-Catenin-GFP plasmids with or with-
out fHipk1 plasmids. Transfected neural stem cells were then
collected after 48 h to measure the expression levels of
p16INK4a by RT-PCR. Standard RT-PCR analysis revealed that
the presence of both Nes-D90-b-Catenin-GFP and fHipk1
increased the expression levels of p16INK4a (Fig. 6C, upper
panel). Accordingly, RT-PCR analysis confirmed that p16INK4a
was upregulation of in cells receiving Nes-D90-b-Catenin-GFPand fHipk1 plasmids (Fig. 6C, lower panel). However, in these
experimental conditions, the simple overexpression ofNes-D90-
Figure 5. fHipk1 and Nes-D90-bCatenin-GFP mediated the premature exit of exit from cell cycle of cortical progenitor cells. E13.5 forebrains were electroporated with Nes-D90-bCatenin-GFP (A, n 5 5), fHipk1 (B, n 5 4), or with both plasmids (C, n 5 6), then brains were collected at E16.5 and labeled for RC2 and GFP. By confocal sectioning, wecounted double positive cells in the VZ of each experimental group, and mean numbers (±standard deviation [SD]) of RC2/GFP cells per section are plotted in panel (D). E13.5embryos were further electroporated with pCAG-GFP (E, J, n 5 3), Nes-D90-bCatenin-GFP (F, K, n 5 3), fHipk1 (G, L, n 5 3) or with Nes-D90-bCatenin-GFP and fHipk1plasmids (H, M, n 5 3), brains were then collected at E15.5, and fractions of differentiating cells in each electroporated area were calculated by staining sections for TuJ1 andGFP (E--H) to calculate. Mean values (±SD) are plotted in panel (I). Electroporated embryos received the EdU tracer for 1 h at the day of the sacrifice. Sections were doublelabeled for EdU and GFP (J--M). Percentages (±SD) of double positive cells were calculated in each electroporated area and plotted in panel (N). E13.5 embryos wereelectroporated with control RNAi plasmids (panel O, n 5 3) or Hipk1-RNAi plasmids (panel P, n 5 3) along with pCAG-GFP plasmids. Mice were pulsed with EdU for 2 h at E16.5and then sacrificed. Percentages (±SD) of GFP/EdU double positive cells above the total number of SVZ-restricted GFPþ cells are shown in panel (Q). *P \ 0.05, **P \ 0.01,and ***P \ 0.001. For panels (D, I, and N), we used ANOVA pretest, followed by t-test. Scale bar 50 lm.
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b-Catenin-GFP failed to increase p16INK4a levels, possible
because neurospheres were maintained in the presence of high
concentration of FGF2 that, per se, is capable to inhibit
b-Catenin signaling (Israsena et al. 2004). However, the addition
of fHipk1 forced the upregulation of p16INK4a, suggesting that
Hipk1 is a limiting factor for p16INK4a expression in aNPCs.
SVZ Restricted b-Catenin Loss of Function Affects theExpression of Hipk1
A large number of genes implicated in the control of Wnt/
b-Catenin signaling are often regulated by b-Catenin through
positive or negative feedback loops (Kazanskaya et al. 2004;
Logan and Nusse 2004; Chamorro et al. 2005; Khan et al. 2007),
we asked whether Hipk1 expression might be modulated by
the b-Catenin. We firstly induced the b-Catenin depletion by
crossing b-Cateninflox/flox mice (Brault et al. 2001) with
GlastCreERT2 transgenic mice (Mori et al. 2006). Cells express-
ing Glast, Gfap, and Nestin are presumably high hierarchical
aNPCs of the adult SVZ that are capable to self-renew in vitro
and to generate multipotent neurosphere cultures (Ninkovic
et al. 2007; Beckervordersandforth et al.). Adult GlastCreERT2/
b-Cateninflox/flox and their controls—that is, GlastCreERT2
/
b-Cateninflox/+—were fed with Tamoxifen and sacrificed
4 weeks after Tamoxifen treatment. Efficiency of recombina-
tion was tested by including the Rosa26YFP allele. Virtually, all
cell or the periventricular lining expressed robust yellow
fluorescent protein (YFP) levels (not shown). Robust Hipk1
expression levels were detected in controls (Supplementary
Fig. 6A) but significantly reduced in conditional KO mice
(Supplementary Fig. 6B). Accordingly, neurospheres deprived
of b-Catenin significantly lowered Hipk1 expression levels
(Supplementary Fig. 5C). We also examined Hipk1 in brains
overexpressing the stabilized form of b-Catenin. Nestin-
CreERT2/b-CateninEx3 mice administered with Tamoxifen for
5 consecutive days were collected 1 week after the last Tam
injection and probed forHipk1 detection. In parallel, the SVZ of
some of themwasmicrodissected for RT-PCR analysis. However,
the stabilization of b-Catenin produced a slight, but not
significant, increase of Hipk1 levels as measured by RT-PCR
(Supplementary Fig. 6D--F). Thus, the inactivation of the
b-Catenin signaling inhibited Hipk1 expression, while the
upregulation of this molecule did not.
