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RESEARCH ARTICLE

The extracellular matrix glycoprotein tenascin-R regulatesneurogenesis during development and in the adult dentate gyrusof mice

Jin-Chong Xu1, Mei-Fang Xiao1, Igor Jakovcevski1, Elena Sivukhina1, Gunnar Hargus1, Yi-Fang Cui1,Andrey Irintchev1,2, Melitta Schachner1,3,4,* and Christian Bernreuther1,5

ABSTRACT

Abnormal generation of inhibitory neurons that synthesize

c-aminobutyric acid (GABAergic) is characteristic of

neuropsychological disorders. We provide evidence that the

extracellular matrix molecule tenascin-R (TNR) – which is

predominantly expressed by a subpopulation of interneurons –

plays a role in the generation of GABAergic and granule neurons in

the murine dentate gyrus by regulating fate determination of neural

stem or progenitor cells (NSCs). During development, absence of

TNR in constitutively TNR-deficient (TNR2/2) mice results in

increased numbers of dentate gyrus GABAergic neurons,

decreased expression of its receptor b1 integrin, increased

activation of p38 MAPK and increased expression of the

GABAergic specification gene Ascl1. Postnatally, increased

GABAergic input to adult hippocampal NSCs in TNR2/2 mice is

associated not only with increased numbers of GABAergic and,

particularly, parvalbumin-immunoreactive neurons, as seen during

development, but also with increased numbers of granule neurons,

thus contributing to the increased differentiation of NSCs into

granule cells. These findings indicate the importance of TNR in the

regulation of hippocampal neurogenesis and suggest that TNR acts

through distinct direct and indirect mechanisms during development

and in the adult.

KEY WORDS: Extracellular matrix, Tenascin-R, Neural stem cells,

Neurogenesis, Dentate gyrus, Mouse

INTRODUCTIONProportionately small numbers of GABAergic interneurons in theneocortex and hippocampus provide inhibitory input and regulate

the activity of large populations of principal excitatory cells.Deficits in the balance of excitation and inhibition lead to braindysfunction and appear to be pivotal in the pathogenesis of majorneuropsychiatric disorders, such as schizophrenia and autism

(Chao et al., 2010; Kehrer et al., 2008; Uhlhaas and Singer,

2012). Abnormal inhibition of neuronal excitability in

neuropsychiatric disorders is often related to abnormalities in

the generation of GABAergic neurons (Lee et al., 2011; Levitt

et al., 2004; Volk et al., 2000). Identifying the molecular basis

underlying the generation of interneurons is therefore expected to

augment our understanding of psychiatric disorders.

The ability of neural stem or progenitor cells (NSCs) to

produce different types of neurons is determined by intrinsic

genetic programs (Martynoga et al., 2012; Miyata et al., 2010;

Shen et al., 2006). Cell fate can also be specified by signals from

the local environment in the nervous system (Borello and Pierani,

2010; Guillemot and Zimmer, 2011). For example, extracellular

matrix molecules can interact with cell surface receptors and

regulate the generation of neurons by promoting or impeding the

self-renewal and fate determination of NSCs (Barros et al., 2011;

Ma et al., 2008; Tanentzapf et al., 2007).

Among the extracellular matrix molecules involved in

neurogenesis is tenascin-R (TNR), a large glycoprotein

expressed in the central nervous system by subpopulations of

interneurons, motoneurons and oligodendrocytes (Norenberg

et al., 1996; Wintergerst et al., 1993). TNR, with its epidermal-

growth-factor- and fibronectin-type-III-homologous repeats

(EGFL and FNIII, respectively) and fibrinogen knob, is a

multifunctional molecule implicated in cell adhesion and

repulsion, and the promotion and inhibition of neuritogenesis,

as well as synaptic and ion channel activity (Chiquet-Ehrismann

and Tucker, 2011; Dityatev et al., 2010; Pesheva and

Probstmeier, 2000). TNR also interacts with the GABA(B)

receptor through its associated carbohydrate, HNK-1, and can

thus directly influence signaling in cells expressing this receptor

(Saghatelyan et al., 2004b). Furthermore, TNR is important for

activity-dependent recruitment of neuroblasts in the adult mouse

forebrain (Saghatelyan et al., 2004a). In vitro, the EGFL and

FNIII domains 6–8 of TNR have been shown to modulate NSC

proliferation and differentiation through interactions with the

cell-surface receptor b1 integrin (Liao et al., 2008). Deficiency in

TNR leads to increased anxiety and severe cognitive deficits in

mice (Freitag et al., 2003; Montag-Sallaz and Montag, 2003).

The present study was designed to analyze the role of TNR

in differentiation of NSCs in the murine dentate gyrus. We

provide evidence that TNR deficiency leads to increased

numbers of GABAergic neurons in the dentate gyrus, when

compared with wild-type (TNR+/+) littermates early during

development, and to increased numbers of GABAergic neurons

and granule cells in the adult. In addition, we propose signaling

pathways that might underlie these phenotypes during

development and in the adult.

1Zentrum fur Molekulare Neurobiologie, Universitatsklinikum Hamburg-Eppendorf, 20246 Hamburg, Germany. 2Department of Otorhinolaryngology,Friedrich-Schiller-University Jena, 07743 Jena, Germany. 3Center forNeuroscience, Shantou University Medical College, Shantou, Guandong 515041,China. 4Keck Center for Collaborative Neuroscience and Department of CellBiology and Neuroscience, Rutgers University, Piscataway, NJ 08854, USA.5Institute of Neuropathology, University Medical Center Hamburg-Eppendorf,20246 Hamburg, Germany.

*Author for correspondence (schachner@zmnh.uni-hamburg.de)

Received 1 July 2013; Accepted 14 November 2013

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 641–652 doi:10.1242/jcs.137612

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RESULTSTenascin-R deficiency leads to increased numbers of granule cellsand GABAergic neurons in the dentate gyrusWe first analyzed the morphology of the dentate gyrus in adultmice. TNR2/2 mice showed an increase in the volume of thegranule cell layer of the dentate gyrus at 3 months of age and

throughout adulthood when compared with TNR+/+ littermatemice (Fig. 1A,B). Although the volume of the granule cell layerof the dentate gyrus remained stable in TNR+/+ mice, it increased

between 3 and 18 months of age in TNR2/2 mice. Thisabnormality of the granule cell layer was not due to changes incell density (143,00063000 cells/mm3 in TNR+/+ mice versus

149,00066000 cells/mm3 in TNR2/2 mice in 3-month-oldanimals), but was a result of an increase in the total number ofgranule cells (Fig. 1A–C). Previous work has shown that adult (4-

to 6-month-old) TNR2/2 mice have more parvalbuminimmunoreactive (PV+) inhibitory neurons in all subfields of thehippocampus compared with TNR+/+ littermates (Morellini et al.,2010). A likely explanation of this abnormality is that TNR is

involved in regulating the generation of inhibitory interneuronsduring embryonic development. To test this notion, we analyzednumbers of GAD65/67+ (GABAergic) neurons (Fig. 1D) in the

dentate gyrus at embryonic day (E) 18, an age at which thegeneration of inhibitory interneurons is in its final stage (Danglotet al., 2006). The number of GAD65/67+ interneurons was twice

as high in TNR2/2 mice when compared with TNR+/+ littermates(Fig. 1E). A similar difference was found at postnatal days (P) 7

and 90 (Fig. 1E). These findings suggest that TNR is involved inthe regulation of the generation of GABAergic inhibitory

interneurons during embryogenesis.

