Efficient regeneration by activation of neurogenesis in ...brain regeneration can occur by activation of neurogenesis in quiescent brain regions. KEY WORDS: 6-OHDA, Adult neurogenesis,

Efficient regeneration by activation of neurogenesis in homeostatically quiescent regionsof the adult vertebrate brainDaniel 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 138, 180 (2011) doi:10.1242/dev.061754

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4127DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE

INTRODUCTIONAbundant production of new neurons in the adult mammalian brainis limited to the dentate gyrus of the hippocampus and thesubventricular zone of the lateral ventricles in the forebrain(Alvarez-Buylla and Lim, 2004; Thored et al., 2007; Frielingsdorfet al., 2004; Hermann et al., 2009; Zhao et al., 2003). Neurogenesismay be evoked in quiescent regions, but the number of persistingnew neurons that are generated remains low and consequently thefunctional recovery of the animals limited (Lindvall et al., 2004).

By contrast, the adult brain in non-mammalian vertebrates ofcertain fish and salamander species repairs damage via processesfuelled by neurogenesis (Zupanc, 2009). Previous studies haverevealed several proliferation hotspots in the adult zebrafish brainfrom which neurons are continuously derived (Adolf et al., 2006;Chapouton et al., 2007; Grandel et al., 2006). From these and otherobservations it was hypothesised that the broad distribution ofhomeostatic neurogenesis in the brain is an underlying componentof the extensive regenerative ability in these animals (Kaslin et al.,2008; Zupanc, 2009). This link, however, needs further testing, asit would have important implications for the possibility of engagingnon-germinal zones for functional neuronal replacement in specieswhere it naturally does not occur.

Cells that give rise to new neurons in mammals are of glialcharacter in terms of morphology and gene expression pattern(Doetsch, 2003; Gotz and Barde, 2005; Kempermann et al., 2004).

Data indicated that glial cells are neural stem cells also in non-mammalian vertebrates (Benraiss et al., 1999; Pellegrini et al.,2007) but the glial origin of brain neurons has however not beendirectly demonstrated. A recent report suggested that stem cells inthe adult non-mammalian brain have neuroepithelial rather thanglial features (Kaslin et al., 2009).

Here, we have addressed whether the presence of constitutivelyactive neurogenic niches is a prerequisite for extensive neuronalregeneration, and revisited the identity of cells that produce newneurons. We studied an aquatic salamander, the red spotted newt,which has the widest regenerative repertoire among vertebrates.Adult newts regenerate among other structures limbs, cardiacmuscle, ocular tissues and tails. Central nervous system (CNS)regeneration in newts has mostly been studied after spinal cordtransection, 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 byintraventricular injection of 6-hydroxydopamine (6-OHDA) (Parishet al., 2007). 6-OHDA acts as a selective neurotoxin and is used tomodel aspects of Parkinson’s disease in many species by theelimination of midbrain dopamine (DA) neurons. Similar to otherspecies, midbrain DA neurons in newts express the evolutionarilyconserved markers, tyrosine hydroxylase (TH) and Nurr1 (Marinet al., 1997; Parish et al., 2007; Wallen and Perlmann, 2003).Newts respond uniquely to the loss of midbrain DA neurons by fullregeneration within 4 weeks. Regeneration of DA neurons dependson cellular proliferation and is characterised by the gradual birth ofnew neurons leading to complete histological and locomotorperformance recovery (see Fig. S4 in the supplementary material)(Parish et al., 2007).

In the present study we show, unexpectedly, that the adult newtmidbrain is essentially quiescent. Proliferation zones are normallyrestricted to the telencephalon and the most rostral areas of thediencephalon in the newt brain. We see no sign of caudalmigration from the constitutively active germinal zones towards

Development 137, 4127-4134 (2010) doi:10.1242/dev.055541© 2010. Published by The Company of Biologists Ltd

1Karolinska Institute, Department for Cell and Molecular Biology, Stockholm 17177,Sweden. 2Center for regenerative therapies, Dresden 01307, Germany. 3Max-PlanckInstitute, Dresden 01307, Germany. 4Karolinska Institute, Department forNeurosciences, Stockholm 17177, Sweden.

*These authors contributed equally to this work†Author for correspondence ([email protected])

Accepted 7 October 2010

SUMMARYIn contrast to mammals, salamanders and teleost fishes can efficiently repair the adult brain. It has been hypothesised thatconstitutively active neurogenic niches are a prerequisite for extensive neuronal regeneration capacity. Here, we show that thehighly regenerative salamander, the red spotted newt, displays an unexpectedly similar distribution of active germinal niches withmammals 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 inducedependymoglia cells in the normally quiescent midbrain to proliferate and to undertake full dopamine neuron regeneration. Usingoligonucleotide 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 thatbrain regeneration can occur by activation of neurogenesis in quiescent brain regions.

