ERRATUM
Development 138, 180 (2011) doi:10.1242/dev.061754
Efficient regeneration by activation of neurogenesis in homeostatically quiescent regions
of the adult vertebrate brain
Daniel A. Berg, Matthew Kirkham, Anna Beljajeva, Dunja Knapp, Bianca Habermann, Jesper Ryge,
Elly M. Tanaka and András Simon
There was an error in the ePress version of Development 137, 4127-4134 published on 10 November 2010.
On p. 4133, the title of Fig. 6 was incorrect. The correct title appears below:
Adult midbrain DA regeneration depends on hedgehog signalling.
The online issue and print copy are correct.
We apologise to authors and readers for this mistake.
DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 4127
Development 137, 4127-4134 (2010) doi:10.1242/dev.055541
© 2010. Published by The Company of Biologists Ltd
Efficient regeneration by activation of neurogenesis in
homeostatically quiescent regions of the adult vertebrate
brain
Daniel A. Berg1,*, Matthew Kirkham1,*, Anna Beljajeva1, Dunja Knapp2, Bianca Habermann3, Jesper Ryge4,
Elly M. Tanaka2 and András Simon1,†
SUMMARY
In contrast to mammals, salamanders and teleost fishes can efficiently repair the adult brain. It has been hypothesised that
constitutively active neurogenic niches are a prerequisite for extensive neuronal regeneration capacity. Here, we show that the
highly regenerative salamander, the red spotted newt, displays an unexpectedly similar distribution of active germinal niches with
mammals under normal physiological conditions. Proliferation zones in the adult newt brain are restricted to the forebrain,
whereas all other regions are essentially quiescent. However, ablation of midbrain dopamine neurons in newts induced
ependymoglia cells in the normally quiescent midbrain to proliferate and to undertake full dopamine neuron regeneration. Using
oligonucleotide microarrays, we have catalogued a set of differentially expressed genes in these activated ependymoglia cells.
This strategy identified hedgehog signalling as a key component of adult dopamine neuron regeneration. These data show that
brain regeneration can occur by activation of neurogenesis in quiescent brain regions.
INTRODUCTION
Abundant production of new neurons in the adult mammalian brain
is limited to the dentate gyrus of the hippocampus and the
subventricular zone of the lateral ventricles in the forebrain
(Alvarez-Buylla and Lim, 2004; Thored et al., 2007; Frielingsdorf
et al., 2004; Hermann et al., 2009; Zhao et al., 2003). Neurogenesis
may be evoked in quiescent regions, but the number of persisting
new neurons that are generated remains low and consequently the
functional recovery of the animals limited (Lindvall et al., 2004).
By contrast, the adult brain in non-mammalian vertebrates of
certain fish and salamander species repairs damage via processes
fuelled by neurogenesis (Zupanc, 2009). Previous studies have
revealed several proliferation hotspots in the adult zebrafish brain
from which neurons are continuously derived (Adolf et al., 2006;
Chapouton et al., 2007; Grandel et al., 2006). From these and other
observations it was hypothesised that the broad distribution of
homeostatic neurogenesis in the brain is an underlying component
of the extensive regenerative ability in these animals (Kaslin et al.,
2008; Zupanc, 2009). This link, however, needs further testing, as
it would have important implications for the possibility of engaging
non-germinal zones for functional neuronal replacement in species
where it naturally does not occur.
Cells that give rise to new neurons in mammals are of glial
character in terms of morphology and gene expression pattern
(Doetsch, 2003; Gotz and Barde, 2005; Kempermann et al., 2004).
1
Karolinska Institute, Department for Cell and Molecular Biology, Stockholm 17177,
Sweden. 2Center for regenerative therapies, Dresden 01307, Germany. 3Max-Planck
Institute, Dresden 01307, Germany. 4Karolinska Institute, Department for
Neurosciences, Stockholm 17177, Sweden.
*These authors contributed equally to this work
†
Author for correspondence ([email protected])
Accepted 7 October 2010
Data indicated that glial cells are neural stem cells also in nonmammalian vertebrates (Benraiss et al., 1999; Pellegrini et al.,
2007) but the glial origin of brain neurons has however not been
directly demonstrated. A recent report suggested that stem cells in
the adult non-mammalian brain have neuroepithelial rather than
glial features (Kaslin et al., 2009).
Here, we have addressed whether the presence of constitutively
active neurogenic niches is a prerequisite for extensive neuronal
regeneration, and revisited the identity of cells that produce new
neurons. We studied an aquatic salamander, the red spotted newt,
which has the widest regenerative repertoire among vertebrates.
Adult newts regenerate among other structures limbs, cardiac
muscle, ocular tissues and tails. Central nervous system (CNS)
regeneration in newts has mostly been studied after spinal cord
transection, tail amputation, or by removing a piece of brain tissue
(Chernoff et al., 2003; Okamoto et al., 2007).
Recently, we developed a chemical ablation model in newts by
intraventricular injection of 6-hydroxydopamine (6-OHDA) (Parish
et al., 2007). 6-OHDA acts as a selective neurotoxin and is used to
model aspects of Parkinson’s disease in many species by the
elimination of midbrain dopamine (DA) neurons. Similar to other
species, midbrain DA neurons in newts express the evolutionarily
conserved markers, tyrosine hydroxylase (TH) and Nurr1 (Marin
et al., 1997; Parish et al., 2007; Wallen and Perlmann, 2003).
Newts respond uniquely to the loss of midbrain DA neurons by full
regeneration within 4 weeks. Regeneration of DA neurons depends
on cellular proliferation and is characterised by the gradual birth of
new neurons leading to complete histological and locomotor
performance recovery (see Fig. S4 in the supplementary material)
(Parish et al., 2007).
In the present study we show, unexpectedly, that the adult newt
midbrain is essentially quiescent. Proliferation zones are normally
restricted to the telencephalon and the most rostral areas of the
diencephalon in the newt brain. We see no sign of caudal
migration from the constitutively active germinal zones towards
DEVELOPMENT
KEY WORDS: 6-OHDA, Adult neurogenesis, Dopamine, Midbrain, Neuronal stem cell, Salamander
4128 RESEARCH ARTICLE
MATERIALS AND METHODS
Animals
Adult red spotted newts Notophthalmus viridescens (Charles Sullivan,
Nashville, TN, USA) were maintained in a humidified room at 15-20°C.
