1268 Research Article

IntroductionPostnatal neurogenesis in mammals persists in two brain regions:the subgranular zone (SGZ) of the dentate gyrus in the hippocampusand the subventricular zone (SVZ) of the lateral ventricles (Lledoet al., 2006; Ninkovic and Gotz, 2007; Zhao et al., 2008). Underpathological conditions, new neuroblasts also migrate into injuredcortex and striatum (Kreuzberg et al., 2010; Parent et al., 2002).Neural stem cells (NSCs) in the SGZ and SVZ divide slowly andare preserved throughout life. Little is known about the cellularsignaling that guarantees that NSCs are maintained in anundifferentiated state. Postnatal NSCs divide asymmetrically anddifferentiate into transit-amplifying precursors, which are fast-dividing cells that, in turn, give rise to more differentiatedneuroblasts (Abrous et al., 2005; Lledo et al., 2006; Zhao et al.,2008). Neuroblasts in the hippocampal SGZ migrate into thegranule cell layer of dentate gyrus and integrate into previouslyestablished neural circuits, where they are thought to play a role incertain forms of hippocampal-dependent learning and memory(Aimone et al., 2009; Kempermann et al., 2004). Neuroblastsoriginating in the SVZ migrate over long distances via the rostralmigratory stream (RMS) to the olfactory bulb, where they matureinto granule or periglomerular neurons and contribute to olfactoryinformation processing (Alonso et al., 2006; Lledo and Saghatelyan,2005).

Ephrins and their Eph tyrosine kinase receptors are importantregulators of many developmental processes (Klein, 2004; Pasquale,2005). Receptors and ligands alike are grouped into two subclasses:type A and type B. Although, in general, ephrin A ligands bind toEphA receptors and ephrin B ligands to EphB receptors, cross-specificity has been described for some members (Klein, 2004;Pasquale, 2004). Several ephrins and their Eph receptors regulatedifferent stages of neurogenesis and are differentially expressed ondistinct cell types in the neurogenic niches (Chumley et al., 2007;

Conover et al., 2000; Depaepe et al., 2005; Furne et al., 2009;Holmberg et al., 2005; Jiao et al., 2008; Qiu et al., 2008; Ricard etal., 2006). For instance, ephrins B2 and B3 and their receptorEphB1 cooperatively regulate proliferation and survival of neuralprogenitor cells and migration of neuroblasts in the SVZ/RMS andSGZ (Chumley et al., 2007; Conover et al., 2000; Ricard et al.,2006). Two other Eph receptors, namely, EphB2 and EphB3, haveopposite function in the adult SVZ. Whereas EphB2 inducesproliferation in the SVZ (Katakowski et al., 2005), EphB3suppresses progenitor cell proliferation via a p53-dependentmechanism (Theus et al., 2010). In the SVZ, the complementaryexpression of ephrin A2 in transit-amplifying cells and neuroblastsand that of EphA7 on ependymal cells and NSCs controls neuralprogenitor cell proliferation (Holmberg et al., 2005). In anotherstudy, EphA7 was shown to be involved in the modulation ofapoptosis of neural progenitors during embryonic development(Depaepe et al., 2005). Also, ephrin B1 has been demonstrated tocontribute to the maintenance of NSCs in an undifferentiated state(Qiu et al., 2008).

We recently described the generation of transgenic mice inwhich EGFP (enhanced green fluorescent protein) is expressed inthe entire RMS (Inta et al., 2008), and optimized a procedure forRNA isolation from in vivo fluorescent RMS neuroblasts(Khodosevich et al., 2007). The RMS is a robust and hence anideal structure in the postnatal brain that allows the harvesting ofsufficient cells to perform microarray analysis. Using transgenic5HT3A-EGFP mice (where EGFP is expressed under the controlof the serotonin receptor 3A promoter) with clearly EGFP-labeledRMS, we isolated neuroblasts from two distinct locations: one inthe immediate vicinity of the SVZ (posterior RMS, pRMS), andthe other more rostral, closer to the bulb (anterior RMS, aRMS)(Khodosevich et al., 2009). It is safe to assume that upregulatedgenes in cells from the aRMS are most probably involved in