KEY WORDS: 6-OHDA, Adult neurogenesis, Dopamine, Midbrain, Neuronal stem cell, Salamander

Efficient regeneration by activation of neurogenesis inhomeostatically quiescent regions of the adult vertebratebrainDaniel A. Berg1,*, Matthew Kirkham1,*, Anna Beljajeva1, Dunja Knapp2, Bianca Habermann3, Jesper Ryge4,Elly M. Tanaka2 and András Simon1,†

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quiescent regions. Elimination of midbrain DA neurons by 6-OHDA injection on the other hand leads to cell cycle re-entry bymidbrain ependymoglia cells that line the ventricular lumen. Usingcell-type specific labelling, we observed that many of these cellsexited from the ventricular layer and underwent neurogenesisto replace the lost TH-expressing neurons. By contrast,intraventricular sham injection induced a mitotic response but themajority of these cells remained locally in their ventricular niche.We further gained molecular insight into the processes thataccompany ependymoglia activation during DA neurogenesisusing a cross-species oligonucleotide based microarray strategy.This approach identified a large set of candidate genes and alsoled to the demonstration that hedgehog signalling is required foradult DA regeneration.

MATERIALS AND METHODSAnimalsAdult 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 andlocal ethics committee guidelines.

ImmunohistochemistryAnimals were anaesthetised by immersion in an aqueous solution of 0.1%MS-222 (Sigma) and perfused with 4% formaldehyde in PBS. Animalswere dissected and the brains were rapidly placed in 4% formaldehyde.After 1 hour of post fixation, brains were cryoprotected in 20% sucrose inPBS for 12 hours and then embedded in OCT compound. Coronal sections(20 m) were collected alternating on five slides. Sections were thenincubated with one of the following antibodies: mouse anti-PCNA (1:500,Chemicon), rat anti-BrdU (1:500, Accurate Chemical and ScientificCorporation), 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 anti-MCM2 antibody (1:200, Abcam). The following day, sections wereincubated with appropriate secondary antibody: Alexa 594 or Alexa 448IgG (1:1000; Molecular Probes). Cells were observed using a Zeiss uprightmicroscope, and pictures were captured by a colour CCD camera. Forconfocal microscopy, an LSM 510 Meta laser microscope with LSM 5Image Browser software (both Carl Zeiss MicroImaging) was used. Allimages were manipulated using Adobe Photoshop according to theguidelines in Development.

BrdU pulse labellingBrdU (Sigma, 20 mg/kg) was injected intraperitoneally five times with 12hour intervals. Animals were sacrificed 3 and 15 days after the first BrdUinjection and brains processed for immunohistochemistry.

6-OHDA and sham ablationsNewts were anaesthetised by placing them in an aqueous solution of 0.1%MS-222 for 20 minutes. Animals were placed in a neonatal stereotaxichead frame. 6-OHDA (200 nl of 6 g/l) was injected into the thirdventricle 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 animalswere left to recover overnight in a shallow container of water before beingplaced back into a 25°C water environment. For BrdU-tracing experiments,BrdU was injected intraperitoneally five times with 12 hours intervalstarting 48 hours after lesion. Locomotion performance was assessed aspreviously described (Parish et al., 2007).

ElectroporationpCMV H2BYFP (a kind gift from Claire Acquaviva, Welcome Trust/CRUK Gurdon Institute, UK) was modified as described previously (Loof etal., 2007; Morrison et al., 2007). pGFAP Gal4-VP16-Gal4UAS GFP(Echeverri and Tanaka, 2002) was purified using high purity maxiprepsystem (Marligen) and was resuspended in 10 mM Tris HCl (pH 8.5) at 5g/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 50msecond pulses at 150 V/cm with 950 ms intervals and current of 0.1-0.15A were used.