TNR is expressed in mouse dentate gyrus in embryos and adults, butnot in neural stem cellsGABAergic neurons in the dentate gyrus are mainly generatedbetween E13 and E14 (Danglot et al., 2006). Given the increase in thenumber of GABAergic inhibitory interneurons at E18 in TNR2/2

mice, it appeared most likely that TNR deficiency influences the cellfate determination of NSCs at early embryonic stages. To address thisissue, we first analyzed whether TNR is expressed in the ganglionic

eminence, the origin of hippocampal neurons during embryonicdevelopment (Fig. 2A). Indeed, TNR was expressed in close proximityto proliferating (Ki67+) cells (Fig. 2A). The expression of TNR in

mouse embryos was confirmed by western blot analysis of total brainhomogenate (Fig. 2B). However, in vitro TNR was not detectable incultured nestin+ NSCs. Two days after the induction of differentiationby withdrawal of FGF-2 and EGF, immunoreactivity for TNR was

detected in a subset of differentiating cells (Fig. 2C). Taken together,our results demonstrate that TNR is not expressed by nestin+ NSCs, butis expressed by differentiating or differentiated cells.

To determine whether TNR is expressed in the adulthippocampus, immunohistochemical analysis of TNR expressionwas performed and showed prominent expression of TNR in the

sub-granular layer of the dentate gyrus. In particular, TNR wasdetectable in close proximity to Ki67+ progenitor cells (Fig. 2D).

Fig. 1. Deficiency in TNR leads to increased numbers of neurons in the dentate gyrus of mice. (A) Confocal images of the dentate gyrus (DG) of adult (90-day-old) TNR-deficient (TNR2/2) and wild-type (TNR+/+) littermates stained with an antibody against NeuN (green). Images of the granule cell layer (GCL)outlined in the top panel are shown at higher magnification in the lower panel. Note the increased thickness of the GCL in TNR2/2 mice. Scale bars: 200 mm(top panel) and 20 mm (bottom panel). (B) Quantitative analysis of the volume of the GCL in TNR2/2 and TNR+/+ mice reveals an increased volume of theGCL in TNR2/2 mice at postnatal (P) days P90 and P540 when compared with TNR+/+ controls. Means 6 s.e.m. are shown (n55). Two-way ANOVA withfactors ‘age’ and ‘genotype’ followed by Tukey post-hoc was used for statistical analysis; *P,0.05. (C) Quantitative analysis of the number of granule cells in theGCL shows increased numbers of granule neurons in the GCL of TNR2/2 mice at P90 and P540. Means 6 s.e.m. are shown (n55). Two-way ANOVAwith Tukey post-hoc was used for statistical analysis; *P,0.05. (D) Confocal images of coronal slices of the posterior part of the DG of P7 TNR2/2 andTNR+/+ mice stained with an antibody against GAD65/67 (red). Scale bar: 100 mm. (E) Quantitative analysis at embryonic day 18 (E18) and P7 and P90 revealsan increase in GAD65/67-positive neurons in the DG of TNR2/2 from day E18 to P90. Means 6 s.e.m. are shown (n55). Two-way ANOVA with Tukey post-hocwas used for statistical analysis; *P,0.01.

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Western blot analysis of the hippocampus of TNR+/+ mice showedsustained expression of TNR in the hippocampus at bothembryonic and adult stages (Fig. 2E).

TNR deficiency reduces the expression of b1 integrin and increasesthe activation of p38 MAPKMolecules of the extracellular matrix can interact with integrinreceptors (Tanentzapf et al., 2007). By immunohistochemicalanalysis, we determined the distribution of TNR and b1 integrin

in the ganglionic eminence of TNR+/+ mice at E14, the time whengeneration of interneurons is most prominent. In line withprevious observations (Campos et al., 2004), integrin expressionwas detected in the germinal neuroepithelium throughout the

ganglionic eminence (Fig. 3A). In TNR+/+ mice, TNR and b1integrin were co-expressed in the ganglionic eminence (Fig. 3A).

In addition, TNR could be co-immunoprecipitated with b1integrin from brain homogenate at embryonic day E14(Fig. 3B) indicating that TNR and b1 integrin are associated

during mouse brain development.We then examined whether the expression of b1 integrin is

altered in TNR2/2 mice using neurospheres as a model system

because these three-dimensional mixtures of stem cells,committed precursors and differentiated cells are similar toembryonic brain tissue in cell and extracellular matrix

composition (Campos et al., 2004). We showed that b1 integrinis expressed and partially colocalizes with TNR (Fig. 3C) andthat cells expressing TNR are in the vicinity of Ki67+

proliferating cells (Fig. 3E). We also probed for the expression

of b1 integrin in neurospheres by western blot analysis andobserved a 30% decrease of b1 integrin expression in TNR2/2

Fig. 2. TNR is expressed in the medialganglionic eminence of mouse embryosand in the adult dentate gyrus of mice.(A) Confocal images of the medialganglionic eminence (MGE) of E14 mouseembryos stained with antibodies againstTNR (red) and Ki67 (green). The boxedarea is shown at higher magnification in theright column. Note the close association ofTNR and Ki67+ (proliferating) cells. Scalebar: 200 mm. (B) Western blot analysis ofbrain lysates of mouse embryos at E10,E12, E14, E16 and E18 with an antibodyagainst TNR. Actin was used to control forprotein loading. (C) Immunocytochemicalanalysis shows the homogeneity of nestin-immunoreactive (red) NSCs cultured underthe influence of FGF-2 and EGF in vitro,whereas TNR is not expressed in nestin-positive NSCs, TNR (red) is detected in asubset of differentiated cells 7 days afterinduction of differentiation by growth factorwithdrawal. Nuclei were counterstained withDAPI. Scale bar: 20 mm. (D) Confocalimages of the subgranular zone of the DG ofadult mice stained with antibodies againstTNR (red) and Ki67 (green). Note theexpression of TNR (red dots) in theneurogenic area of the hippocampuscontaining Ki67+ neural stem cells (NSCs).Scale bar: 20 mm. (E) Western blot analysisof lysates of the hippocampus of mice atE16, E18, P0, P7, P15 and P90 with anantibody against TNR. Actin was used tocontrol for protein loading.