All experiments were performed according to European Community and
local ethics committee guidelines.
Immunohistochemistry
Animals were anaesthetised by immersion in an aqueous solution of 0.1%
MS-222 (Sigma) and perfused with 4% formaldehyde in PBS. Animals
were dissected and the brains were rapidly placed in 4% formaldehyde.
After 1 hour of post fixation, brains were cryoprotected in 20% sucrose in
PBS for 12 hours and then embedded in OCT compound. Coronal sections
(20 m) were collected alternating on five slides. Sections were then
incubated with one of the following antibodies: mouse anti-PCNA (1:500,
Chemicon), rat anti-BrdU (1:500, Accurate Chemical and Scientific
Corporation), mouse anti-GFAP (1:500, Chemicon), rabbit anti-GFAP
(1:500, Chemicon), mouse anti-NeuN (1:500, AbCam), mouse anti-GFP
(1:200, Millipore), rabbit anti-TH (1:500, Chemicon) and rabbit antiMCM2 antibody (1:200, Abcam). The following day, sections were
incubated with appropriate secondary antibody: Alexa 594 or Alexa 448
IgG (1:1000; Molecular Probes). Cells were observed using a Zeiss upright
microscope, and pictures were captured by a colour CCD camera. For
confocal microscopy, an LSM 510 Meta laser microscope with LSM 5
Image Browser software (both Carl Zeiss MicroImaging) was used. All
images were manipulated using Adobe Photoshop according to the
guidelines in Development.
BrdU pulse labelling
BrdU (Sigma, 20 mg/kg) was injected intraperitoneally five times with 12
hour intervals. Animals were sacrificed 3 and 15 days after the first BrdU
injection and brains processed for immunohistochemistry.
6-OHDA and sham ablations
Newts were anaesthetised by placing them in an aqueous solution of 0.1%
MS-222 for 20 minutes. Animals were placed in a neonatal stereotaxic
head frame. 6-OHDA (200 nl of 6 g/l) was injected into the third
ventricle with a glass micropipette through a small hole drilled in the skull.
Sham ablated animals were injected with 200 nl 0.9% saline. Subsequently,
the surgery cavity in the scull was sealed with dental cement and animals
were left to recover overnight in a shallow container of water before being
placed back into a 25°C water environment. For BrdU-tracing experiments,
BrdU was injected intraperitoneally five times with 12 hours interval
starting 48 hours after lesion. Locomotion performance was assessed as
previously described (Parish et al., 2007).
Electroporation
pCMV H2BYFP (a kind gift from Claire Acquaviva, Welcome Trust/CR
UK Gurdon Institute, UK) was modified as described previously (Loof et
al., 2007; Morrison et al., 2007). pGFAP Gal4-VP16-Gal4UAS GFP
(Echeverri and Tanaka, 2002) was purified using high purity maxiprep
system (Marligen) and was resuspended in 10 mM Tris HCl (pH 8.5) at 5
g/l. Plasmid solution (400-500 nl) was injected into the third ventricle
of the newt, as previously described for the injection of 6-OHDA above.
Electroporation was carried out using round (0.5 cm diameter; TR Tech)
electrodes and an electroporator CUY21 EDIT (TR Tech). Five 50
msecond pulses at 150 V/cm with 950 ms intervals and current of 0.1-0.15
A were used.
Array analyses
Ambystoma mexicanum and Ambystoma tigrinum sequences available in
EST databases were assembled and annotated using the TIGR assembler
and by blasting against the Refseq database. Oligonucleotides (60 mer)
derived from these sequences and from available Notophthalmus viridscens
cDNA sequences were designed by e-Array (Agilent). To optimise
oligonucleotide selection for Notophthalmus viridescens, total RNA was
prepared from Notophthalmus viridescens whole brains or laser
microdissected ventral midbrain. RNA quality was assessed using
Bioanalyzer (Agilent), before amplified RNA (aRNA) was synthesised
using two rounds of linear amplification using Amino allyl message amp
II aRNA amplication kit (Ambion) according to the manufacturer’s
recommendations. Hybridisations were carried out on duplicate arrays.
Oligonucleotides that had a signal higher than background after
normalisation (Gry et al., 2009; Smyth, 2004) were selected and from this
set two oligonucleotides for each EST were printed on 44 K arrays. RNA
from laser microdissected ventral ventriclurar zone of the midbrain from
four control and four 6-OHDA-injected brains were individually analysed.
Principal component analysis (Owzar et al., 2008) was used to group
individual samples, which led to the exclusion of the data from one control
and one 6-OHDA injected sample (see Fig S6 in the supplementary
material).
Cloning, qRTPCR and in situ hybridisation
cDNA fragments were generated from whole brain RNA extracts using the
following primers.
nRAD: Forw, GCG TTV TCY TCT TTG CTR TC; Rev, TWY GAC
ATW TGG GAR CAG GA
Annexin1: Forw, TAR TCT CCT TTG GTK TCA TCC A; Rev, TAY
GAA GCW GGA GAA ARG AGA
ODC1: Forw, TTG AYT GTG CMA GYA AGA CTG; Rev, RGC CCC
CAR ATC AAA GAC AM
Jarid2: Forw, GAT TTC CTY ACG TTT CTV TGY C; Rev, TTC CTT
AAG WCC TGA GCC GA
HNRNPK: Forw, AAT GCC AGT GTT TCA GTC CC; Rev, CGA TCA
GTC GAA TGA GGR CAR
Shh: Forw, GAG CGC TTC AAG GAG CTA AC; Rev, ACC AGT
GGA CTC CCT CTG AC
FGF2: Forw, AAG MGG CTS TAC TGC AAR AA; Rev, GTT CKY
TTY AGH GCC ACA TAC CA
Sox1: Forw, TTY TTV AGC AGS GTC TTG GT; Rev, CCY ATG AAC
GCC TTY ATG GT
qRTPCRs were performed on 7500 fast real-time PCR system (Applied
Biosystems), using experimental design template for comparative CT
experiment where the reference sample was uninjured tissue and the
endogenous control was 40S ribosomal protein S21. Out of the 14 primer
sets used, seven gave products that could be accurately analysed based on
CT value and fragment size, and these are marked with an asterisk. The
following primers were used.