Array analysesAmbystoma mexicanum and Ambystoma tigrinum sequences available inEST databases were assembled and annotated using the TIGR assemblerand by blasting against the Refseq database. Oligonucleotides (60 mer)derived from these sequences and from available Notophthalmus viridscenscDNA sequences were designed by e-Array (Agilent). To optimiseoligonucleotide selection for Notophthalmus viridescens, total RNA wasprepared from Notophthalmus viridescens whole brains or lasermicrodissected ventral midbrain. RNA quality was assessed usingBioanalyzer (Agilent), before amplified RNA (aRNA) was synthesisedusing two rounds of linear amplification using Amino allyl message ampII aRNA amplication kit (Ambion) according to the manufacturer’srecommendations. Hybridisations were carried out on duplicate arrays.Oligonucleotides that had a signal higher than background afternormalisation (Gry et al., 2009; Smyth, 2004) were selected and from thisset two oligonucleotides for each EST were printed on 44 K arrays. RNAfrom laser microdissected ventral ventriclurar zone of the midbrain fromfour control and four 6-OHDA-injected brains were individually analysed.Principal component analysis (Owzar et al., 2008) was used to groupindividual samples, which led to the exclusion of the data from one controland one 6-OHDA injected sample (see Fig S6 in the supplementarymaterial).

Cloning, qRTPCR and in situ hybridisationcDNA fragments were generated from whole brain RNA extracts using thefollowing primers.

nRAD: Forw, GCG TTV TCY TCT TTG CTR TC; Rev, TWY GACATW TGG GAR CAG GA

Annexin1: Forw, TAR TCT CCT TTG GTK TCA TCC A; Rev, TAYGAA GCW GGA GAA ARG AGA

ODC1: Forw, TTG AYT GTG CMA GYA AGA CTG; Rev, RGC CCCCAR ATC AAA GAC AM

Jarid2: Forw, GAT TTC CTY ACG TTT CTV TGY C; Rev, TTC CTTAAG WCC TGA GCC GA

HNRNPK: Forw, AAT GCC AGT GTT TCA GTC CC; Rev, CGA TCAGTC GAA TGA GGR CAR

Shh: Forw, GAG CGC TTC AAG GAG CTA AC; Rev, ACC AGTGGA CTC CCT CTG AC

FGF2: Forw, AAG MGG CTS TAC TGC AAR AA; Rev, GTT CKYTTY AGH GCC ACA TAC CA

Sox1: Forw, TTY TTV AGC AGS GTC TTG GT; Rev, CCY ATG AACGCC TTY ATG GT

qRTPCRs were performed on 7500 fast real-time PCR system (AppliedBiosystems), using experimental design template for comparative CT

experiment where the reference sample was uninjured tissue and theendogenous control was 40S ribosomal protein S21. Out of the 14 primersets used, seven gave products that could be accurately analysed based onCT value and fragment size, and these are marked with an asterisk. Thefollowing primers were used.

40S_S21: Forw, AAG TAA CCA TGC AGA ACG ATG; Rev, GGCTAA CCG AAG GAT AGA GTC

*nRad: Forw, ACG AGG ATG ATT GGA ATG T; Rev, TAT GGG AGCAGG ATG AGA C

*Collagenase: Forw, CTG GGC ACT TAA TGG GTA CG; Rev, ATGGTG CGG GTA CTT TCA TC

*Cytokeratin 8: Forw, GGA GGC AGC ACT GAA TAA GG; Rev, TCCAGC AGC TTC CTG TAG GT

*Annexin1: Forw, CGC TTG TAA TGT GCC TTG A; Rev, CCT GACCGC TAT TGT GAA A

*JARID2: Forw, TGG GTA CAG CAA ATC ACC AA; Rev, TAT GTGGAG CAG GAG TGT GG

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*ODC1: Forw, ACA GAT TGT GCA GAG CAT CG; Rev, CTG GATGGT TTC TTG CCA CT

*HNRNPK: Forw, CCC ACC TTG GAA GAG TAC CA; Rev, AGTCGA ATG AGG GCA ACA CT

MMP9: Forw, TCC AAC TGC ACT CAG ACG AC; Rev, CAT CGGGAC TCT CTG TGG TT

FGF2: Forw, TCC GTG ATC GGT ATG TGT TG; Rev, TGC AGCTTC AAG CAG AAG AG

FGF4: Forw, TTG GGA GAG CGA CTC TGT ACT; Rev, GTC TCCCAA CTC CAT TCC AG

Sox1: Forw, CGG GGC CTG TAC TTG TAG TC; Rev, TGA TGATGG AGA CGG ACT TG

FGFR1: Forw, ACA CTG GGT GGC TCT CCT T; Rev, ACT CGGTTG GGT TTC TCC TT

Shh: Forw, ACC GGG ACC GCA GCA AGT AT; Rev, GGA AGCAGC CTC CCG ATT T

BMP2/4: Forw, ACA AGA GGG AGA AGC GAC AG; Rev, GTT GGTGGA GTT CAG GTG GT

For in situ hybridisation, probes were generated that corresponded tothe entire Notophthalmus viridescens Shh fragment (see Fig. S7 in thesupplementary material), DIG-labelled with standard reagents (Roche) andused for hybridisations according to recommendations of themanufacturer.