SummaryIn the postnatal brain, new neurons continue to be generated in two neurogenic areas, the subventricular zone of the lateral ventricles(SVZ) and the subgranular zone of the hippocampus. There is evidence that ephrins and their Eph receptors belong to a signalingnetwork that regulates neurogenesis. On the basis of previous data, we have identified Eph receptor A4 (EphA4) as a potential regulatorof neurogenesis. We showed by immunohistochemistry that in adult neurogenic niches EphA4 is expressed only by neural stem cells(NSCs). Using in vitro and in vivo assays, we demonstrated that EphA4 expression maintains NSCs in an undifferentiated state.Specifically, in neurosphere cultures Epha4 knockdown resulted in a decrease of NSC proliferation and premature differentiation. Inpostnatal and adult brain, Epha4 knockdown caused a decrease in NSCs in the SVZ, eventually resulting in a reduced number ofpostnatally generated neuroblasts. Both in vitro and in vivo effects were rescued by co-infection with a modified EphA4 that wasresistant to Epha4 shRNA.

Key words: Differentiation, Ephrins, Neural stem cells, Postnatal neurogenesis

Accepted 22 November 2010Journal of Cell Science 124, 1268-1279 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.076059

EphA4 preserves postnatal and adult neural stemcells in an undifferentiated state in vivoKonstantin Khodosevich1,2,*, Yasuhito Watanabe1,2 and Hannah Monyer1,2

1Department of Clinical Neurobiology, Heidelberg University Medical Center, 69120 Heidelberg, Germany2Department of Clinical Neurobiology/A230, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany*Author for correspondence (k.khodosevich@dkfz-heidelberg.de)

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migration and differentiation, whereas upregulated genes in cellsfrom the pRMS that is closer to the SVZ might affect cellgeneration.

We identified Epha4, the gene coding for the Eph receptor A4,as one of the upregulated genes in the pRMS. The upregulationwas comparable with that of other genes known to be expressed inNSCs (e.g. GFAP, Nr2e1, Nes). Epha4 mRNA expression levelswere below background expression in samples collected from theaRMS or from the periglomerular region of the olfactory bulb.

EphA4 plays an important role in the developing and postnatalbrain, including axon guidance (Egea et al., 2005; Gallarda et al.,2008; Wegmeyer et al., 2007), synaptic plasticity (Bourgin et al.,2007; Deininger et al., 2008; Fu et al., 2007; Inoue et al., 2009)and proliferation of precursor cells during cortical neurogenesis(North et al., 2009). Furthermore, expression of Epha4 was foundin the adult SVZ (Conover et al., 2000; Liebl et al., 2003). Indeed,there is an increase in the number of neuroblasts in the SVZ andRMS in EphA4 knockout animals (Furne et al., 2009). Interestingly

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the number of proliferating cells in the SVZ (identified by Ki67expression) is not changed and there is a decrease in TUNEL-positive (i.e. apoptotic) cells in the adult SVZ in these animals(Furne et al., 2009; Ricard et al., 2006).

In this study we provide evidence that EphA4 is expressed inthe adult neurogenic niches exclusively by NSCs, but not by othercell types. In neurosphere cultures and in vivo, knockdown ofEpha4 expression caused reduction in cell proliferation andpremature differentiation of NSCs.

ResultsAnalysis of EphA4 expression in the postnatal brainTo establish the cellular expression of EphA4 in the postnatalbrain, we employed double- and triple-immunohistochemistry inbrain sections from 3-month-old animals. In both adult neurogenicniches, the SVZ and SGZ, EphA4 co-localizes with GFAP (Fig.1A–C). Furthermore, GFAP–nestin double-positive cells that arebona fide NSCs, at least in the SGZ (Lledo et al., 2006; Zhao et

Fig. 1. EphA4 is expressed inpostnatal NSCs of the SVZ andSGZ, but not in transit-amplifyingprogenitors and neuroblasts.(A)EphA4 is expressed in GFAP-positive NSCs along the lateralventricle. (B)EphA4 is expressed innestin and GFAP-double positiveNSCs in the SVZ. Arrowhead labels aGFAP/nestin/EphA4-triple positivecell that is magnified on the right sideshowing two stacks 2-m apart.(C,C�) EphA4 is expressed in SGZradial glia-like cells. (D)EphA4 isexpressed in Sox2-positive cells.(E,F)EphA4 is expressed in slow-proliferating cells (F), but is notexpressed in fast-proliferating cells(E). In E, the arrowhead indicatesBrdU-negative magnified cell shownon the right. Lv, lateral ventricle. Scalebars: 20m (A,B,E) and 10m(C,D,F).