40S_S21: Forw, AAG TAA CCA TGC AGA ACG ATG; Rev, GGC
TAA CCG AAG GAT AGA GTC
*nRad: Forw, ACG AGG ATG ATT GGA ATG T; Rev, TAT GGG AGC
AGG ATG AGA C
*Collagenase: Forw, CTG GGC ACT TAA TGG GTA CG; Rev, ATG
GTG CGG GTA CTT TCA TC
*Cytokeratin 8: Forw, GGA GGC AGC ACT GAA TAA GG; Rev, TCC
AGC AGC TTC CTG TAG GT
*Annexin1: Forw, CGC TTG TAA TGT GCC TTG A; Rev, CCT GAC
CGC TAT TGT GAA A
*JARID2: Forw, TGG GTA CAG CAA ATC ACC AA; Rev, TAT GTG
GAG CAG GAG TGT GG
DEVELOPMENT
quiescent regions. Elimination of midbrain DA neurons by 6OHDA injection on the other hand leads to cell cycle re-entry by
midbrain ependymoglia cells that line the ventricular lumen. Using
cell-type specific labelling, we observed that many of these cells
exited from the ventricular layer and underwent neurogenesis
to replace the lost TH-expressing neurons. By contrast,
intraventricular sham injection induced a mitotic response but the
majority of these cells remained locally in their ventricular niche.
We further gained molecular insight into the processes that
accompany ependymoglia activation during DA neurogenesis
using a cross-species oligonucleotide based microarray strategy.
This approach identified a large set of candidate genes and also
led to the demonstration that hedgehog signalling is required for
adult DA regeneration.
Development 137 (24)
*ODC1: Forw, ACA GAT TGT GCA GAG CAT CG; Rev, CTG GAT
GGT TTC TTG CCA CT
*HNRNPK: Forw, CCC ACC TTG GAA GAG TAC CA; Rev, AGT
CGA ATG AGG GCA ACA CT
MMP9: Forw, TCC AAC TGC ACT CAG ACG AC; Rev, CAT CGG
GAC TCT CTG TGG TT
FGF2: Forw, TCC GTG ATC GGT ATG TGT TG; Rev, TGC AGC
TTC AAG CAG AAG AG
FGF4: Forw, TTG GGA GAG CGA CTC TGT ACT; Rev, GTC TCC
CAA CTC CAT TCC AG
Sox1: Forw, CGG GGC CTG TAC TTG TAG TC; Rev, TGA TGA
TGG AGA CGG ACT TG
FGFR1: Forw, ACA CTG GGT GGC TCT CCT T; Rev, ACT CGG
TTG GGT TTC TCC TT
Shh: Forw, ACC GGG ACC GCA GCA AGT AT; Rev, GGA AGC
AGC CTC CCG ATT T
BMP2/4: Forw, ACA AGA GGG AGA AGC GAC AG; Rev, GTT GGT
GGA GTT CAG GTG GT
For in situ hybridisation, probes were generated that corresponded to
the entire Notophthalmus viridescens Shh fragment (see Fig. S7 in the
supplementary material), DIG-labelled with standard reagents (Roche) and
used for hybridisations according to recommendations of the
manufacturer.
Inhibition of hedgehog and TGFb signalling
Animals were kept in water supplemented with 1 mM cyclopamine
(Sigma) or 25 M 2-(5-Benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4yl)-6-methylpyridine (Sigma) for 7 days by daily water replacement.
Regeneration of TH+ neurons was related to the degree of DA ablation,
which was assessed by counting the remaining TH+ neurons cells in
animals that were sacrificed 3 days after 6-OHDA injection.
RESEARCH ARTICLE 4129
RESULTS
Constitutive mitotic activity is restricted to the
forebrain
To reveal actively dividing cells in the adult newt brain, we first
identified cells that express the proliferating cell nuclear antigen
(PCNA). In general, we saw that PCNA+ cells were in contact with
the ventricles, had radial glia-like morphology and expressed the glial
fibrillary acidic protein (GFAP). These cells are generally referred to
as ependymoglia cells in newts and provide the only cell type
expressing GFAP given the lack of astrocytes (Benraiss et al., 1996;
Lazzari et al., 1997). A detailed analysis of proliferation zones
revealed distinct patterns of clusters of actively dividing cells, which
were restricted to regions located rostral to the ventral thalamic
region. The telencephalon harbours several mitotic clusters located
caudal to the rostral part of the olfactory bulb (OB) parenchyma. The
rostral OB is devoid of mitotic cells (Fig. 1B). Along the rostrocaudal
axis, starting from the accessory olfactory bulb, proliferating cells are
present in the lateral walls of the lateral ventricles (Fig. 1C).
Proliferating cells are also found in the dorsolateral wall of the lateral
ventricle throughout the telencephalon, situated adjacent to the border
between the dorsal pallium and the lateral pallium (Fig. 1D-E). A
ventrally located accumulation of PCNA+ cells is visible in the region
of the bed nucleus of stria terminalis. This area was dense in PCNA+
cells, stretching ~300 m along the rostral-caudal axis of the brain
(Fig. 1F,G, lower arrows). An accumulation of proliferating cells is
also apparent in the lateral wall of the lateral ventricles adjacent to
the lateral and medial amygdala (Fig. 1F,G, upper arrows). The walls
of the third ventricle were found to contain two proliferation zones
Fig. 1. Proliferation zones are restricted to the
forebrain. (A,A’) Schematic representation of the newt
brain. (B-J)Sections through (B-G) the telencephalon, (H)
the diencephalon, (I) the mesencephalon and (J) the
hindbrain. (B)No proliferating cells are detected in the
rostral olfactory bulb (OB). (C)PCNA+ cells line the medial
wall of the lateral ventricles in the accessory olfactory bulb.