Inhibition of hedgehog and TGFb signallingAnimals 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-4-yl)-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 inanimals that were sacrificed 3 days after 6-OHDA injection.

RESULTSConstitutive mitotic activity is restricted to theforebrainTo reveal actively dividing cells in the adult newt brain, we firstidentified cells that express the proliferating cell nuclear antigen(PCNA). In general, we saw that PCNA+ cells were in contact withthe ventricles, had radial glia-like morphology and expressed the glialfibrillary acidic protein (GFAP). These cells are generally referred toas ependymoglia cells in newts and provide the only cell typeexpressing GFAP given the lack of astrocytes (Benraiss et al., 1996;Lazzari et al., 1997). A detailed analysis of proliferation zonesrevealed distinct patterns of clusters of actively dividing cells, whichwere restricted to regions located rostral to the ventral thalamicregion. The telencephalon harbours several mitotic clusters locatedcaudal to the rostral part of the olfactory bulb (OB) parenchyma. Therostral OB is devoid of mitotic cells (Fig. 1B). Along the rostrocaudalaxis, starting from the accessory olfactory bulb, proliferating cells arepresent in the lateral walls of the lateral ventricles (Fig. 1C).Proliferating cells are also found in the dorsolateral wall of the lateralventricle throughout the telencephalon, situated adjacent to the borderbetween the dorsal pallium and the lateral pallium (Fig. 1D-E). Aventrally located accumulation of PCNA+ cells is visible in the regionof 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 isalso apparent in the lateral wall of the lateral ventricles adjacent tothe lateral and medial amygdala (Fig. 1F,G, upper arrows). The wallsof the third ventricle were found to contain two proliferation zones

4129RESEARCH ARTICLENeurogenesis in the adult midbrain

Fig. 1. Proliferation zones are restricted to theforebrain. (A,A’) Schematic representation of the newtbrain. (B-J)Sections through (B-G) the telencephalon, (H)the diencephalon, (I) the mesencephalon and (J) thehindbrain. (B)No proliferating cells are detected in therostral olfactory bulb (OB). (C)PCNA+ cells line the medialwall of the lateral ventricles in the accessory olfactory bulb.(D,E)Proliferating cells line the lateral walls of the lateralventricle adjacent to the dorsal and lateral pallium (Dp andLp, respectively). (F,G)Ventral accumulation of proliferationsituated ventrally to the striatum (Str) in the region of thebed nucleus of the stria terminalis (Bst). (H)A ventrallylocated proliferation zone in the area of suprachiasmaticnucleus (Sc), and a medial zone situated adjacent to theventral thalamic nucleus (Vtn). In the most caudal region ofthe lateral ventricles, proliferating cells are scattered onboth the medial and lateral wall. Note unspecificcytoplasmic labelling between medial and ventralproliferation zone (arrowhead; see Fig. S1 in thesupplementary material). (I,J)Lack of proliferating cells inthe midbrain and hindbrain, respectively. Tm, midbraintegmentum; Tc, tectum; LDT, laterodorsal tegmentalnucleus; Ra, Raphe nuclei. Scale bar: 100m

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situated in the rostral diencephalon. The most ventral of these twozones starts at the optic recess and extends caudally through to theventricular cells medial to the suprachiasmatic nucleus (Fig. 1H,lower arrow). The more dorsally located proliferation zone is foundin the ventricular layer bordering to the ventral thalamus (Fig. 1H,upper arrow). We did not find any other accumulation of proliferatingcells in the adult newt brain located caudal to this region. With theexception of a maximum 3±2 scattered PCNA+ cells/brain, the caudaldiencephalon, the entire mesencephalon, hindbrain and cerebellumwere found to be essentially quiescent (Fig. 1I,J). To confirm theconclusions based on the distribution of the PCNA+ cells, we lookedfor cells positive for minichromosome maintenance protein, Mcm2,which is involved in DNA replication (Maslov et al., 2007). Asshown in Fig. S2 in the supplementary material, although themidbrain is devoid of MCM2+ cells, the forebrain harbours numerousMCM2+ cells. In addition most cells that are MCM2+ also expressPCNA (data not shown). These results together show that duringnormal homeostatic conditions, dividing cells are restricted to thetelencephalon and rostral diencephalon.