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al., 2008), express EphA4 (Fig. 1B for SVZ; Fig. 1C,C� forSGZ). In SVZ, ependymal cells also exhibit coexpression ofGFAP and nestin (Doetsch et al., 1997). However, EphA4expression does not co-localize with the ependymal cell markerS100 (supplementary material Fig. S1). Because the EphA4antibody almost exclusively labels cell processes, EphA4 co-labeling with other nuclear or cell body markers was determinedby analyzing series of confocal stacks (supplementary materialFig. S2A). To avoid cross-reactivity between secondaryantibodies, we used rabbit anti-EphA4, mouse anti-GFAP andchicken anti-nestin antibodies (see Materials and Methods).Furthermore, stainings on separate sections with anti-EphA4and GFAP antibodies resulted in similar labeling patterns(supplementary material Fig. S2B,C). EphA4-positive cellscoexpress Sox2, another neural stem cell marker (Fig. 1D andsupplementary material Fig. S3A–D).

EphA4 is not present in transit-amplifying precursors and glialprecursors or oligodendrocytes, as revealed by the lack ofcoexpression with Mash1 and Olig2, respectively (supplementarymaterial Fig. S4A,B). EphA4 is also not co-localized with theneuroblast markers DCX and PSA-NCAM (supplementary materialFig. S4C,D).

We analyzed the proliferating capacity of EphA4-positive cellstaking recourse to labeling with 5-bromo-2�-deoxyuridine (BrdU),a marker of dividing cells. After a single injection of BrdU, noneof the EphA4-positive cells retained BrdU (Fig. 1E). However,EphA4-positive cells were labeled when BrdU was administeredfor 1 week followed by 3 weeks with no treatment before sacrificingthe animals (Fig. 1F; supplementary material Fig. S3E–H), whichindicates that EphA4 is expressed in slow-dividing cells, but not infast-dividing cells.

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The phenotype of EphA4-positive cells in the SVZ issummarized in supplementary material Table S1.

EphA4 is involved in NSC proliferation and differentiationin vitroTo study the cellular function of EphA4 in NSCs of the postnatalSVZ, we conducted knockdown experiments using two Epha4short hairpin RNA (shRNA) constructs (shRNA Epha4-1 andshRNA Epha2) (see Fig. 2E; supplementary material Fig. S5) andanalyzed NSC proliferation in neurosphere cultures after Epha4knockdown. We used neurosphere cultures obtained from the SVZof P4 mice that were infected with retroviruses expressing redfluorescent protein and either control shRNA (scrambled) or oneof the two Epha4 shRNAs. After 5 days in culture, BrdU wasadded to the culture media and cells were fixed after another 12hours. In the presence of either of the two Epha4 shRNAs, therewas a significant decrease in the number of infected proliferatingcells in neurospheres (green and red double-positive cells in Fig.2A,B, quantification in Fig. 2D). Details regarding cell counts inneurospheres are described in Materials and Methods. Knockdownof Epha4 in neurospheres was already pronounced at 4 days invitro (DIV4) (supplementary material Fig. S6A,B) and by DIV5was at most 25% of that in control neurospheres (see Fig. 2E forwestern-blot analysis and supplementary material Fig. S6C,D forimmunocytochemistry).

The effect of Epha4 knockdown was completely rescued bycoexpression of a modified EphA4, containing three silentmutations that make it resistant to shRNA Epha4-1 (Fig. 2C, seedouble-infected BrdU-positive cells). To visualize the EphA4mutant it was linked to copGFP via the T2A peptide sequence (fordetails on copGFP detection see Materials and Methods).