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neurospheres when compared with neurospheres derived fromTNR+/+ mice (Fig. 3D).

Because b1 integrin is an important part of the signalingmechanisms that modulate NSC maintenance and differentiation

through the MAPK pathways (Campos et al., 2004), we analyzedwhether the reduction of b1 integrin expression would affectsignal transduction through the MAPK pathway. Western blot

analysis revealed that phosphorylation of p38 MAPK wassignificantly increased in TNR2/2 versus TNR+/+ neurospheres,whereas no changes were observed in the overall p38 MAPK

protein levels. No difference was detected between TNR2/2 andTNR+/+ neurospheres in the levels of p44/42 MAPK and in thephosphorylation of p44/42 MAPK (Fig. 3F).

TNR deficiency is associated with increased ASCL1 expressionProneural basic helix-loop-helix factors play important roles inthe specification of neural cell types (Guillemot, 2007). Ablation

of b1 integrin in embryonic stem cells leads to an increasedexpression of the lineage-specific gene Ascl1 (also known as

Mash1), which encodes a basic helix-loop-helix transcriptionfactor that plays an important role in the specification ofGABAergic interneurons and favors increased neuronaldifferentiation (Rohwedel et al., 1998). Given that TNR2/2 mice

have increased numbers of GABAergic interneurons and reducedexpression of b1 integrin, we hypothesized that the effects of TNRdeficiency on neuronal differentiation are mediated by ASCL1.

Thus, we first studied the localization of ASCL1 and b1 integrin inE14 mouse brains. ASCL1-expressing cells in the wall of the lateralventricles at the levels of the ganglionic eminence co-expressed b1

integrin (Fig. 4A). Many of the ASCL1-immunoreactive cells co-expressed Ki67 in the proliferative zones of the ganglionic eminence(Fig. 4B), where TNR is also expressed (Fig. 2A). Colocalization of

ASCL1 with b1 integrin was also confirmed in neurospheres derivedfrom E14 mouse brains (Fig. 4C). Western blot analysis of celllysates of neurospheres derived from the E14 brains showed a70% increase of ASCL1 expression in TNR2/2 versus TNR+/+

neurospheres (Fig. 4D). Immunohistochemical analysis revealed thatthis increase in ASCL1 expression in TNR2/2 neurospheres was

Fig. 3. TNR deficiency is associated with decreased expression of b1 integrin and activation of p38 MAPK. (A) Confocal images of the medial ganglioniceminence (MGE) of a TNR+/+ mouse at E14, stained with antibodies against TNR (red) and b1 integrin (green). Nuclei were counterstained with DAPI. Note theco-distribution of TNR and b1 integrin in the MGE. Scale bar: 20 mm. (B) Immunoprecipitation (IP) of brain lysates from E14 TNR+/+ mice with an antibodyagainst b1 integrin (left) or non-specific goat IgG as negative control (right) followed by western blot (WB) for TNR. (C) Confocal images of neurospheres derivedfrom the E14 TNR+/+ mouse brain maintained in the presence of EGF and FGF2 stained with antibodies against TNR (red) and b1 integrin (green). Nuclei werecounterstained with DAPI. Note the close association of TNR with b1 integrin. Scale bar: 20 mm. (D) Western blot analysis of cell lysates of neurospheresderived from TNR2/2 and TNR+/+ mice at E14 with antibodies against b1 integrin. GAPDH was used to control for protein loading. Means 6 s.e.m. are shown.Values in TNR+/+ mice were set to 100%. Student’s t-test was used for statistical analysis; *P,0.01. (E) Confocal images of neurospheres derived fromthe E14 TNR+/+ mouse brain stained with antibodies against TNR (red) and Ki67 (green). Nuclei were counterstained with DAPI. Note the close association ofTNR with Ki67-positive proliferating cells. Scale bar: 20 mm. (F) Western blot analysis of cell lysates from neurospheres derived from TNR2/2 and TNR+/+ mice atday E14 with antibodies against p38 MAPK, phosphorylated p38 MAPK, p44/42 MAPK and phosphorylated p44/42 MAPK. Band intensities of phosphorylatedproteins were normalized to the total protein levels. Values in TNR+/+ mice were set to 100%. Note the increased phosphorylation of p38 MAPK in TNR2/2

neurospheres. No difference in the phosphorylation of p44/42 MAPK was observed between TNR2/2 and TNR+/+ neurospheres. Means 6 s.e.m. are shown.Student’s t-test was used for statistical analysis; *P,0.01.

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accompanied by enhanced nuclear localization of ASCL1

(supplementary material Fig. S1).

TNR deficiency is associated with an inhibition of the proliferation ofNSCs and an acceleration of GABAergic neuronal differentiation inthe ganglionic eminence of the embryonic mouse brainGiven the conspicuous increase in the number of inhibitory

interneurons in the dentate gyrus of TNR2/2 mice, we examinedhow the lack of TNR affected neurogenesis in the ganglioniceminence. TNR2/2 and TNR+/+ animals were analyzed at E14–

E16 – at the peak of the generation of inhibitory interneurons.Increased neurogenesis in the absence of TNR could be caused

by decreased cell death, increased NSC proliferation, increasedneuronal differentiation at the expense of glial differentiation or a

combination of these factors. Immunohistochemical analysis foractivated caspase-3 at E15.5 and E18 did not show differences inapoptosis between TNR2/2 and TNR+/+ mice, indicating that a

lack of TNR did not affect cell death during embryonicdevelopment in vivo (supplementary material Fig. S2).

By contrast, proliferation in the ganglionic eminence as

determined by counting of Ki67+ cells was reduced by 30% inTNR2/2 mice when compared with TNR+/+ mice (Ki67+ cells:TNR+/+, 113,900611,100; TNR2/2, 79,20065200) (Fig. 5A).This decreased proliferation was confirmed in NSCs derived

from E14 TNR2/2 brains versus NSCs derived from TNR+/+ brainsin vitro (Fig. 5A). Thus, deficiency in TNR leads to decreasedproliferation of NSCs during development both in vitro and in vivo.

To examine the cellular mechanism that leads to decreasedproliferation of NSCs in TNR2/2 mice, we assessed cell-cycle exitand re-entry by BrdU-incorporation experiments. At E14, mice of

both genotypes were labeled with a pulse of BrdU, which isincorporated into the DNA during the S-phase of the cell cycle. At

E15, 24 hours after the BrdU pulse labeling, immunohistochemical

analysis with antibodies against Ki67 and BrdU was performed.TNR2/2 mice had a 27% higher ratio of cells that had exited thecell cycle (BrdU+Ki672 cells / BrdU+ cells) when compared with

TNR+/+ controls (Fig. 5B). This finding suggests that TNR favorscell cycle re-entry and impedes differentiation of NSCs.