Constitutive neurogenesis in the adult newtforebrainIn order to trace the progeny of the proliferating cells, we firstperformed a pulse-chase experiment using the nucleotide analogue5-bromo-2-deoxyuridine (BrdU), which incorporates into thereplicating DNA. Animals received five pulses of BrdU for 3 daysand 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+ cellswere also PCNA+ (see Fig. S2 in the supplementary material), andthe majority (74.5±4.5%) of the BrdU+ cells were immunoreactivefor the GFAP and had a radial glia-like morphology (Fig. 2B). At thistime 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 closeproximity (within two cell layers) to the lateral ventricles. Fifteendays after the first BrdU pulse, the majority (81.3±1.9%) of theBrdU+ cells were found away from the lateral ventricles, in theparenchyma of the lateral pallium and dorsal pallium, suggesting thatthe progeny of the dividing cells had migrated laterally. 72±6.6% ofthe BrdU+ nuclei were positive for the pan-neuronal marker NeuN(Fig. 2C,E; see Fig. S3A in the supplementary material), showingthat the progeny of the ventricular GFAP+ cells entered a neuronaldifferentiation program. At this time point, ~1.3% of the ventricular,PCNA+ cells were retaining BrdU-labelling, suggesting that theymight represent a label retaining stem cell population (Fig. 2D).Several BrdU+ cells expressing NeuN were also observed in therostral olfactory bulb (Fig. 2F; see Fig. S3B in the supplementarymaterial), where no cycling cells could be detected at the earlier timepoint (Fig. 1B), suggesting a rostral migration and neuronaldifferentiation of cells originating from the walls of the lateralventricles. By contrast, analysing four brains we could not detect anyBrdU+ cells after the chase periods in the caudal diencephalon,midbrain, hindbrain or cerebellum showing the lack of caudalmigration. These results show that constitutive proliferation andneurogenesis are essentially restricted to the forebrain under normalhomeostatic conditions.

Activation of ependymoglia cells and ablation-responsive exit from normally quiescent midbrainnichesWe previously showed that selective ablation of diencephalic andmesencephalic DA neurons by stereotaxic injection of 6-OHDAleads to complete regeneration in adult newts (Parish et al., 2007)

(see Fig S4 in the supplementary material). Following the death ofDA neurons, regeneration involves extensive neurogenesis anddepends on cellular proliferation (Parish et al., 2007). In contrast tothe non-ablated animals, ependymoglia cells in the midbrain exitquiescence and proliferate (Parish et al., 2007). Both sham and 6-OHDA injections evoked mitotic activity with higher proliferationindex after 6-OHDA injection compared with the sham control(Parish et al., 2007). We compared the cellular dynamics of theproliferation response by BrdU pulse-chase experiments. Weobserved a marked difference between 6-OHDA- and sham-injectedanimals after a 3-day chase. 91±6% of the BrdU-labelled cells werefound distant from the ventricles and were GFAP– following6-OHDA injection, and only 35±16% following sham injection (Fig.3B; see Fig. S5 in the supplementary material). 6-OHDA injectionsignificantly shifted the equal distribution of the BrdU label towardsGFAP– non-ventricular cells when comparing 1-day and 3-day chaseperiods. By contrast, we did not see any difference in the distributionof the BrdU label in sham injected midbrains (Fig. 3B). These resultsshowed that neuron ablation stimulated the activation and exit ofependymoglia cells from quiescent niches.

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Fig. 2. Forebrain-derived ependymoglia cells undergoconstitutive neurogenesis. (A)Timeline of BrdU administration andsubsequent analyses. (B,B�) BrdU+ cells are seen in the GFAP+

ependymoglia layer (arrowheads) after a 1-day chase. (C,C�) After a 13-day chase, BrdU+ cells distant from the lateral ventricles express thepan-neuronal marker NeuN (see also Fig. S3 in the supplementarymaterial). (D)Quantification of BrdU+/PCNA+ cells. (E)Quantification ofNeuN-expressing BrdU+ cells. Error bars represent s.e.m.; n3-5.(F-F�) Examples of BrdU+/NeuN+ cells (arrowheads) in the olfactory bulbafter a 13-day chase. Boxed area in F is magnified in F� and F� (see alsoFig. S3 in the supplementary material). Student’s t-test was used.***P<0.001. Scale bar: 50m.