(D,E)Proliferating cells line the lateral walls of the lateral
ventricle adjacent to the dorsal and lateral pallium (Dp and
Lp, respectively). (F,G)Ventral accumulation of proliferation
situated ventrally to the striatum (Str) in the region of the
bed nucleus of the stria terminalis (Bst). (H)A ventrally
located proliferation zone in the area of suprachiasmatic
nucleus (Sc), and a medial zone situated adjacent to the
ventral thalamic nucleus (Vtn). In the most caudal region of
the lateral ventricles, proliferating cells are scattered on
both the medial and lateral wall. Note unspecific
cytoplasmic labelling between medial and ventral
proliferation zone (arrowhead; see Fig. S1 in the
supplementary material). (I,J)Lack of proliferating cells in
the midbrain and hindbrain, respectively. Tm, midbrain
tegmentum; Tc, tectum; LDT, laterodorsal tegmental
nucleus; Ra, Raphe nuclei. Scale bar: 100m
DEVELOPMENT
Neurogenesis in the adult midbrain
4130 RESEARCH ARTICLE
Development 137 (24)
situated in the rostral diencephalon. The most ventral of these two
zones starts at the optic recess and extends caudally through to the
ventricular cells medial to the suprachiasmatic nucleus (Fig. 1H,
lower arrow). The more dorsally located proliferation zone is found
in the ventricular layer bordering to the ventral thalamus (Fig. 1H,
upper arrow). We did not find any other accumulation of proliferating
cells in the adult newt brain located caudal to this region. With the
exception of a maximum 3±2 scattered PCNA+ cells/brain, the caudal
diencephalon, the entire mesencephalon, hindbrain and cerebellum
were found to be essentially quiescent (Fig. 1I,J). To confirm the
conclusions based on the distribution of the PCNA+ cells, we looked
for cells positive for minichromosome maintenance protein, Mcm2,
which is involved in DNA replication (Maslov et al., 2007). As
shown in Fig. S2 in the supplementary material, although the
midbrain is devoid of MCM2+ cells, the forebrain harbours numerous
MCM2+ cells. In addition most cells that are MCM2+ also express
PCNA (data not shown). These results together show that during
normal homeostatic conditions, dividing cells are restricted to the
telencephalon and rostral diencephalon.
Activation of ependymoglia cells and ablationresponsive exit from normally quiescent midbrain
niches
We previously showed that selective ablation of diencephalic and
mesencephalic DA neurons by stereotaxic injection of 6-OHDA
leads to complete regeneration in adult newts (Parish et al., 2007)
Fig. 2. Forebrain-derived ependymoglia cells undergo
constitutive neurogenesis. (A)Timeline of BrdU administration and
subsequent analyses. (B,B⬘) BrdU+ cells are seen in the GFAP+
ependymoglia layer (arrowheads) after a 1-day chase. (C,C⬘) After a 13day chase, BrdU+ cells distant from the lateral ventricles express the
pan-neuronal marker NeuN (see also Fig. S3 in the supplementary
material). (D)Quantification of BrdU+/PCNA+ cells. (E)Quantification of
NeuN-expressing BrdU+ cells. Error bars represent s.e.m.; n3-5.
(F-F⬙) Examples of BrdU+/NeuN+ cells (arrowheads) in the olfactory bulb
after a 13-day chase. Boxed area in F is magnified in F⬘ and F⬙ (see also
Fig. S3 in the supplementary material). Student’s t-test was used.
***P<0.001. Scale bar: 50m.
(see Fig S4 in the supplementary material). Following the death of
DA neurons, regeneration involves extensive neurogenesis and
depends on cellular proliferation (Parish et al., 2007). In contrast to
the non-ablated animals, ependymoglia cells in the midbrain exit
quiescence and proliferate (Parish et al., 2007). Both sham and 6OHDA injections evoked mitotic activity with higher proliferation
index after 6-OHDA injection compared with the sham control
(Parish et al., 2007). We compared the cellular dynamics of the
proliferation response by BrdU pulse-chase experiments. We
observed a marked difference between 6-OHDA- and sham-injected
animals after a 3-day chase. 91±6% of the BrdU-labelled cells were
found distant from the ventricles and were GFAP– following
6-OHDA injection, and only 35±16% following sham injection (Fig.
3B; see Fig. S5 in the supplementary material). 6-OHDA injection
significantly shifted the equal distribution of the BrdU label towards
GFAP– non-ventricular cells when comparing 1-day and 3-day chase
periods. By contrast, we did not see any difference in the distribution
of the BrdU label in sham injected midbrains (Fig. 3B). These results
showed that neuron ablation stimulated the activation and exit of
ependymoglia cells from quiescent niches.
DEVELOPMENT
Constitutive neurogenesis in the adult newt
forebrain
In order to trace the progeny of the proliferating cells, we first
performed a pulse-chase experiment using the nucleotide analogue
5-bromo-2-deoxyuridine (BrdU), which incorporates into the
replicating DNA. Animals received five pulses of BrdU for 3 days
and were analysed either 1 day later or after a 13 days chase period
(Fig. 2A). One day after the BrdU pulses 96±0.7% of the BrdU+ cells
were also PCNA+ (see Fig. S2 in the supplementary material), and
the majority (74.5±4.5%) of the BrdU+ cells were immunoreactive
for the GFAP and had a radial glia-like morphology (Fig. 2B). At this
time point, 62±1.5% of the PCNA+ cells were still positive for BrdU
(Fig. 2D) and these cells were either lining or were found in close
proximity (within two cell layers) to the lateral ventricles. Fifteen
days after the first BrdU pulse, the majority (81.3±1.9%) of the
BrdU+ cells were found away from the lateral ventricles, in the
parenchyma of the lateral pallium and dorsal pallium, suggesting that
the progeny of the dividing cells had migrated laterally. 72±6.6% of
the BrdU+ nuclei were positive for the pan-neuronal marker NeuN
(Fig. 2C,E; see Fig. S3A in the supplementary material), showing
that the progeny of the ventricular GFAP+ cells entered a neuronal
differentiation program. At this time point, ~1.3% of the ventricular,
PCNA+ cells were retaining BrdU-labelling, suggesting that they
might represent a label retaining stem cell population (Fig. 2D).
Several BrdU+ cells expressing NeuN were also observed in the
rostral olfactory bulb (Fig. 2F; see Fig. S3B in the supplementary
material), where no cycling cells could be detected at the earlier time
point (Fig. 1B), suggesting a rostral migration and neuronal
differentiation of cells originating from the walls of the lateral
ventricles. By contrast, analysing four brains we could not detect any
BrdU+ cells after the chase periods in the caudal diencephalon,
midbrain, hindbrain or cerebellum showing the lack of caudal
migration. These results show that constitutive proliferation and
neurogenesis are essentially restricted to the forebrain under normal
homeostatic conditions.