Discussion
In the forebrain, early ectopic activation of the canonical Wnt
signaling fosters RG to proliferate thus promoting an excessive
tangential expansion of the cortical field (Chenn and Walsh
2002, 2003). By contrary, later time point activation of Wnt
signaling—via stabilization of the active form of b-Catenin—fosters RG to differentiate (Hirabayashi et al. 2004). This latter
effect is due to the positive regulation of the proneural gene
Ngn1 expression (Hirabayashi et al. 2004). By studying cell
proliferation of GfapCre/b-CateninEx3 brains, we found that the
persistent activation of Wnt signaling promotes RG proliferation
also at later time points of forebrain development. RG pro-
liferation increased in E13.5 embryos once the expression of
the activated form of b-Catenin was transgenically induced.
Consistently with this finding, the electroporation of Nes-D90-b-Catenin-GFP plasmid at E16.5 induces targeted RG to pro-
liferate and to inhibit the expression of TuJ1 and Tbr2.
Figure 6. Hipk1 can directly interact with b-Catenin. Co-IP experiments were performed on 1 3 106 Hek293T cells transfected with TOP-Flash (2 lg), Nes-D90-bCatenin-GFP (4lg) plasmids, with or without fHipk1 (4 lg) plasmids (n 5 3 independent experiments). Cells were collected after 48 h, and total extracts were immunoprecipitated with a a-Flagantibody. D90-bCatenin/GFP chimeric protein was detected by using specific a a-GFP antibody, and data form a representative experiment are plotted in panel (A). Hek293T (5 3105) cells were transfected with TOP-Flash (0.5 lg), prlTK (0.1 lg), Nes-D90-bCatenin-GFP (0.5 lg), and increasing concentrations of fHipk1 (0.1, 0.5, 0.7, 1, and 2 lg). Controlexperiments were done by transfecting cells with Fop-Flash (0.5 lg), prlTK (0.1 lg), Nes-D90-bCatenin-GFP (0.5 lg), and fHipk1 (2 lg) plasmids. Cell lysates were prepared 24h after transfection and were analyzed for luciferase activity. The mean luciferase activities (±standard deviation [SD]) of 3 independent experiments, each performed intriplicate, are plotted in panel (B). Neural precursor cells (3 3 105) derived from dissociated primary neurospheres cultures (n 5 3) were nucleofected with Nes-D90-bCatenin-GFP (1 lg) and fHipk1 (1 and 2 lg). Cells were lysated after 24 h, and RNA extracts were used to detected p16INK4a by standard RT-PCR (upper panel C). Upper lanes representp16INK4a amplicons (166 bp), while the housekeeping gene H3(190 bp) is shown in lower lanes. Total mRNAs were also used to detect p16INK4a by RT-PCR analysis. Each datawere normalized on the expression of the housekeeping gene H3, and percentages (±SD) are plotted on histogram in C. *P \ 0.05, **P \ 0.01, ***P \ 0.001, t-test.
Hipk1 and b-Catenin Regulate Proliferation of Cortical Precursor Cells d Marinaro et al.2424
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Interestingly, we also observed a severe alteration of RG
morphology in our transgenicmice consistingwith the presence
of a significant high number of abventricular mitoses in the VZ/
SVZ. At this stage, we can only speculate that the active form of
b-Catenin might have directly influenced the positioning of
pH3+mitoses by regulating nuclear interkinesis.
It is well known that Wnts are expressed by aNPCs residing
within neurogenic niches (Lie et al. 2005; Adachi et al. 2007).
However, their role is still partially unclear. As a matter of fact,
the presence of a stabilized form of b-Catenin increases
proliferation of Mash1 expressing aNPCs placed in both the
ventral and the dorsal SVZ (Adachi et al. 2007). On the other
hand, Wnt signaling induces differentiation of aNPCs residing
within the dentate gyrus of the hippocampus (Lie et al. 2005)
by activating the expression of NeuroD1 (Kuwabara et al.
2009). We found that Wnt signaling is activated only in the
aNPCs residing within the dorsal SVZ by using BAT-Gal
reporter mice. As such, BAT-Gal--expressing cells of the dorsal
SVZ incorporate the S-phase tracer BrdU and a large part of
them express molecular markers of SVZ-restricted aNPCs.
Similarly to the results shown by Li and Wang (2008), we found
that Wnt signaling significantly reduced the number of
proliferating aNPCs in the SVZ: a 2-fold increase of type-A
neuroblasts was measured in NestinCreERT2/b-CateninEx3 mice.