Fig. 2. EphA4 is involved in NSC proliferation in vitro. (A–D) Neurospheres were prepared from the SVZ of P4 animals and were infected with retrovirusesexpressing tdTomato and control shRNA (scrambled) (A), shRNA Epha4-1 (B) or Epha4-2, and shRNA Epha4-1 together with a modified Epha4 resistant toEpha4-1 (C). After 5 days, BrdU was added to the culture media. Fixation and staining ensued 12 hours later. Arrowheads indicate BrdU-positive infected cells;arrows indicate BrdU-negative infected cells. (D)Both Epha4 shRNA-expressing viruses caused a significant decrease in the number of infected proliferative cellscompared with control or rescue conditions (*P<0.01, n6). In A and B red fluorescent BrdU-positive cells were counted, and in C red and magenta double-fluorescent BrdU-positive cells were counted. (E)Western-blot analysis of EphA4 expression in neurospheres after infection with different retroviruses. BothEpha4 shRNAs knocked down EphA4 expression by at least 75%. Scale bars: 20m.

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Next, we investigated the effect of Epha4 knockdown onprecursor cell maintenance in neurospheres. We cultured infectedneurospheres, and after 6 days we transferred them into medialacking growth factors. After 2 days following growth factorremoval, Epha4 knockdown caused a prominent decrease in thepercentage of nestin-positive cells relative to the total number ofinfected cells (Fig. 3A–C). After 4 days of incubation in mediawithout growth factors, the percentage of astrocytes (i.e. numberof GFAP-positive infected cells relative to the total number ofinfected cells) was much higher in neurospheres expressing Epha4shRNAs than in control neurospheres (Fig. 3D–F). Whereas, invivo, GFAP labels NSCs and astrocytes, in neurospheres GFAPonly labels astrocytes. Nestin- and GFAP-positive cells do notoverlap in neurosphere cultures (supplementary material Fig. S7)(see also Campos, 2004; Campos et al., 2004). Although GFAPlabels cells in the center of neurospheres (mature astrocytes), nestinlabels precursor cells at the periphery. Using another marker formature astrocytes, namely S100, we detected a similar increasein infected astrocytes (relative to the total number of infected cells)after Epha4 knockdown as that observed in experiments whereGFAP stainings were performed (Fig. 3J–L). Also, in experimentsin which differentiation was assessed using the neuronal markerTuj1, there was an increase in differentiated cells in conditions ofEpha4 knockdown (Fig. 3G–I). The Epha4 knockdown phenotypecould be rescued when modified EphA4 was coexpressed, whereasa kinase-dead EphA4 mutant was not able to rescue the Epha4knockdown (Fig. 3C,F,I,L).

To confirm that knockdown of Epha4 indeed affects NSCdifferentiation, we analyzed the formation of secondaryneurospheres derived from primary neurospheres infected byretroviruses expressing control or Epha4 shRNAs. The formationof primary neurospheres was not affected by Epha4 knockdown,except for the slightly smaller average size (data not shown) thatwas most probably due to a decrease in the number of infectedproliferating cells (see Fig. 2A–D). However, the formation ofsecondary neurospheres was dramatically reduced after Epha4knockdown in primary neurospheres (Fig. 3M–O). Each secondaryneurosphere is a descendant of a single cell from a primaryneurosphere. Thus, it can be inferred that, after Epha4 knockdownin primary neurospheres, a decrease in the number ofundifferentiated precursor cells would result in fewer secondaryneurospheres. Conversely, the number of non-infected secondaryneurospheres was similar for control and Epha4 knockdownconditions (Fig. 3P). Thus, in neurosphere cultures, Epha4knockdown accelerates NSC differentiation, which might accountat least in part for the observed decrease in proliferation.

EphA4 is involved in NSC proliferation of the postnataland adult SVZ in vivoTo extend the results obtained in vitro, we silenced EphA4expression in the postnatal SVZ of wild-type mice using lentivirusescontaining shRNAs against Epha4. We injected lentivirusesexpressing EGFP and control or Epha4 shRNAs into the SVZ ofpostnatal day 6 (P6) mice (for details on viral titers see Materialsand Methods) that were sacrificed at 4, 7, 15, 25, 40, 60, 80 and100 days post-injection (Fig. 4). Because the number of infectedcells in the SVZ can vary from one injection to another (even whenthe viral titer is constant), data were normalized and expressed asrelative to the number of all infected cells in the SVZ (for furtherdetails on the quantification index see Materials and Methods).There was no difference in the number of migrating RMS

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neuroblasts when comparing control and Epha4 knockdown miceat any time point up to 25 days post-injection. However, at 40 dayspost-injection and later there was a dramatic decrease of infectedneuroblasts in the RMS (Fig. 4A,B,D) of Epha4 knockdownanimals. Concomitantly, there was a reduction in the number ofinfected neurons in the olfactory bulb of Epha4 knockdown animalsat 80 days post-injection and later (Fig. 4E,F).