The influence of TNR on neuronal differentiation was further

assessed by counts of newly generated neurons in the ganglioniceminence. At E14, TNR2/2 mice and TNR+/+ littermates werepulse-labeled with BrdU. Two days after BrdU administration (E16),

the number of newly generated neurons, identified as Tuj1 and BrdUdouble-labeled cells, was increased by 25% in TNR2/2 mice whencompared with TNR+/+ mice (BrdU+/Tuj1+ cells: TNR+/+, 552622;TNR2/2, 669615) (Fig. 5C). These findings were confirmed in

NSCs cultured in vitro (Fig. 5C), indicating that TNR deficiencyincreases neuronal differentiation of NSCs both in vitro and in vivo.

To specifically assess whether the number of newly generated

cells committed to the GABAergic cell fate was increasedby TNR deficiency, we labeled newly generated cells withantibodies against GAD65/67 and BrdU 2 days after BrdU

injection. Quantification showed that there was an about 20%increase in the number of GAD65/67+/BrdU+ double-labeled cellsin the dentate gyrus of TNR2/2 mice compared with TNR+/+

littermate mice (GAD65/67+/BrdU+ cells: TNR+/+, 299616;

TNR2/2, 373628) (Fig. 5D), indicating that TNR deficiencyincreases GABAergic differentiation in the developing brain.

Increased GABAergic innervation of PSA-immunoreactivehippocampal NSCs in adult TNR2/2 miceIt has been suggested that adult NSCs are influenced by GABAergic

interneurons that are immunoreactive for the calcium-bindingprotein PV (Danglot et al., 2006). To gain more information on

Fig. 4. TNR deficiency is associated withincreased expression of ASCL1.(A) Confocal images of the MGE of a TNR+/+

mouse at E14, stained with antibodies againstASCL1 (red) and b1 integrin (green). Theboxed areas are shown at higher magnificationin the right panel. Note the co-expression ofASCL1 and b1 integrin in cells of the MGE.Scale bar: 20 mm. (B) Confocal images of theganglionic eminence (GE) of a TNR+/+ mouseat E14, stained with antibodies against ASCL1(red) and Ki-67 (green). The arrow shows oneASCL1 and Ki67 double-labeled cell. Note theco-expression of Ki67 and ASCL1 in a subsetof cells. Scale bar: 20 mm. (C) Confocal imageof a neurosphere derived from the GE of E14TNR+/+ embryos stained with antibodiesagainst ASCL1 (red) and b1 integrin (green).Scale bar: 20 mm. (D) Western blot analysis ofcell lysates of neurospheres derived from E14TNR2/2 and TNR+/+ mice with antibodiesagainst ASCL1 and GAPDH. Values in celllysates of TNR+/+ neurospheres were set to100%. GAPDH was used to control for proteinloading. Note the increased expression ofASCL1 in TNR2/2 mice. Means 6 s.e.m. areshown. Student’s t-test was used for statisticalanalysis; *P,0.01.

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the features of GABAergic interneurons that potentially interact with

hippocampal NSCs, we performed double immunofluorescencelabeling with antibodies against PV and Ki67, and found that PV+

axon terminals were closely associated with Ki67+ NSCs(Fig. 6A,B). Stereological analysis showed a 67% increase in the

total number of PV+ interneurons in adult TNR2/2 mice comparedwith TNR+/+ littermates (Fig. 6C). To test whether an increase inPV+ interneurons leads to alteration of the GABAergic perisomatic

coverage of dividing hippocampal NSCs immunoreactive forPSA-NCAM, immunohistochemical analysis was performed todistinguish GABAergic terminals estimated by the expression of the

vesicular GABA transporter VGAT on PSA-NCAM+ NSCs.Analysis of GABAergic perisomatic inputs to PSA-NCAM+ NSCsshowed a 20% increase in TNR2/2 mice compared with TNR+/+

mice (Fig. 6D). Thus, TNR deficiency is associated with increasednumbers of PV+ interneurons in the dentate gyrus, leading toincreased GABAergic input to hippocampal NSCs.

TNR deficiency promotes neuronal differentiation and inhibitsproliferation of NSCs in the adult dentate gyrusGABAergic signaling increases neuronal differentiation of NSCs

in the adult dentate gyrus and decreases proliferation of NSCs in

the adult subventricular zone (Liu et al., 2005; Tozuka et al.,

2005). Thus, we first evaluated proliferation by counting thenumber of Ki67+ cells in the dentate gyrus. TNR2/2 mice showeda 70% reduction in the overall number of proliferating cellscompared with TNR+/+ mice (Ki67+ cells: TNR+/+, 36006800;

TNR2/2, 11206300) (Fig. 7A). Apoptotic cells were rarelydetected in the adult dentate gyrus and, thus, there was nodifference in the number of cells undergoing apoptosis in the

adult dentate gyrus of TNR2/2 mice and TNR+/+ littermates atpostnatal days 120 and 540 (supplementary material Fig. S2).

To evaluate whether increased numbers of PV+ interneurons

and enhanced GABAergic innervation led to an alteration inneuronal differentiation of adult hippocampal progenitor cells inTNR2/2 mice, proliferating NSCs were labeled with BrdU. Mice

were sacrificed 28 days after BrdU injection. The neuronalprogeny of hippocampal NSCs immunostained with antibodiesagainst BrdU and NeuN was determined. Compared with TNR+/+

mice, TNR2/2 mice showed increased numbers of BrdU+/NeuN+

neurons (BrdU+/NeuN+ cells: TNR+/+, 11346126; TNR2/2,16926189) (Fig. 7B). Thus, TNR deficiency decreasedproliferation and increased neuronal differentiation of NSCs in

the adult dentate gyrus.