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Neurogenesis by ependymoglia progenyThe above results suggested but did not prove that activatedependymoglia cells give rise to neurons after neuron ablation.Hence, we genetically labelled quiescent ependymoglia cells priorto the elimination of DA neurons by 6-OHDA. We marked GFAP+

cells lining the ventricle by in vivo electroporation of a DNAconstruct encoding for a histone2b-yellow fluorescent fusionprotein (H2BYFP) under the control of the ubiquitously expressedCMV promoter. H2BYFP was stereotaxically injected into the thirdventricle and an electrical current was applied via externalelectrodes. Fifteen hours after electroporation, a clear unilateralexpression of YFP was observed in ventricular cells throughout thedorsoventral axis of the third ventricle. YFP expression wasnuclear, and 952 out of the 960 YFP+ cells (n3) were found in theventricular GFAP+ cell layer (Fig. 3D-D�). Four days afterelectroporation, we injected 6-OHDA into the third ventricle. After14 days, 26±3% of the H2BYFP-labelled cells were non-ventricular and GFAP– compared with 7.3±3.5% in the controls(Fig. 3E-G). In accordance with this finding, we found 16 timesmore H2BYFP/NeuN cells in 6-OHDA-injected animals comparedwith the controls (Fig. 3H-I). These results show that ependymogliacells enter a neurogenic differentiation programme after the loss ofDA neurons.

To specifically test whether ependymoglia cells can give rise tonew TH+ neurons in the midbrain tegmentum, we electroporated ina dorsal to ventral direction a construct encoding the greenfluorescent protein (GFP) under the control of a human GFAPpromoter via the GAL4-VP16 enhancer. Using this strategy, welabel ependymoglia cells in a way that allows sustained expressionof the transgene also in progeny that had shifted lineage (Echeverriand Tanaka, 2002). Twenty-four hours after electroporation, GFPexpression was observed in cells lining the third ventricle (Fig.4B,B�). We injected 6-OHDA 4 days after electroporation and

analysed animals after 2 weeks. Because of the gradualneurogenesis of TH+ cells during regeneration (Parish et al., 2007)it is likely that the time frame under which the progeny ofindividual ependymoglia cells mature into neurons is substantiallyshorter than 14 days. In contrast to sham-injected animals, whichwere lacking double-labelled cells (n4), we found in average5±1.6 (n5) GFP+/TH+ cells in the 6-OHDA injected animals,which corresponds to 5% of the new TH+ cells that are producedin the midbrain during 14 days (Fig. 4C-C�). However, given thatonly a fraction of ependymoglia cells are labelled byelectroporation, these results are best interpreted in qualitativeterms. These data show that the progeny of the activated GFAP+

ependymoglia have migrated away from the ventricle anddifferentiated into TH+ neurons.

Molecular characterisation of ependymoglia cellsNext we looked at molecular events occurring in ependymogliacells after DA ablation by analysing changes in gene expression.We prepared RNA and subsequently cDNA from lasermicrodissected tissue corresponding 80-120 ventricular cells in themidbrain, and analysed amplified RNA (aRNA) on oligonucleotidemicroarrays, which were derived from salamander EST and cDNAdatabases. Two oligonucleotide features represented each EST. Theanalyses revealed 1063 oligonucleotides out of 20,839, which weredifferentially regulated (P≤0.01) in 6-OHDA-injected comparedwith control animals (data deposited in http://www.ebi.ac.uk/arrayexpress/; Accession Number, E-MEXP-2752; see Table S1 inthe supplementary material). Out of these 1063 oligonucleotides,739 were upregulated and 324 were downregulated. Six-hundredand forty-five oligonucleotides could be annotated to 444 openreading frames (ORFs), out of which 121 ORFs had multiple probehits, i.e. they were represented by several ESTs. Probesrepresenting 113 out of the 121 ORFs with multiple hits showed

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Fig. 3. Ablation-responsive activation, exit andneuronal differentiation of ependymoglia progeny.(A)Timeline of selective or sham ablation, BrdUadministration and subsequent analyses. (B)Quantificationof BrdU+ cells in the ventricular layer or in the neuronallayers, away from the ventricle. Error bars represent s.e.m.;n3-7. (C)Time line of genetic labelling of ependymogliacells and selective ablation of TH+ neurons. (D-D�) Labellingof ependymoglia cells lining the 3rd ventricle by unilateralelectroporation of a H2B2-YFP expression plasmid.(E-G)Ablation of TH+ neurons (see Fig S4 in thesupplementary material) causes the shift of labelling fromventricular GFAP+ (E) to GFAP– (F) in non-ventricular neuronallayers. Quantification of labelled cells expressing or notexpressing GFAP (G). (H-H�) NeuN/YFP double-labelled cellsin the caudal diencephalon at day 18. V indicates theventricle; arrows indicate ventricular YFP+ cells; arrowheadsindicate NeuN+/YFP+ cells. (I)Quantification of YFP+/NeuN+

cells. Error bars represent s.e.m.; n3-7. Student’s t-test wasused. *P<0.05; **P<0.01. Scale bars: 50m.