Interestingly, although the effect of Epha4 knockdown could befirst detected only as late as 40 days post-injection, the decrease ofEphA4 expression levels around the SVZ was already visible 3days post-injection (supplementary material Fig. S8A,B) and wasmore pronounced along the whole SVZ at 6 days post-injection(supplementary material Fig. S8C–E).

For rescue experiments in vivo, we used lentivirus expressing amodified EphA4 that contains three silent mutations, making itresistant to shRNA Epha4-1. Cells infected with the ‘rescue’ viruscan be visualized by copGFP expression whereas infection withthe shRNA Epha4 virus is monitored by EGFP expression. Toobtain a significant number of double-infected cells, we used afivefold higher titer for the ‘rescue’ virus. Co-injection into theSVZ of shRNA Epha4-1 lentivirus (also expressing EGFP, green)together with lentivirus expressing modified EphA4 (also positivefor copGFP, magenta) rescued the Epha4 knockdown effect (Fig.4C,D,F). At 40 days post-injection and later, most RMS neuroblastsexpressing shRNA Epha4-1 also contained the ‘rescuing’ modifiedEphA4 virus (Fig. 4C). Furthermore, at 80 days post-injection andlater, there was a significant increase in the number of cells double-infected by Epha4 knockdown and ‘rescue’ lentiviruses incomparison with the control (i.e. gain-of-function effect) (Fig. 4D).Cells infected by the virus expressing the modified EphA4 did notexhibit morphological alterations.

We determined the number of proliferating cells in the SVZ byadministering two BrdU pulses to label dividing cells(supplementary material Fig. S9A–C). Although at 4 and 25 dayspost-injection there was no difference between the numbers ofinfected proliferating cells in control and Epha4 knockdownconditions, at 40 days post-injection there was a 50% reduction inthe number of BrdU-positive infected cells around the SVZ inEpha4 knockdown animals.

The reduction of infected neuroblasts in the RMS after Epha4knockdown cannot be explained by a change in the number ofapoptotic cells in the SVZ, as indicated by the unaltered caspase-3 activation (supplementary material Fig. S9D–F). Thus, both invitro and in vivo experiments provided evidence that Epha4knockdown resulted in a decrease of precursor cell proliferationthat was not a consequence of altered survival. The observed effectof EphA4 on NSCs is cell autonomous and is mediated byintracellular signaling of EphA4 because it occurs directly in theinfected cells. Also, a kinase-dead EphA4 mutant (with a lack offorward signaling) was not able to rescue the Epha4 knockdownphenotype (Fig. 4D).

The immunohistochemistry results indicated that EphA4 isexpressed only in NSCs, but not in transit-amplifying progenitorsor neuroblasts (Fig. 1). Hence, we expect that the knockdown ofEphA4 forward signaling (in the same cell expressing EphA4)decreases proliferation of NSCs but not of other cell types in SVZ.To this end, we analyzed the effect of Epha4 knockdown in theSVZ using retroviruses known to infect only dividing cells. Incomparison with neurosphere cultures where NSCs divide fast(Campos, 2004; Qian et al., 1998), in postnatal SVZ the NSCsdivide very slowly (Doetsch et al., 1999; Morshead et al., 1994;