Neurogenesis in the adult midbrain
RESEARCH ARTICLE 4131
Fig. 3. Ablation-responsive activation, exit and
neuronal differentiation of ependymoglia progeny.
(A)Timeline of selective or sham ablation, BrdU
administration and subsequent analyses. (B)Quantification
of BrdU+ cells in the ventricular layer or in the neuronal
layers, away from the ventricle. Error bars represent s.e.m.;
n3-7. (C)Time line of genetic labelling of ependymoglia
cells and selective ablation of TH+ neurons. (D-D⬙) Labelling
of ependymoglia cells lining the 3rd ventricle by unilateral
electroporation of a H2B2-YFP expression plasmid.
(E-G)Ablation of TH+ neurons (see Fig S4 in the
supplementary material) causes the shift of labelling from
ventricular GFAP+ (E) to GFAP– (F) in non-ventricular neuronal
layers. Quantification of labelled cells expressing or not
expressing GFAP (G). (H-H⬙) NeuN/YFP double-labelled cells
in the caudal diencephalon at day 18. V indicates the
ventricle; arrows indicate ventricular YFP+ cells; arrowheads
indicate NeuN+/YFP+ cells. (I)Quantification of YFP+/NeuN+
cells. Error bars represent s.e.m.; n3-7. Student’s t-test was
used. *P<0.05; **P<0.01. Scale bars: 50m.
analysed animals after 2 weeks. Because of the gradual
neurogenesis of TH+ cells during regeneration (Parish et al., 2007)
it is likely that the time frame under which the progeny of
individual ependymoglia cells mature into neurons is substantially
shorter than 14 days. In contrast to sham-injected animals, which
were lacking double-labelled cells (n4), we found in average
5±1.6 (n5) GFP+/TH+ cells in the 6-OHDA injected animals,
which corresponds to 5% of the new TH+ cells that are produced
in the midbrain during 14 days (Fig. 4C-C⵮). However, given that
only a fraction of ependymoglia cells are labelled by
electroporation, these results are best interpreted in qualitative
terms. These data show that the progeny of the activated GFAP+
ependymoglia have migrated away from the ventricle and
differentiated into TH+ neurons.
Molecular characterisation of ependymoglia cells
Next we looked at molecular events occurring in ependymoglia
cells after DA ablation by analysing changes in gene expression.
We prepared RNA and subsequently cDNA from laser
microdissected tissue corresponding 80-120 ventricular cells in the
midbrain, and analysed amplified RNA (aRNA) on oligonucleotide
microarrays, which were derived from salamander EST and cDNA
databases. Two oligonucleotide features represented each EST. The
analyses revealed 1063 oligonucleotides out of 20,839, which were
differentially regulated (P≤0.01) in 6-OHDA-injected compared
with control animals (data deposited in http://www.ebi.ac.uk/
arrayexpress/; Accession Number, E-MEXP-2752; see Table S1 in
the supplementary material). Out of these 1063 oligonucleotides,
739 were upregulated and 324 were downregulated. Six-hundred
and forty-five oligonucleotides could be annotated to 444 open
reading frames (ORFs), out of which 121 ORFs had multiple probe
hits, i.e. they were represented by several ESTs. Probes
representing 113 out of the 121 ORFs with multiple hits showed
DEVELOPMENT
Neurogenesis by ependymoglia progeny
The above results suggested but did not prove that activated
ependymoglia cells give rise to neurons after neuron ablation.
Hence, we genetically labelled quiescent ependymoglia cells prior
to the elimination of DA neurons by 6-OHDA. We marked GFAP+
cells lining the ventricle by in vivo electroporation of a DNA
construct encoding for a histone2b-yellow fluorescent fusion
protein (H2BYFP) under the control of the ubiquitously expressed
CMV promoter. H2BYFP was stereotaxically injected into the third
ventricle and an electrical current was applied via external
electrodes. Fifteen hours after electroporation, a clear unilateral
expression of YFP was observed in ventricular cells throughout the
dorsoventral axis of the third ventricle. YFP expression was
nuclear, and 952 out of the 960 YFP+ cells (n3) were found in the
ventricular GFAP+ cell layer (Fig. 3D-D⬙). Four days after
electroporation, we injected 6-OHDA into the third ventricle. After
14 days, 26±3% of the H2BYFP-labelled cells were nonventricular and GFAP– compared with 7.3±3.5% in the controls
(Fig. 3E-G). In accordance with this finding, we found 16 times
more H2BYFP/NeuN cells in 6-OHDA-injected animals compared
with the controls (Fig. 3H-I). These results show that ependymoglia
cells enter a neurogenic differentiation programme after the loss of
DA neurons.
To specifically test whether ependymoglia cells can give rise to
new TH+ neurons in the midbrain tegmentum, we electroporated in
a dorsal to ventral direction a construct encoding the green
fluorescent protein (GFP) under the control of a human GFAP
promoter via the GAL4-VP16 enhancer. Using this strategy, we
label ependymoglia cells in a way that allows sustained expression
of the transgene also in progeny that had shifted lineage (Echeverri
and Tanaka, 2002). Twenty-four hours after electroporation, GFP
expression was observed in cells lining the third ventricle (Fig.
4B,B⬘). We injected 6-OHDA 4 days after electroporation and
Fig. 4. Maturation of ependymoglia progeny into TH+ neurons.
(A)Overview of the newt brain. The boxed area indicates the cell bodies
of the TH+ neurons in the midbrain tegmentum. Descending axons are
visible in this sagittal section extending from the cell bodies in a caudal
direction. (B,B⬘) GFP+/GFAP+ ependymoglia cells in the ventral midbrain.
(C-C⵮) Example of GFP+/TH+ in the midbrain after 6-OHDA-injection
(arrow). Arrowhead indicates a GFP+/TH– cell. Scale bars: 50m.
either up- or downregulation but no mixed patterns of regulation.
Furthermore, in no cases were individual ESTs represented by
probes, where one probe was up- and the other downregulated. Fig.
5A shows that the differentially regulated ORFs represented a
broad range of biological processes. To validate the array data, we
set out to analyse the expression of 14 candidates using quantitative
real-time polymerase chain reactions (qRTPCR). Out of those 14,
we obtained products with analysable Ct-values and of expected
size in 7 cases, all of which confirm the array data (Fig. 5C; see
Table S1 in the supplementary material). Notably the array results
identified several genes implicated in remodelling, neuronal stem
cell regulation and DA neurogenesis (Fig. 5B), reinforcing the
validity of the data.