Fig. 5. Deficiency in TNR leads to decreased proliferation of NSCs and enhanced neuronal differentiation in the ganglionic eminence. (A) Confocal images ofthe MGE of E14 TNR2/2 and TNR+/+ mice, stained with an antibody against Ki67 (top panel). In vitro (bottom panel), NSCs were derived from the MGE and cultured as amonolayer in the presence of EGF and FGF2. Confocal images in the bottom panel show NSCs derived from TNR2/2 mice and TNR+/+ controls stained with an antibodyagainst Ki67. Nuclei were counterstained with DAPI. Note the decreased proliferation of NSCs in TNR2/2 mice. Means 6 s.e.m. are shown. Student’s t-test was used forstatistical analysis; *P,0.01. Scale bars: 100 mm (top panel) and 20 mm (bottom panel). (B) Confocal images of theMGEof TNR+/+ and TNR2/2 mice immunostainedwithantibodies against Ki67 (red) and BrdU (green). Cell cycle exit was determined as the ratio of BrdU+/Ki672 cells of all BrdU+ cells 24 hours after BrdU injection in E15mouse embryos. Note the increased exit from the cell cycle indicated by a decreased ratio of BrdU+/Ki672 cells of all BrdU+ cells in TNR2/2 mice. Means 6 s.e.m. areshown (n55). Student’s t-test was used for statistical analysis; *P,0.05. Scale bar: 50 mm. (C) Confocal images of the MGE of E16 TNR+/+ and TNR2/2 mice,immunostained with antibodies against b-tubulin III (Tuj1, red) and BrdU (green) 48 hours after BrdU injection. Note the increased neuronal differentiation (BrdU+/Tuj1+) inTNR2/2 versus TNR+/+ mice 48 hours after BrdU injection. Values from TNR+/+ mice were set to 100% (top panel). The bottom panel shows confocal images of NSCsderived from the E14 mouse brain immunostained with a Tuj-1 antibody after the induction of differentiation by growth factor withdrawal. Nuclei were counterstained withDAPI. Note increased neuronal differentiation in NSCs derived from TNR2/2 versus TNR+/+ mice. Means 6 s.e.m. are shown (n55). Student’s t-test was used forstatistical analysis, *P,0.05. Scale bars: 50 mm (top panel) and 20 mm (bottom panel). (D) Confocal images of the MGE of E16 TNR+/+ and TNR2/2 mice immunostainedwith antibodies against GAD65/67 (red) and BrdU (green). Values from TNR+/+ mice were set to 100%. Note increased differentiation of newborn GABAergic cells (BrdU+/GAD65/67+) in TNR2/2 versus TNR+/+ mice 48 hours after BrdU injection. Means 6 s.e.m. are shown (n53). Student’s t-test was used for statistical analysis; *P,0.05.Scale bar: 50 mm.

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DISCUSSIONIntrinsic programs and extrinsic factors imposed by the local

extracellular environment tightly control the fate determination ofNSCs (Barnabe-Heider et al., 2005; Ma et al., 2008; Seuntjenset al., 2009; Shen et al., 2006; Tanentzapf et al., 2007). In the

present study we identified TNR, an extracellular matrixglycoprotein that is expressed in the developing and adultnervous system, as an important factor in regulating the

generation of GABAergic interneurons and granule neurons inthe developing and adult murine dentate gyrus.

The majority of GABAergic neurons in rodents arise from themedial and caudal eminence (Danglot et al., 2006). We show that

during embryonic development TNR is not only strongly expressedin the developing murine cortex, but also in the medial and caudalganglionic eminence in close proximity to proliferating cells.

Notably, TNR is also expressed in the developing human cerebralcortex in a spatio-temporally controlled pattern (El Ayachi et al.,2011). Ablation of TNR leads to increased numbers of GABAergic

and PV+ interneurons in the dentate gyrus of embryonic mice. It isinteresting in this context that TNR stimulates the detachment ofmigrating neuroblasts in the rostral migratory stream in an activity-dependent manner, and promotes their migration and differentiation

into inhibitory neurons in the murine olfactory bulb (Saghatelyanet al., 2004a). These findings are compatible with our present results

indicating that TNR signals to NSCs in the subgranular zone of thehippocampus to regulate the generation of inhibitory interneurons.Deficiency in TNR has been shown to lead to severe cognitive

deficits in mice (Montag-Sallaz and Montag, 2003) and intellectualimpairment in a human patient (Dufresne et al., 2012). Thesedeficits could be caused by increased GABAergic neurons, and

especially PV+ interneurons, leading to an imbalance betweenexcitatory and inhibitory neurotransmission.

Other extracellular matrix molecules have been shown toparticipate in the regulation of neurogenesis in the neurogenic

niche. Ablation of tenascin-C (TNC) in the neurogenic nichepromotes the generation of neurons and influenced proliferationof oligodendrocyte precursors, radial glial cells, and retinal stem/

progenitor cell by regulating the cell-cycle exit (Besser et al.,2012; Czopka et al., 2009; Garcion et al., 2001; Garcion et al.,2004). Interestingly, TNC2/2 mice show a reduction in

expression of ASCL1 (Mash1) by retinal stem/progenitor cells(Besser et al., 2012), in contrast to our study where TNRdeficiency resulted in increased ASCL1 expression in NSCs inthe ganglionic eminence. Enzymatic degradation of chondroitin

Fig. 6. The density of GABAergic axon terminals adjacent to PSA immunoreactive hippocampal NSCs is increased in adult TNR2/2 mice.(A) Schematic diagram demonstrating an axon terminal of a hippocampal PV+ inhibitory neuron adjacent to a polysialic acid (PSA)/Ki67 double immunoreactiveNSC. (B) Confocal images of the dentate gyrus of TNR+/+ and TNR2/2 mice, immunostained with antibodies against Ki67 (red) and parvalbumin (PV, green).Nuclei were counterstained with DAPI. Arrows indicate association of PV+ axon terminals with Ki67+ cells. Scale bars: 100 mm. (C) Confocal images of thedentate gyrus of adult TNR+/+ and TNR2/2 mice, immunostained with antibodies against PV (red). Note the increased number of PV+ neurons in the dentategyrus of TNR2/2 mice. Means 6 s.e.m. are shown (n55). Student’s t-test was used for statistical analysis, *P,0.01. Scale bar: 200 mm. (D) Confocal images ofthe GCL of the dentate gyrus of adult TNR+/+ and TNR2/2 mice, immunostained with antibodies against PSA (red) and vesicular GABA transporter VGAT(green). Arrows indicate puncta double-labeled with antibodies against VGAT and PSA-NCAM. Note the increased linear density of puncta around the cellsurface membrane of PSA+ cells in TNR2/2 mice. Means 6 s.e.m. are shown (n55). Values in TNR+/+ mice were set to 100%. Student’s t-test was used forstatistical analysis; *P,0.05. Scale bar: 10 mm.

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sulfate expressed by proteoglycans in the stem cell nichepromotes differentiation and migration of neural progenitor

cells (Gu et al., 2009), indicating an influence of theseextracellular matrix molecules on neurogenesis. The importanceof the extracellular matrix in regulating neurogenesis has been

supported by studies on matrix metalloproteinases, which canregulate neurogenesis by proteolytic modulation of extracellularmatrix molecules (Bovetti et al., 2007; Kang et al., 2008; Tontiet al., 2009; Zhang et al., 2007). Deficiency in tenascin-R affects

developmental neurogenesis in the mouse olfactory bulb, but notneurogenesis in the adult, thus highlighting that regulation ofneurogenesis can vary during an animal’s lifetime (David et al.,

2013).In the dentate gyrus of the hippocampus, granule neurons are

generated during development later than inhibitory interneurons

(Danglot et al., 2006). Moreover, in the adult forebrain, thedentate gyrus is one of the few areas where a continuous supplyof newborn neurons occurs (Tozuka et al., 2005). Although the

signaling mechanisms that regulate neurogenesis in the adultdentate gyrus have remained largely unknown, increasingevidence indicates that the proliferation and differentiation ofNSCs in the dentate gyrus are influenced by neurotransmitters.