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Fig. 3. EphA4 is involved in NSC differentiation in vitro. (A–L) Neurospheres were prepared from the SVZ of P4 animals and infected by retrovirusesexpressing tdTomato and control shRNA (scrambled) (A,D,G,J), shRNA Epha4-1 (B,E,H,K) or Epha4-2. In rescue experiments, neurospheres were infected byshRNA Epha4-1 expressing virus (Ep1) together with another virus expressing either modified EphA4 resistant to Epha4-1 (Epha4 mut) or EphA4 kinase-dead(KD) mutant (C,F,I,L). After 6 days, growth factors from the culture medium were removed. Cultures were analyzed 2 days later for nestin- (A–C) and 4 days laterfor GFAP- (D–F), Tuj1- (G–I) or S100- (J–L) positive infected (double-infected for rescue experiments) cells. Arrowheads indicate infected cells that are positivefor the specific marker; arrows indicate infected cells that are negative for the specific marker. Infection with any of the two Epha4 shRNA-expressing virusesresulted in a significant decrease in the number of infected nestin-positive cells (C) and an increase in the number of infected GFAP-positive (F), Tuj1-positive (I)and S100-positive (L) cells compared with control or rescue conditions (*P<0.01, n6). Kinase-dead EphA4 mutant was not able to rescue the Epha4 knockdownphenotype. Note that image panels do not necessarily reflect quantitative differences, but rather qualitative differences. (M–P) Primary neurospheres were infectedby retroviruses expressing control or Epha4 shRNAs and after 6 days secondary neurospheres were prepared. In comparison with control condition (M), afterEpha4 knockdown (N) there were fewer secondary neurospheres derived from infected cells in primary neurospheres (O) (*P<0.001, n6). Note that the number ofsecondary neurospheres derived from non-infected cells in primary neurospheres was comparable between control and Epha4 knockdown (P). Scale bars: 20m(A–K) and 100m (M,N).

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Morshead and van der Kooy, 2004). Indeed it has been establishedthat the majority of infected cells in the SVZ after retrovirusinjection are fast-dividing transit-amplifying precursors orneuroblasts, but not slow-dividing NSCs (Rogelius et al., 2005).Thus, we injected retroviruses expressing tdTomato (a redfluorescent protein) and either control shRNA or shRNA Epha4-1into the SVZ of P6 mice, and determined the number of infectedcells in the RMS or olfactory bulb relative to the total number ofinfected cells in the SVZ at 5, 8, 12, 18, 25, 40 and 60 days post-

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injection (Fig. 5A–C). There was no difference in the number ofinfected cells in the RMS (Fig. 5C) or olfactory bulb (Fig. 5B)between control and Epha4 knockdown mice at any time point.Also, we detected only one or two infected neuroblasts in the RMSper animal at 18 days post-injection and no infected neuroblasts at40 days post-injection and later (Fig. 5C). The number of BrdU-positive infected cells in the SVZ, labeled by two short BrdUpulses, was not different between Epha4 knockdown and controlconditions (Fig. 5D).

Fig. 4. EphA4 affects proliferation of SVZ NSCs in vivo. (A–F) P6 animals were infected with lentiviruses expressing EGFP and control shRNA (scrambled),shRNA Epha4-1, shRNA Epha4-2, shRNA Epha4-1 together with a modified EphA4 resistant to Epha4-1 or the same modified EphA4 containing a ‘kinase-dead’domain. Animals were sacrificed 4, 7, 15, 25, 40, 60, 80 and 100 days post-injection. There were significantly more infected migrating cells in the RMS at 40 dayspost-injection and later in animals expressing the control and rescue constructs (A and C, respectively), compared with animals expressing shRNA Epha4-1 (B) orshRNA Epha4-2 (D). Kinase-dead EphA4 mutant was not able to rescue Epha4 knockdown phenotype (D). Neuroblasts in the RMS were visualized by stainingwith doublecortin (DCX) antibodies. Note that most shRNA Epha4-1-expressing RMS neuroblasts (green) in C are co-infected with the modified EphA4 resistantto Epha4-1 expressing virus (magenta). Examples are shown in C1–C3. The decrease in the number of infected RMS neuroblasts (D) and olfactory bulb neurons(E,F) is visible only as late as 40 and 80 days post-injection, respectively, indicating that Epha4 knockdown affects the proliferation of NSC that are slow-dividingcells (*P<0.05, n≥6). Scale bars: 40m (A–C) and 100m (E).

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Finally, we extended our in vivo injection experiments to adultanimals and injected the SVZ of 3-month-old mice with lentivirusesexpressing EGFP together with either control shRNA or one of theEpha4 shRNAs. Animals were sacrificed at 4, 25, 40 and 60 dayspost-injection (Fig. 6). Although there was no difference in thenumber of infected cells in the RMS of control and Epha4knockdown animals at 4 and 25 days post-injection, at 40 dayspost-injection and later the number of neuroblasts in the RMS wassignificantly decreased after Epha4 knockdown (Fig. 6A–C). Thus,knockdown of Epha4 in adults resulted in the same phenotype asthat observed after injections in P6 animals.