Sonic hedgehog (Shh) was one of the genes whose differential
regulation we could not validate using qRTPCR and hence we used
in situ hybridisation. Shh has been implicated in regulating the fate
of neural stem cells in general, and in DA neurogenesis in
particular (Arenas, 2008; Traiffort et al., 2010). In addition Shh
signalling has been implicated controlling aspects of appendage
and lens regeneration in salamanders (Schnapp et al., 2005; Tsonis
et al., 2004). We first cloned a fragment of Notophthalmus Shh
encoding for a 174 amino acid long ORF (see Fig S7 in the
Development 137 (24)
Fig. 5. Molecular characterisation of ependymoglia response.
(A)Injection of 6-OHDA leads to the regulation of factors representing
a wide range of cellular functions. Dark colours indicate upregulation;
light colours indicate downregulation. (B)6-OHDA injection leads to the
regulation of genes implicated in tissue remodelling, stem cell
maintenance and DA neurogenesis. (C)qRTPCR analyses confirming the
differential regulation revealed by the microarray studies (see also Fig.
5B; see Table S1 in the supplementary material). Seven out of 14 primer
sets generated PCR fragments of expected size and gave products with
consistent CT values. Rad, newt ras associated to diabetes; HnRnp K,
heterogeneous nuclear ribonucleoprotein; ODC-1, ornithine
decarboxylase. Stars, circles, squares and triangles represent individual
data points. Black, control samples; red, 6-OHDA-ablated samples.
supplementary material). Subsequently, we performed in situ
hybridisations, which showed upregulation of Shh after 6-OHDA
injection (Fig. 6A,B). In accordance with these observations,
inhibiting Shh signalling with cyclopamine reduced DA
regeneration, as measured by the regeneration of the number of
TH+ cells (Fig. 6C-E). As a control, we saw that cyclopamine
treatment did not reduce the number of TH+ cells in non-ablated
brains (data not shown). Furthermore, interference with TGFb
signalling using an inhibitor of activin receptor-like kinase did not
inhibit DA regeneration (data not shown). This is consistent with
the array result, which did not show differential regulation of
TGFb1 (see deposited data http://www.ebi.ac.uk/arrayexpress;
Accession Number, E-MEXP-2752). Treatment with cyclopamine
did not reduce the proliferation of ependymoglia cells (data not
shown), nevertheless our data show that hedgehog signalling is
required for adult midbrain DA regeneration.
DISCUSSION
Regenerative therapies aiming to replace lost neurons could be
achieved either by transplantation of exogenous cells or by
stimulating self-repair. The identification of neural stem cells in the
adult brain that continuously give rise to neurons has coaxed the
DEVELOPMENT
4132 RESEARCH ARTICLE
Fig. 6. Adult midbrain DA regeneration depends on hedgehog
signalling. (A,B)In situ hybridisation shows upregulation of Shh 7 days
after 6-OHDA injection. (C-E)Cyclopamine inhibits regeneration of TH+
neurons (C) (n7-10; **P0.003, Student’s t-test). Representative
images showing TH+ cells with (E) and without (D) cyclopamine
treatment. V indicates that ventricle. Scale bars: 50m.
hope for involving these cells in functional repair after injury or in
various degenerative diseases (Lindvall et al., 2004). Germinal
zones are, however, restricted mainly to two areas in the
mammalian brain. Although some neuroblasts derived from these
regions are attracted to injury sites (Brill et al., 2009; Carlen et al.,
2009), the extent of neuronal replacement is modest in regions that
normally do not produce new neurons.
In contrast to mammals, some non-mammalian vertebrate
species regenerate neurons more efficiently in the entire brain
(Becker and Becker, 2008). Careful mapping of the zebrafish
showed that this capacity correlated with the sustained and
widespread production of neurons in the adult brain (Grandel et al.,
2006), which suggested a correlation between homeostatic and
injury-responsive cell replacement (Zupanc, 2009). In this report,
we showed that the adult brain is capable of maintaining as well as
awakening the efficient neurogenic and regenerative potential of
quiescent niches. Neurogenesis is normally limited to the forebrain
in newts, but neuronal loss leads to activation of neuronal stem
cells in otherwise quiescent areas. Thus, the newt is akin to
mammals in terms of the extent of normal homeostatic cell turn
over, but distinct in terms of its injury response, which is
manifested in complete regeneration.
The exact fate of the stem cells in the various constitutively
active proliferation zones is unclear. However, three different
fate-mapping methods identified that ependymoglia cells that
have radial glia morphology and express GFAP give rise to
neurons in the newt brain. Although ependymoglia cells and
neuroepithelial cells are closely related, our results to some extent
contrast a recent study in zebrafish, which suggested that the cells
giving rise to neurons in the adult non-mammalian brain have
neuroepithelial rather than glial characteristics (Kaslin et al.,
2009). It is possible that species variations exist between newts
and zebrafish, not only in terms of the cellular turn over during
normal homeostatic conditions, but also when it comes to the
identity of stem cells. The type of neuronal stem cells that are
retained in adults may also reflect the degree of maturation of the
brain during embryonic development and postnatally. The identity
of neuronal stem cells may also be different in different regions
RESEARCH ARTICLE 4133
of the CNS as spinal cord regeneration studies in zebrafish
indicated radial-glial like cells as a source of new neurons
(Reimer et al., 2008).
A key question is how the cell cycle block in ependymoglia cells
is lifted upon injury and neuronal loss. Quiescence may be an
intrinsic cellular property that is undermined by extrinsic factors
generated upon injury, similar to newt limb regeneration (Tanaka
et al., 1999). Alternatively, stem cells may constantly and actively
be kept out of the cell cycle by extracellular signals that disappear
after injury.