Importantly, NSCs in the dentate gyrus receive GABAergic input(Bhattacharyya et al., 2008) that promotes their differentiationinto neurons (Tozuka et al., 2005). Furthermore, GABAergic

signaling decreases the proliferation of NSCs in thesubventricular zone of the adult brain (Liu et al., 2005).Potential candidates among the diverse populations of

interneurons that may be responsible for the GABAergicinfluence on NSCs are PV+ interneurons (Bhattacharyya et al.,2008; Tozuka et al., 2005). Accumulating evidence indicates thatTNR, a component of perineuronal nets (Dityatev et al., 2010)

influences synaptic plasticity and GABAergic signaling in the

hippocampus (Saghatelyan et al., 2001). Given that the number ofGABAergic neurons, and in particular PV+ interneurons, is

increased in the dentate gyrus of adult TNR2/2 mice, leading toenhanced GABAergic contacts with dividing NSCs, it seemsreasonable to suggest that TNR deficiency promotes neuronal

differentiation and inhibits proliferation of hippocampal NSCs inthe adult dentate gyrus by increased GABAergic signaling, thusexplaining the increase in the number of excitatory granuleneurons in the dentate gyrus of TNR2/2 mice. In view of the

close association of TNR and proliferating cells in the adulthippocampus of TNR+/+ mice, it is likely that TNR directlycontributes to neurogenesis in the adult, similar to what we

observed in embryonic mice. It is noteworthy that no effect ofTNR on adult neurogenesis was observed in the olfactory system(David et al., 2013).

The mechanism by which decreased proliferation of NSCscontributes to increased numbers of excitatory granule neuronsis presently not known. It is interesting in this context that

decreased proliferation of hippocampal NSCs is associated withincreased neuronal differentiation (Chen et al., 2012; Jung et al.,2008; Li et al., 1998; Spella et al., 2011; Theriault et al., 2005;Yabut et al., 2010; Zhao et al., 2010) and that 80% of newly

generated neurons die within the first month after generation inthe adult hippocampus (Kempermann et al., 2003). Thus,decreased apoptosis of newly generated neurons might have

contributed to the increase in numbers of granule neurons in thehippocampus of adult TNR2/2mice. In a recent study, PV+

neurons were shown to regulate fate decision in the adult murine

hippocampus in that optogenetic activation of PV+ interneuronactivity affects fate decisions of NSCs by decreasing theirproliferation (Song et al., 2012). Furthermore, alterations inlong-term potentiation in TNR2/2 mice could be rescued by

pharmacological inhibition with GABAA receptor antagonists

Fig. 7. TNR deficiency leads to increasedneuronal differentiation and decreasedproliferation of hippocampal NSCs in thedentate gyrus of adult mice. (A) Confocalimages of the dentate gyrus of adult(3-month-old) TNR+/+ and TNR2/2 mice,immunostained with antibodies against Ki67(green). Arrows indicate Ki67+ cells. Valuesfrom TNR+/+ mice were set to 100%. Note thereduced proliferation of NSCs in the adultdentate gyrus of TNR2/2 mice. Means 6

s.e.m. are shown (n55). Student’s t-test wasused for statistical analysis; *P,0.01. Scalebar: 200 mm. (B) Confocal images of thedentate gyrus of adult (3-month-old) TNR+/+

and TNR2/2 mice, that had been injectedwith BrdU 28 days before brains wereremoved, immunostained with antibodiesagainst BrdU (red) and NeuN (green). In thetop right, an orthogonal view is shown toconfirm the double labeling with antibodiesagainst NeuN and BrdU. Arrows indicateBrdU+ cells (top panel) and BrdU+/NeuN+

cells (bottom panel). Note the increasedneuronal differentiation in the dentate gyrusof adult TNR2/2 mice. Means 6 s.e.m. areshown (n55). Values in TNR+/+ mice wereset to 100%. Student’s t-test was used forstatistical analysis; *P,0.01. Scale bar:40 mm.

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(Morellini et al., 2010). Thus, we suggest that increased numbersof GABAergic neurons and in particular PV+ interneurons, being

generated in the developing hippocampus of TNR2/2 mice,contribute to the maintenance of increased neurogenesis in theadult TNR2/2 hippocampus, which also leads to the generation ofprincipal neurons.

Extracellular matrix molecules exert their regulatory effect onNSCs through intracellular signaling pathways mediated byinteraction with cell surface receptors (Brown, 2011; Fietz et al.,

2012; Hynes, 2009; Tanentzapf et al., 2007). TNR has beenshown to associate with sodium channel b-subunits (Xiao et al.,1999), the cell surface receptor F3/contactin-1 (Pesheva et al.,

1993), acetylated gangliosides (Probstmeier et al., 1999) andreceptor protein tyrosine phosphatase (RPTP)-zeta/beta (Milevet al., 1998; Xiao et al., 1997). In addition, a cognate receptor

interacting with TNR in NSCs is b1 integrin, which is aprominent sensor of signaling from extracellular matrixcomponents expressed by neural cells (Liao et al., 2008). b1integrins are highly expressed in murine neurospheres (Gu et al.,

2009), and more than 90% of human embryonic-stem-cell-derived NSCs express b1 integrins (Ma et al., 2008). Blocking thefunctions of b1 integrin inhibits proliferation of NSCs (Ma et al.,

2008). In the present study, we show that TNR colocalizes withb1 integrin in the developing medial and caudal ganglioniceminence. Furthermore, we show that TNR and b1 integrin co-

immunoprecipitate, most likely through their EGFL or FN6-8domains (Liao et al., 2008) during development, suggesting thatthey cooperate in determining neurogenesis in the developing

brain. It will be important to analyze which domains determinethe cell fates of hippocampal NSCs and whether there aredifferences in b1 integrin signaling in development and inadulthood. Integrins mediate cellular responses via intracellular

signaling cascades and regulation of gene expression (Camposet al., 2004; Tanentzapf et al., 2007). The influence of b1

integrins on NSC maintenance is regulated, at least to someextent, through the MAPK signaling pathway (Campos et al.,2004). First steps towards analysis of the molecular mechanismsunderlying TNR signaling pathways in TNR2/2 mice revealed

reduced b1 integrin expression that was accompanied by anupregulation of p38 MAPK and an enhanced expression of thetranscription factor ASCL1. This observation is in agreement

with a study showing that loss of b1 integrin function leads toincreased ASCL1 expression (Rohwedel et al., 1998), which inturn modulates GABAergic differentiation (Berninger et al.,

2007; Guillemot, 2007). ASCL1 is expressed by NSCs in the stemcell niches of the medial and caudal ganglionic eminence, andleads to cell cycle exit and neuronal differentiation of NSCs

(Nieto et al., 2001). In the present study, upregulation ofendogenous ASCL1 suggests an activation of this pathway inTNR2/2 mice, thereby leading to decreased proliferation throughincreased levels of ASCL1, increased cell cycle exit and

increased GABAergic neuronal differentiation in the absence ofTNR. It thus seems plausible to suggest that the observed effectsof TNR deficiency during development of the hippocampus are

caused by the reduction of b1 integrin expression, which appearsto be an important component in lineage-specific signalingpathways.