EphA4 is involved in NSC differentiation of the postnataland adult SVZ in vivoWe subsequently tested in vivo whether, in addition to proliferation,EphA4 also affected NCS differentiation, as was suggested by the

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neurosphere experiments. When restricting the analysis to the areaaround the lateral ventricles, there was a significant decrease in thenumber of GFAP-positive cells following Epha4 knockdown (Fig.7A–D), which was already apparent at 15 days post-injection (Fig.7D; see supplementary material Fig. S10A,B for representativeimages when using a higher viral titer). In fact, the decrease in NSCsin the knockdown condition might even be an underestimation if oneconsiders that some GFAP-positive cells in the area analyzed mightbe astrocytes. Similar results were obtained when Sox2 and nestinwere used as NSC markers (Fig. 7E–I; for nestin see alsosupplementary material Fig. S10C,D for representative images wheninjecting a higher viral titer). Thus, at 4 days post-injection thenumber of Sox2-positive infected cells was comparable for allconditions, whereas after 15 days we observed a significant decreasein the Sox2-positive cell number in Epha4 knockdown animals incomparison with control or rescue animals.

Fig. 5. Epha4 knockdown in fast-dividing cells does not affect SVZprogenitor proliferation in vivo.(A–C)P6 animals were infected withretroviruses expressing tdTomato andcontrol shRNA (scrambled) or shRNAEpha4-1 and sacrificed 5, 8, 12, 18, 25,40 and 60 days post-injection.Arrowheads mark infected cells. Epha4knockdown in fast-dividing cells(transit-amplifying progenitors andneuroblasts) did not result in a decreaseof infected cells in the olfactory bulb(B) or RMS (C) (n≥6). (D)P6 animalswere infected with retroviruses asdescribed above and received two BrdUinjections 24 hours prior to perfusion.There was no difference in BrdU-positive infected cells in the SVZbetween control and Epha4 knockdownconditions at any time-point analyzed(n6). GCL, granule cell layer, GL,glomerular layer, epl, externalplexiform layer. Scale bars: 100m.

Fig. 6. EphA4 affects proliferation of adult SVZ NSCs in vivo. (A–C) Three-month-old animals were infected with lentiviruses expressing EGFP and controlshRNA (scrambled), shRNA Epha4-1 or shRNA Epha4-2. Animals were sacrificed 4, 25, 40 and 60 days post-injection. There were significantly more infectedcells migrating through the RMS 40 days post-injection and later in control animals (A), compared with animals expressing either of the two shRNA Epha4 (B,C)(*P<0.05, n6). Neuroblasts in the RMS were visualized by staining with doublecortin (DCX) antibodies. Scale bars: 40m (A,B); insets: 20m.

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Fig. 7. Epha4 knockdown contributes to premature differentiation of SVZ NSCs in vivo. (A–O) P6 animals were infected with lentiviruses expressing EGFPand control shRNA (scrambled), shRNA Epha4-1, shRNA Epha4-2, or shRNA Epha4-1 together with a modified EphA4 resistant to Epha4-1. Animals weresacrificed 4, 7, 15, 25 and 40 days post-injection. All images were acquired along the lateral ventricles. (A–C) There were significantly more GFAP-positiveinfected cells around the lateral ventricles relative to the total number of infected SVZ cells in animals infected with the control and rescue (arrowheads) viruses(A and C, respectively), compared with animals expressing shRNA Epha4-1 (B) or shRNA Epha4-2 (D). (D)A significant decrease in the number of GFAP-positive infected cells (the majority of which are NSCs) around the lateral ventricles in Epha4 knockdown animals, could be detected from 15 days post-injectiononwards (*P<0.05, n≥6). (E,F)A similar decrease in Sox2-positive infected cells (many of which are NSCs) around the SVZ after Epha4 knockdown was observedstarting from 15 days post-injection onwards (*P<0.05, n10). White and orange arrowheads mark Sox2-positive and Sox2-negative infected cells, respectively.(G–I) The number of nestin-positive (arrowheads) infected cells (many of which are NSCs) around the lateral ventricles in Epha4 knockdown animals (H) wassignificantly decreased 40 days post-injection in comparison with control (G) or rescue animals (I) (*P<0.01, n10). (J–L) The number of Mash1-positive(arrowheads) infected cells (transit-amplifying precursors) around the lateral ventricles in Epha4 knockdown animals (K) was significantly decreased 40 days post-injection in comparison with control (J) or rescue animals (L) (*P<0.01, n≥6). (M–O) Conversely, the number of NeuN-positive (arrowheads) infected cells (i.e.mature neurons) around the lateral ventricles in Epha4 knockdown animals (N) was significantly increased 40 days post-injection in comparison with controls (M)(*P<0.01, n10). Note that the magnified cell in N is rotated by 45° in N1. Scale bars in all panels except M and N are 20m; scale bars in M,N are 50m.