Irrespective of how stem cells are activated, we see a specific
response in terms of the cellular dynamics when comparing 6OHDA-ablated animals with sham-ablated animals. We see
increased exit from the niche after DA ablation compared with
sham ablation. Activated ependymoglia cells that do not produce
neurons after sham ablation may participate in the complete
restoration of the ependymoglia layer. We see that the
ependymoglia layer recovers both after sham and DA ablation, and
is able to support a second round of regeneration following
repetitive DA ablation (data not shown). Ependymoglia cells that
remain locally after sham ablation may also undergo higher rate of
cell death than after DA ablation. Although the fate of the activated
ependymoglia cells after sham ablation is not clear at present, the
observations indicate the existence of attractants produced
specifically after the DA ablation. Such signals may act on cells
that are analogous to transit amplifying cells, immature neuroblasts
or on fully differentiated neurons. Alternatively, neuronal stem cells
in the midbrain may undergo one asymmetric division producing
one stem cell and one post-mitotic progenitor that differentiates
into a mature DA neuron as described during embryonic DA
neurogenesis in the mammalian midbrain (Bonilla et al., 2008). In
mammals, neuroblasts are attracted by factors derived from
astrocytes (Mason et al., 2001). The newt brain lacks astrocytes
(Benraiss et al., 1996; Lazzari et al., 1997); thus, such attractant
signals are either different compared with mammals or are
produced by other cell types.
The extent of the genomic information currently available for
newt research is currently limited but rapidly increasing (Borchardt
et al., 2010; Maki et al., 2010). Here, we used a cross-species
approach to start deciphering adult DA regeneration at the
molecular level. The data indicate that DA regeneration in an
existing brain structure demands to some extent similar cues that
are used during embryonic midbrain development. Further analyses
are likely to reveal necessary cues for neuron replacement in the
adult midbrain and thereby contribute to novel restorative strategies
in neurodegenerative diseases, such as Parkinson’s disease.
Acknowledgements
This work was supported by grants from the Swedish Research Council,
Karolinska Institute, Parkinsonfonden, Swedish Foundation for Strategic
Research and Wenner-Gren Foundation to A.S., and by DFG TA274/4-1 in
priority program Pluripotency, DFG TA274/2-2 Collaborative Research Center
655 from Cell to Tissues, and funds from the Max-Planck Institute and the
Center for Regenerative Therapies to E.T. M.K. was supported by a long-term
postdoctoral fellowship from HFSPO.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.055541/-/DC1
DEVELOPMENT
Neurogenesis in the adult midbrain
References
Adolf, B., Chapouton, P., Lam, C. S., Topp, S., Tannhauser, B., Strahle, U.,
Gotz, M. and Bally-Cuif, L. (2006). Conserved and acquired features of adult
neurogenesis in the zebrafish telencephalon. Dev. Biol. 295, 278-293.
Alvarez-Buylla, A. and Lim, D. A. (2004). For the long run: maintaining germinal
niches in the adult brain. Neuron 41, 683-686.
Arenas, E. (2008). Foxa2: the rise and fall of dopamine neurons. Cell Stem Cell 2,
110-112.
Becker, C. G. and Becker, T. (2008). Adult zebrafish as a model for successful
central nervous system regeneration. Restor. Neurol. Neurosci. 26, 71-80.
Benraiss, A., Caubit, X., Arsanto, J. P., Coulon, J., Nicolas, S., Le Parco, Y. and
Thouveny, Y. (1996). Clonal cell cultures from adult spinal cord of the
amphibian urodele Pleurodeles waltl to study the identity and potentialities of
cells during tail regeneration. Dev. Dyn. 205, 135-149.
Benraiss, A., Arsanto, J. P., Coulon, J. and Thouveny, Y. (1999). Neurogenesis
during caudal spinal cord regeneration in adult newts. Dev. Genes Evol. 209,
363-369.
Bonilla, S., Hall, A. C., Pinto, L., Attardo, A., Gotz, M., Huttner, W. B. and
Arenas, E. (2008). Identification of midbrain floor plate radial glia-like cells as
dopaminergic progenitors. Glia 56, 809-820.
Borchardt, T., Looso, M., Bruckskotten, M., Weis, P., Kruse, J. and Braun, T.
(2010). Analysis of newly established EST databases reveals similarities between
heart regeneration in newt and fish. BMC Genomics 11, 4.
Brill, M. S., Ninkovic, J., Winpenny, E., Hodge, R. D., Ozen, I., Yang, R.,
Lepier, A., Gascon, S., Erdelyi, F., Szabo, G. et al. (2009). Adult generation of
glutamatergic olfactory bulb interneurons. Nat. Neurosci. 12, 1524-1533.
Carlen, M., Meletis, K., Goritz, C., Darsalia, V., Evergren, E., Tanigaki, K.,
Amendola, M., Barnabe-Heider, F., Yeung, M. S., Naldini, L. et al. (2009).
Forebrain ependymal cells are Notch-dependent and generate neuroblasts and
astrocytes after stroke. Nat. Neurosci. 12, 259-267.
Chapouton, P., Jagasia, R. and Bally-Cuif, L. (2007). Adult neurogenesis in nonmammalian vertebrates. BioEssays 29, 745-757.
Chernoff, E. A., Stocum, D. L., Nye, H. L. and Cameron, J. A. (2003). Urodele
spinal cord regeneration and related processes. Dev. Dyn. 226, 295-307.
Doetsch, F. (2003). The glial identity of neural stem cells. Nat. Neurosci. 6, 11271134.
Echeverri, K. and Tanaka, E. M. (2002). Ectoderm to mesoderm lineage
switching during axolotl tail regeneration. Science 298, 1993-1996.
Frielingsdorf, H., Schwarz, K., Brundin, P. and Mohapel, P. (2004). No
evidence for new dopaminergic neurons in the adult mammalian substantia
nigra. Proc. Natl. Acad. Sci. USA 101, 10177-10182.
Gotz, M. and Barde, Y. A. (2005). Radial glial cells defined and major intermediates
between embryonic stem cells and CNS neurons. Neuron 46, 369-372.
Grandel, H., Kaslin, J., Ganz, J., Wenzel, I. and Brand, M. (2006). Neural stem
cells and neurogenesis in the adult zebrafish brain: origin, proliferation
dynamics, migration and cell fate. Dev. Biol. 295, 263-277.
Gry, M., Rimini, R., Stromberg, S., Asplund, A., Ponten, F., Uhlen, M. and
Nilsson, P. (2009). Correlations between RNA and protein expression profiles in
23 human cell lines. BMC Genomics 10, 365.