Taken together, we propose that TNR functions as a negative-feedback signaling molecule that regulates the generation ofappropriate numbers of GABAergic neurons in the dentate gyrus

during development. TNR secreted by inhibitory interneuronsmight influence the fate of NSCs by binding to b1 integrin,regulating intracellular p38 MAPK signaling and expression ofASCL1, thereby controlling GABAergic neuronal differentiation

Fig. 8. Model of a potential mechanism showing the influence of TNR on embryonic and adult neurogenesis in the dentate gyrus. Prenatally, TNR issecreted by niche cells, most probably by GABAergic interneurons located adjacent to the MGE and CGE. Binding of TNR to b1 integrins in the neurogenicniche influences intracellular p38 MAPK signaling in NSCs and modulates the expression and nuclear localization of the transcription factor ASCL1,which is involved in GABAergic differentiation of NSCs. These processes facilitate self-renewal of NSCs and inhibit their differentiation into GABAergic neurons.During adulthood, the processes of PV+ interneurons, a major subtype of GABAergic inhibitory interneurons in the dentate gyrus (DG), mediateGABAergic input to hippocampal progenitor cells regulating the differentiation of hippocampal NSCs and generation of granule neurons.

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during development. In the adult, direct input to NSCs fromGABAergic neurons, and in particular PV+ neurons that are

increased in TNR2/2 mice, promotes the differentiation of NSCsinto excitatory granule neurons (Tozuka et al., 2005), anobservation that might explain the increased numbers of granuleneurons in TNR2/2 mice. Furthermore, direct effects of TNR as

shown during development could contribute to the regulation ofadult neurogenesis in the hippocampus. A schematic view of ourproposed model is shown in Fig. 8. On the basis of this model,

further studies should identify whether TNR is a potential target formimetic small molecules that might beneficially influence theabnormal development of interneurons in the mammalian dentate

gyrus in diverse neurological disorders.

MATERIALS AND METHODSAnimalsThe generation of TNR-deficient (TNR2/2) mice and wild-type (TNR+/+)

littermates used in this study has been described (Weber et al., 1999).

Numbers and age of the animals studied in different experiments are

given in the figure legends. Experiments were conducted in accordance

with the German and European Community laws on protection of

experimental animals and approved by the local authorities of the City of

Hamburg.

Culture of NSCsNeural stem cells (NSCs), defined in this article as neural stem or

progenitor cells, were isolated from the brains of C57BL/6J mice at

embryonic day 14 (E14) as described (Dihne et al., 2003). Briefly,

ganglionic eminences were removed, mechanically dissociated using a

pipette, and maintained in a defined medium composed of a 1:1 mixture

of Dulbecco’s Modified Eagle’s Medium and F-12 supplemented

with glucose (0.6%, Sigma-Aldrich, Deisenhofen, Germany), sodium

bicarbonate (3 mM, Invitrogen, Karlsruhe, Germany), B27 (2%,

Invitrogen), glutamine (2 mM; Invitrogen) and HEPES buffer (5 mM,

Sigma-Aldrich). The medium was supplemented with epidermal growth

factor (EGF; 20 ng/ml; PreproTech, Rocky Hill, NY), and basic

fibroblast growth factor (FGF-2; 20 ng/ml; PreproTech). The cells

were maintained as neurospheres which were then passaged by

mechanical dissociation approximately every fifth day and reseeded at

a density of 50,000 cells/ml. Experiments were performed with

neurospheres taken from passages 3 and 6.

Determination of proliferation and differentiation of neural stemcells in vitroFor measurement of NSC proliferation, dissociated NSCs were plated

onto 15 mm glass coverslips coated with 0.01% poly-L-lysine (PLL,

Sigma-Aldrich). After 5 days in culture, 5-bromo-2-deoxyuridine (BrdU)

(10 mM; Sigma-Aldrich) was administered in EGF and FGF-2 containing

culture medium 8 hours before the cells were fixed with 4%

formaldehyde. The percentage of BrdU-positive cells was determined

by immunocytochemical analysis with monoclonal rat antibody against

BrdU (1:100; Abcam, Cambridge, MA). For analysis of differentiation,

neurospheres were mechanically dissociated and plated at a density of

50,000 cells/ml onto PLL-coated coverslips. Precursor cells were first

maintained in an undifferentiated state for 2 days after plating in EGF and

FGF2 containing serum-free culture medium. Growth factors were then

removed, and precursor cells were allowed to differentiate for an

additional 7 days. Then, coverslips were washed in phosphate-buffered

saline, pH 7.3 (PBS), and cells were fixed for 30 minutes with 4%

formaldehyde in PBS.

Immunocytochemistry and immunohistochemistryFor immunocytochemical analysis of fixed cells, cultured cells were

washed in PBS, and fixed in 4% formaldehyde. For

immunohistochemical analysis of hippocampus, postnatal and adult

mice were anesthetized and transcardially perfused with fixative

consisting of 4% formaldehyde and 0.1% CaCl2 in 0.1 M cacodylate

buffer (Sigma-Aldrich), pH 7.3, for 15 minutes at room temperature.

Brains were removed, incubated in 4% formaldehyde for 24 hours

followed by incubation in 15% sucrose in PBS for 48 hours at 4 C. 25-

mm-thick serial coronal sections were cut in a cryostat (CM3050; Leica,

Nussloch, Germany) and collected on SuperFrost Plus glass slides (Roth,

Karlsruhe, Germany). Mouse embryos were fixed in ice-cold 4%

formaldehyde for 2 hours. After fixation, the tissue was washed

extensively in ice-cold PBS and transferred to 15% sucrose solution

overnight at 4 C.