Finally, we observed that the active form of b-Catenin induces
the expression of the p16INK4a in aNPCs of the SVZ (Lukas
et al. 1995). Reduced expression level of p16INK4a was
observed upon inactivation of the expression of b-Catenin in
SVZ-derived neurosphere cultures derived from b-Cateninflox/
flox mice. This was attributed to the capability of b-Catenin to
engage the promoter region of the mouse p16INK4a (Saegusa
et al. 2006). While technical hurdles (i.e., retroviral vector-
based cell tracing vs. inducible transgenes) might explain the
different results we obtained from Adachi et al. (2007), it is
reasonable to think that the activation of the Wnt signaling in
different SVZ cell populations might lead to different pheno-
types. Future studies must address the relative contribution of
Wnts in each specific cell population belonging to the SVZ.
The opposite effects produced by b-Catenin signaling in
aNPCs may be due to signals capable of positively or negatively
regulate b-Catenin target genes. Among such proteins those
coded by Hipk genes—Hipk1, Hipk2, Hipk3, and Hipk4—
have been shown to be reliable modulators of the b-Cateninsignaling (Rinaldo et al. 2007). Hipk1 can regulate the
transcription of Wnt/b-Catenin targets during X. levis gastru-
lation (Louie et al. 2009). Hipk2 interacting with b-Cateninand repressing b-Catenin/Lef1 target genes impairs self-
renewal of skin stem cells (Wei et al. 2007): This suppressive
effect does not require the phosphorylation of b-Catenincritical residues (Wei et al. 2007). The constitutive inactivation
of Hipk1 and Hipk2 induces early exencephaly, which is
characterized by an exaggerate overgrowth of forebrain/
midbrain neural tissue (Isono et al. 2006). While the expression
pattern of Hipk1 has been deeply investigated in early embryos
(Isono et al. 2006), still few data are available at later time
points during forebrain maturation. We found that Hipk1 is
expressed within developing and adult neurogenic germinal
niches: Its expression levels are generally low during forebrain
development but significantly increased in aNPCs of the adult
SVZ, when Wnts induced aNPCs differentiation. We also found
that overexpression of Hipk1 during embryonic develop-
ment—when Hipk1 expression levels are low—slightly aff-
ected RG proliferation. However, the coelectroporation of
Hipk1 with the constitutive active form of b-Cateninsignificantly reduced the number of proliferating cells and
a significant number of RG receiving both constructs differen-
tiated into TuJ1+cells. While excluding that our findings could
be due to the exaggerated rate of cells death induced by
overexpression of Hipk1 (D’Orazi et al. 2002; Doxakis et al.
2004), we finally demonstrated that Hipk1 was able to
physically interact with b-Catenin and that Hipk1 is able to
regulate the b-Catenin--mediated expression of the reporter
plasmid TOP-Flash.
Since we demonstrated that b-Catenin is capable to engage
the promoter region of the mouse p16INK4a and that Hipk
genes are able to physically interact with b-Catenin, we finally
explore the possibility that Hipk1-b-Catenin interaction
regulates p16INK4a expression. In neurosphere cultures, the
overexpression of b-Catenin slightly modulated the expression
of p16INK4a. However, increased expression of p16INK4a was
obtained in the presence of both Hipk1 and the active form of
b-Catenin. Our data, showing that Hipk1might cooperate with
the canonical Wnt signaling in the adult SVZ, parallel the above-
mentioned previous results obtained showing that Hipk2 and
b-Catenin act jointly as cell cycle repressors in skin stem cells
(Wei et al. 2007). However, we cannot exclude that Hipk genes
can interact with other molecules to regulate the cell cycle as
Hipk2 can interact with Axin to stimulate the expression of
P53 (Rui et al. 2004).
In conclusion, our data further reiterate the strong in-
volvement of the Wnt signaling pathway in the regulation and
orchestration of the cell cycle in embryonic and adult neural
progenitors.
Supplementary Material
Supplementary material can be found at: http://www.cercor.
oxfordjournals.org/
Funding
BMW Italy group and Italian Association for Multiple Sclerosis
(Fism/Aism) grant number ‘‘Stem cells 2008.’’
Notes
We deeply thank Dr Furlan and Dr P. Brown for their helpful
discussions and support. We thank Dr Hiroyoshi Ariga for providing
the Hipk1 expressing construct, Dr Stefano Piccolo for providing BAT-
Gal transgenic mice, Dr R. Kageyama for providing NestinCreERT2
transgenic mice, Dr C. Walsh for providing the Nestin-D90-b-Catenin-GFP construct, and Dr U. Borello for the Cre-ires-GFP construct. This
work is dedicated to the memory of Dr G. Corte. Conflict of Interest :
None declared.
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