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either up- or downregulation but no mixed patterns of regulation.Furthermore, in no cases were individual ESTs represented byprobes, where one probe was up- and the other downregulated. Fig.5A shows that the differentially regulated ORFs represented abroad range of biological processes. To validate the array data, weset out to analyse the expression of 14 candidates using quantitativereal-time polymerase chain reactions (qRTPCR). Out of those 14,we obtained products with analysable Ct-values and of expectedsize in 7 cases, all of which confirm the array data (Fig. 5C; seeTable S1 in the supplementary material). Notably the array resultsidentified several genes implicated in remodelling, neuronal stemcell regulation and DA neurogenesis (Fig. 5B), reinforcing thevalidity of the data.

Sonic hedgehog (Shh) was one of the genes whose differentialregulation we could not validate using qRTPCR and hence we usedin situ hybridisation. Shh has been implicated in regulating the fateof neural stem cells in general, and in DA neurogenesis inparticular (Arenas, 2008; Traiffort et al., 2010). In addition Shhsignalling has been implicated controlling aspects of appendageand lens regeneration in salamanders (Schnapp et al., 2005; Tsoniset al., 2004). We first cloned a fragment of Notophthalmus Shhencoding for a 174 amino acid long ORF (see Fig S7 in the

supplementary material). Subsequently, we performed in situhybridisations, which showed upregulation of Shh after 6-OHDAinjection (Fig. 6A,B). In accordance with these observations,inhibiting Shh signalling with cyclopamine reduced DAregeneration, as measured by the regeneration of the number ofTH+ cells (Fig. 6C-E). As a control, we saw that cyclopaminetreatment did not reduce the number of TH+ cells in non-ablatedbrains (data not shown). Furthermore, interference with TGFbsignalling using an inhibitor of activin receptor-like kinase did notinhibit DA regeneration (data not shown). This is consistent withthe array result, which did not show differential regulation ofTGFb1 (see deposited data http://www.ebi.ac.uk/arrayexpress;Accession Number, E-MEXP-2752). Treatment with cyclopaminedid not reduce the proliferation of ependymoglia cells (data notshown), nevertheless our data show that hedgehog signalling isrequired for adult midbrain DA regeneration.

DISCUSSIONRegenerative therapies aiming to replace lost neurons could beachieved either by transplantation of exogenous cells or bystimulating self-repair. The identification of neural stem cells in theadult brain that continuously give rise to neurons has coaxed the

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Fig. 4. Maturation of ependymoglia progeny into TH+ neurons.(A)Overview of the newt brain. The boxed area indicates the cell bodiesof the TH+ neurons in the midbrain tegmentum. Descending axons arevisible in this sagittal section extending from the cell bodies in a caudaldirection. (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: 50m.

Fig. 5. Molecular characterisation of ependymoglia response.(A)Injection of 6-OHDA leads to the regulation of factors representinga wide range of cellular functions. Dark colours indicate upregulation;light colours indicate downregulation. (B)6-OHDA injection leads to theregulation of genes implicated in tissue remodelling, stem cellmaintenance and DA neurogenesis. (C)qRTPCR analyses confirming thedifferential regulation revealed by the microarray studies (see also Fig.5B; see Table S1 in the supplementary material). Seven out of 14 primersets generated PCR fragments of expected size and gave products withconsistent CT values. Rad, newt ras associated to diabetes; HnRnp K,heterogeneous nuclear ribonucleoprotein; ODC-1, ornithinedecarboxylase. Stars, circles, squares and triangles represent individualdata points. Black, control samples; red, 6-OHDA-ablated samples.

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hope for involving these cells in functional repair after injury or invarious degenerative diseases (Lindvall et al., 2004). Germinalzones are, however, restricted mainly to two areas in themammalian brain. Although some neuroblasts derived from theseregions are attracted to injury sites (Brill et al., 2009; Carlen et al.,2009), the extent of neuronal replacement is modest in regions thatnormally do not produce new neurons.

In contrast to mammals, some non-mammalian vertebratespecies regenerate neurons more efficiently in the entire brain(Becker and Becker, 2008). Careful mapping of the zebrafishshowed that this capacity correlated with the sustained andwidespread production of neurons in the adult brain (Grandel et al.,2006), which suggested a correlation between homeostatic andinjury-responsive cell replacement (Zupanc, 2009). In this report,we showed that the adult brain is capable of maintaining as well asawakening the efficient neurogenic and regenerative potential ofquiescent niches. Neurogenesis is normally limited to the forebrainin newts, but neuronal loss leads to activation of neuronal stemcells in otherwise quiescent areas. Thus, the newt is akin tomammals in terms of the extent of normal homeostatic cell turnover, but distinct in terms of its injury response, which ismanifested in complete regeneration.