Antigen retrieval was performed by incubation of the tissue sections in

0.01 M sodium citrate solution, pH 9.0, for 30 minutes at 80 C followed

by blocking with PBS containing 0.2% v/v Triton X-100, 0.02% w/v

sodium azide (Sigma-Aldrich) and 5% v/v appropriate non-immune

serum (Jackson ImmunoResearch via Dianova, Hamburg, Germany) for

30 minutes. Primary antibodies diluted in PBS containing 0.5% w/v l-

carrageenan (Sigma-Aldrich) were applied for 48 hours at 4 C. After

washing in PBS, secondary antibodies were applied for 1 hour at room

temperature. Finally, coverslips were washed twice with PBS. To

determine total cell numbers in vitro, cells were counterstained with

DAPI (Sigma-Aldrich) for 2 minutes and the ratio of cell type-specific

marker-positive cells of all DAPI-positive cells was calculated. For BrdU

staining, DNA was denatured with 2 M HCl for 30 minutes at 37 C.

Monoclonal rat antibody against BrdU (1:100; Abcam) was applied

overnight at 4 C. For negative controls, primary antibody was omitted.

For cryosectioning of neurospheres, they were fixed in 4%

formaldehyde in PBS for 30 minutes at room temperature. After

fixation, the neurospheres were incubated in 30% sucrose in PBS for

4 hours at 4 C. Finally, 14-mm-thick cryosections were prepared.

Primary antibodies used were mouse monoclonal antibodies against

ASCL1 (1:200, BD Pharmingen, CA), NeuN (1:1,000, Millipore, MA),

nestin (1:50; Developmental Studies Hybridoma Bank, Iowa City,

IA), parvalbumin (PV) (clone PARV-19, 1:1000, Sigma-Aldrich),

polysialylated neural cell adhesion molecule [PSA-NCAM, 1:1000,

Millipore), TNR (clone 619 (Morganti et al., 1990)], polyclonal rabbit

antibodies against b1 integrin (1:200, Chemicon, Temecula, CA),

caspase-3 (1:2000, R&D Systems, Minneapolis, MN), glutamate

decarboxylase (GAD) 65 and 67 (1:500, Sigma-Aldrich), Ki67 (1:500,

Abcam), neuronal class III b-tubulin (Tuj1) (1:2000; Covance, Berkeley,

CA) and VGAT (anti-vesicular GABA transporter, 1:1000, VGAT,

Synaptic Systems, Gottingen, Germany). Cyanine 2 (Cy2)-, Cy3- and

Cy5-conjugated secondary antibodies (Dianova) were used to detect

primary antibodies.

Stereological analysisNumerical densities were estimated using the optical dissector method as

described (Nikonenko et al., 2006). Counting was performed on an

Axioskop microscope (Carl Zeiss Microimaging) equipped with a

motorized stage and Neurolucida software-controlled computer system

(MicroBrightField Europe, Magdeburg, Germany). The volume of the

granule cell layer was estimated using spaced serial sections (250 mm

interval) and the Cavalieri principle.

Analysis of cell cycle exitAnalysis of cell cycle exit was performed as described previously (Lien

et al., 2006). Briefly, mice received an intraperitoneal injection of

100 mg/kg BrdU in PBS. 24 hours later, mice were perfused and

cryosections were cut as described above and immunostained with

antibodies against Ki67 and BrdU. BrdU+ cells that had left the cell cycle

after the BrdU injection did not express Ki67 and could thus be identified

as BrdU+/Ki672, whereas the cells that remained in an active cell cycle

could be identified as BrdU+/Ki67+.

Light-microscopic analysis of perisomatic terminalsEstimation of perisomatic puncta in the dentate gyrus was performed as

described (Nikonenko et al., 2006) with a slight modification. Stacks of

images of 1 mm thickness were obtained using sections double-stained

for PSA-NCAM and VGAT using a TCS SP2 confocal microscope

(Leica). One merged image (red and green channel) per cell at the level

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of the largest cross-sectional area of the cell body was used to measure

soma area and area of perisomatic puncta. Area of VGAT-positive puncta

around each PSA-NCAM positive cell was measured. All measurements

were performed using the Image Tool 2.0 software (University of Texas

Health Science Center, San Antonio, TX).

ImmunoprecipitationBrain protein extracts were generated by lysing brain with RIPA buffer

for 1 hour at 4 C. Protein extracts were cleared with protein A/G agarose

beads (Santa Cruz Biotechnology) for 3 hours at 4 C and then incubated

with anti-b1-integrin antibody (1:1000, Chemicon) or non-immune IgG

(1:1000, Chemicon) overnight at 4 C. Protein-A/G agarose was added to

capture the immunocomplexes for 6 hours at 4 C under constant

agitation. Immunoprecipitated proteins were eluted from agarose beads

by 26 SDS sample buffer (125 mM Tris-HCl, 4% SDS, 30% glycerol,

10% b-mercaptoethanol and 0.00625% Bromophenol Blue, pH 6.8).

Western blot analysisSDS-PAGE and transfer were performed according to standard laboratory

protocols. Membranes were probed with antibodies against ASCL1 (1:500,

BD Pharmingen), glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

(1:1000; Millipore), TNR [clone 619 (Morganti et al., 1990)], actin (1:1000,

Sigma-Aldrich), b1 integrin (1:1000, Chemicon), phospho-p38 (Thr180/

Tyr182), phospho-p44/42 (Thr202/Tyr204) or total p38 and total p44/42

antibodies (1:1000; Cell Signaling Technology, Danvers, MA, USA).

Immunoreactivity was visualized using the enhanced chemiluminescence

detection system (ECL, Thermo Fisher Scientific, Bonn, Germany).

Statistical analysisAll numerical data are presented as means 6 s.e.m. Statistical analyses

were performed as indicated in the text and figures using the SPSS 11.5

software package (SPSS, Chicago, IL).

AcknowledgementsThe authors are grateful to Eva Kronberg for animal care, Dr Fabio Morellini andAchim Dahlmann for animal care and genotyping. Melitta Schachner is NewJersey Professor of Spinal Cord Research and is supported by the Li KashingFoundation at Shantou University Medical College, China.

Competing interestsThe authors declare no competing financial interests.

Author contributionsJ.-C.X.: conception and design, provision of study material, collection of data,data analysis and interpretation, manuscript writing, final approval of manuscript;M.-F.X.: collection of data, provision of study material, final approval ofmanuscript; E.S., G.H., Y.-F.C. and I.J.: collection of data, interpretation and finalapproval of manuscript; A.I.: conception and design, data analysis andinterpretation, final approval of manuscript; M.S.: conception and design, dataanalysis and interpretation, manuscript writing, financial support, administrativesupport, final approval of manuscript; C.B.: conception and design, provision ofstudy material, collection of data, data analysis and interpretation, manuscriptwriting, administrative support, final approval of manuscript.

FundingThis work was supported by Deutsche Forschungsgemeinschaft [grant numberSCHA 185/29-3, 4 to M.S.].

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.137612/-/DC1

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