Hermann, A., Suess, C., Fauser, M., Kanzler, S., Witt, M., Fabel, K., Schwarz,
J., Hoglinger, G. U. and Storch, A. (2009). Rostro-caudal gradual loss of
cellular diversity within the periventricular regions of the ventricular system.
Stem Cells 27, 928-941.
Kaslin, J., Ganz, J. and Brand, M. (2008). Proliferation, neurogenesis and
regeneration in the non-mammalian vertebrate brain. Philos. Trans. R. Soc. Lond.
B. Biol. Sci. 363, 101-122.
Kaslin, J., Ganz, J., Geffarth, M., Grandel, H., Hans, S. and Brand, M. (2009).
Stem cells in the adult zebrafish cerebellum: initiation and maintenance of a
novel stem cell niche. J. Neurosci. 29, 6142-6153.
Kempermann, G., Jessberger, S., Steiner, B. and Kronenberg, G. (2004).
Milestones of neuronal development in the adult hippocampus. Trends Neurosci.
27, 447-452.
Lazzari, M., Franceschini, V. and Ciani, F. (1997). Glial fibrillary acidic protein
and vimentin in radial glia of Ambystoma mexicanum and Triturus carnifex: an
immunocytochemical study. J. Hirnforsch. 38, 187-194.
Development 137 (24)
Lindvall, O., Kokaia, Z. and Martinez-Serrano, A. (2004). Stem cell therapy for
human neurodegenerative disorders-how to make it work. Nat. Med. 10, S42S50.
Loof, S., Straube, W. L., Drechsel, D., Tanaka, E. M. and Simon, A. (2007).
Plasticity of mammalian myotubes upon stimulation with a thrombin-activated
serum factor. Cell Cycle 6, 1096-1101.
Maki, N., Martinson, J., Nishimura, O., Tarui, H., Meller, J., Tsonis, P. A. and
Agata, K. (2010). Expression profiles during dedifferentiation in newt lens
regeneration revealed by expressed sequence tags. Mol. Vis. 16, 72-78.
Marin, O., Smeets, W. J. and Gonzalez, A. (1997). Basal ganglia organization in
amphibians: catecholaminergic innervation of the striatum and the nucleus
accumbens. J. Comp. Neurol. 378, 50-69.
Maslov, A. Y., Bailey, K. J., Mielnicki, L. M., Freeland, A. L., Sun, X., Burhans,
W. C. and Pruitt, S. C. (2007). Stem/progenitor cell-specific enhanced green
fluorescent protein expression driven by the endogenous Mcm2 promoter. Stem
Cells 25, 132-138.
Mason, H. A., Ito, S. and Corfas, G. (2001). Extracellular signals that regulate the
tangential migration of olfactory bulb neuronal precursors: inducers, inhibitors,
and repellents. J. Neurosci. 21, 7654-7663.
Morrison, J. I., Loof, S., He, P., Alestrom, P., Collas, P. and Simon, A. (2007).
Targeted gene delivery to differentiated skeletal muscle: a tool to study
dedifferentiation. Dev. Dyn. 236, 481-488.
Okamoto, M., Ohsawa, H., Hayashi, T., Owaribe, K. and Tsonis, P. A. (2007).
Regeneration of retinotectal projections after optic tectum removal in adult
newts. Mol. Vis. 13, 2112-2118.
Owzar, K., Barry, W. T., Jung, S. H., Sohn, I. and George, S. L. (2008).
Statistical challenges in preprocessing in microarray experiments in cancer. Clin.
Cancer Res. 14, 5959-5966.
Parish, C. L., Beljajeva, A., Arenas, E. and Simon, A. (2007). Midbrain
dopaminergic neurogenesis and behavioural recovery in a salamander lesioninduced regeneration model. Development 134, 2881-2887.
Pellegrini, E., Mouriec, K., Anglade, I., Menuet, A., Le Page, Y., Gueguen, M.
M., Marmignon, M. H., Brion, F., Pakdel, F. and Kah, O. (2007).
Identification of aromatase-positive radial glial cells as progenitor cells in the
ventricular layer of the forebrain in zebrafish. J. Comp. Neurol. 501, 150-167.
Reimer, M. M., Sorensen, I., Kuscha, V., Frank, R. E., Liu, C., Becker, C. G. and
Becker, T. (2008). Motor neuron regeneration in adult zebrafish. J. Neurosci. 28,
8510-8516.
Schnapp, E., Kragl, M., Rubin, L. and Tanaka, E. M. (2005). Hedgehog
signaling controls dorsoventral patterning, blastema cell proliferation and
cartilage induction during axolotl tail regeneration. Development 132, 32433253.
Smyth, G. K. (2004). Linear models and empirical bayes methods for assessing
differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3,
Article 3.
Tanaka, E. M., Drechsel, D. N. and Brockes, J. P. (1999). Thrombin regulates Sphase re-entry by cultured newt myotubes. Curr. Biol. 9, 792-799.
Thored, P., Wood, J., Arvidsson, A., Cammenga, J., Kokaia, Z. and Lindvall,
O. (2007). Long-term neuroblast migration along blood vessels in an area with
transient angiogenesis and increased vascularization after stroke. Stroke 38,
3032-3039.
Traiffort, E., Angot, E. and Ruat, M. (2010). Sonic Hedgehog signaling in the
mammalian brain. J. Neurochem. 113, 576-590.
Tsonis, P. A., Vergara, M. N., Spence, J. R., Madhavan, M., Kramer, E. L., Call,
M. K., Santiago, W. G., Vallance, J. E., Robbins, D. J. and Del Rio-Tsonis, K.
(2004). A novel role of the hedgehog pathway in lens regeneration. Dev. Biol.
267, 450-461.
Wallen, A. and Perlmann, T. (2003). Transcriptional control of dopamine neuron
development. Ann. NY Acad. Sci. 991, 48-60.
Zhao, M., Momma, S., Delfani, K., Carlen, M., Cassidy, R. M., Johansson, C.
B., Brismar, H., Shupliakov, O., Frisen, J. and Janson, A. M. (2003). Evidence
for neurogenesis in the adult mammalian substantia nigra. Proc. Natl. Acad. Sci.
USA 100, 7925-7930.
Zupanc, G. K. (2009). Towards brain repair: insights from teleost fish. Semin. Cell
Dev. Biol. 20, 683-690.
DEVELOPMENT
4134 RESEARCH ARTICLE