The exact fate of the stem cells in the various constitutivelyactive proliferation zones is unclear. However, three differentfate-mapping methods identified that ependymoglia cells thathave radial glia morphology and express GFAP give rise toneurons in the newt brain. Although ependymoglia cells andneuroepithelial cells are closely related, our results to some extentcontrast a recent study in zebrafish, which suggested that the cellsgiving rise to neurons in the adult non-mammalian brain haveneuroepithelial rather than glial characteristics (Kaslin et al.,2009). It is possible that species variations exist between newtsand zebrafish, not only in terms of the cellular turn over duringnormal homeostatic conditions, but also when it comes to theidentity of stem cells. The type of neuronal stem cells that areretained in adults may also reflect the degree of maturation of thebrain during embryonic development and postnatally. The identityof neuronal stem cells may also be different in different regions

of the CNS as spinal cord regeneration studies in zebrafishindicated 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 cellsis lifted upon injury and neuronal loss. Quiescence may be anintrinsic cellular property that is undermined by extrinsic factorsgenerated upon injury, similar to newt limb regeneration (Tanakaet al., 1999). Alternatively, stem cells may constantly and activelybe kept out of the cell cycle by extracellular signals that disappearafter injury.

Irrespective of how stem cells are activated, we see a specificresponse in terms of the cellular dynamics when comparing 6-OHDA-ablated animals with sham-ablated animals. We seeincreased exit from the niche after DA ablation compared withsham ablation. Activated ependymoglia cells that do not produceneurons after sham ablation may participate in the completerestoration of the ependymoglia layer. We see that theependymoglia layer recovers both after sham and DA ablation, andis able to support a second round of regeneration followingrepetitive DA ablation (data not shown). Ependymoglia cells thatremain locally after sham ablation may also undergo higher rate ofcell death than after DA ablation. Although the fate of the activatedependymoglia cells after sham ablation is not clear at present, theobservations indicate the existence of attractants producedspecifically after the DA ablation. Such signals may act on cellsthat are analogous to transit amplifying cells, immature neuroblastsor on fully differentiated neurons. Alternatively, neuronal stem cellsin the midbrain may undergo one asymmetric division producingone stem cell and one post-mitotic progenitor that differentiatesinto a mature DA neuron as described during embryonic DAneurogenesis in the mammalian midbrain (Bonilla et al., 2008). Inmammals, neuroblasts are attracted by factors derived fromastrocytes (Mason et al., 2001). The newt brain lacks astrocytes(Benraiss et al., 1996; Lazzari et al., 1997); thus, such attractantsignals are either different compared with mammals or areproduced by other cell types.

The extent of the genomic information currently available fornewt research is currently limited but rapidly increasing (Borchardtet al., 2010; Maki et al., 2010). Here, we used a cross-speciesapproach to start deciphering adult DA regeneration at themolecular level. The data indicate that DA regeneration in anexisting brain structure demands to some extent similar cues thatare used during embryonic midbrain development. Further analysesare likely to reveal necessary cues for neuron replacement in theadult midbrain and thereby contribute to novel restorative strategiesin neurodegenerative diseases, such as Parkinson’s disease.

AcknowledgementsThis work was supported by grants from the Swedish Research Council,Karolinska Institute, Parkinsonfonden, Swedish Foundation for StrategicResearch and Wenner-Gren Foundation to A.S., and by DFG TA274/4-1 inpriority program Pluripotency, DFG TA274/2-2 Collaborative Research Center655 from Cell to Tissues, and funds from the Max-Planck Institute and theCenter for Regenerative Therapies to E.T. M.K. was supported by a long-termpostdoctoral fellowship from HFSPO.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.055541/-/DC1

4133RESEARCH ARTICLENeurogenesis in the adult midbrain

Fig. 6. Adult midbrain DA regeneration depends on hedgehogsignalling. (A,B)In situ hybridisation shows upregulation of Shh 7 daysafter 6-OHDA injection. (C-E)Cyclopamine inhibits regeneration of TH+

neurons (C) (n7-10; **P0.003, Student’s t-test). Representativeimages showing TH+ cells with (E) and without (D) cyclopaminetreatment. V indicates that ventricle. Scale bars: 50m.

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