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A decrease in the number of NSCs would eventually result in adecreased number of their progeny cells. Indeed, there was adecrease in the number of transit-amplifying precursors andneuroblasts around the SVZ, as revealed by immunolabeling withMash1 and DCX, respectively. At 40 days post-injection, thepercentage of Mash1-positive infected cells in Epha4 knockdownanimals was half that of controls (Fig. 7J–L). The decrease inDCX-positive infected cells around the SVZ corresponded to thedecrease in infected neuroblasts in RMS (see Fig. 4D).

If Epha4 knockdown reduces the number of cells expressingstem cell markers and drives direct differentiation, one wouldexpect an increase in neuronal markers in the knockdown cells.Indeed, this was the case. However, to test this, an injection sitelocated slightly more dorso-medially was used to avoid the infectionof many striatal neurons that occurred when injections were closeto the lateral ventricles. When using these injection coordinates,Epha4 knockdown resulted in an increase of NeuN-positive infectedcells around the SVZ in comparison with control animals at 40days post-injection (Fig. 7M–O).

A similar approach was used to analyze cell differentiation inthe SVZ using 3- to 4-month-old mice (supplementary materialFig. S11). Animals were injected into the SVZ with lentivirusesexpressing EGFP together with either control shRNA or one of theEpha4 shRNAs and sacrificed at 4, 15 and 40 days post-injection.Similar to early postnatal injections, Epha4 knockdown decreasedthe number of GFAP- (supplementary material Fig. S11A–C),BrdU- (supplementary material Fig. S11D–F) and Mash1-positive(supplementary material Fig. S11G–I) infected cells around theSVZ. Conversely, there was an increase in NeuN-positive cells(supplementary material Fig. S11J–M).

Finally, we performed BrdU-retaining experiments to directlydemonstrate that Epha4 knockdown indeed reduces the number of

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slow-dividing cells. The SVZ of P6 or adult animals was injectedwith lentiviruses expressing EGFP together with either controlshRNA or one of the Epha4 shRNAs. To label slow-dividing cells,at 40 days post-injection BrdU was administered in the drinkingwater for 1 week followed by 3 weeks with no treatment beforesacrificing the animals. Both early postnatal (Fig. 8A–C) and adult(Fig. 8D–F) Epha4 knockdown animals showed a 50% reductionin the number of infected slow-dividing cells around the SVZcompared with controls. Thus, several lines of evidence indicatethat Epha4 expression preserves NSCs in an undifferentiated state.

DiscussionEphrins and their Eph receptors regulate many aspects of neuralsystem development during embryogenesis (Klein, 2004; Pasquale,2005). They also participate in the control of neurogenesis in bothpostnatal neurogenic regions, the SVZ of the lateral ventricles, andthe hippocampal SGZ (Chumley et al., 2007; Conover et al., 2000;Holmberg et al., 2005; Jiao et al., 2008; Qiu et al., 2008; Ricard etal., 2006). In the SVZ, ephrin signaling has been shown to controlprogenitor cell proliferation (Chumley et al., 2007; Conover et al.,2000; Katakowski et al., 2005; Ricard et al., 2006; Theus et al.,2010) and apoptosis of excessively generated cells (Holmberg etal., 2005; Jiao et al., 2008). In this study, we have shown that Ephreceptor A4 is expressed specifically in NSCs, and is necessary topreserve NSCs in an undifferentiated state. Interaction of Ephreceptors with ephrins activates both forward signaling (in the cellwhere the receptor is expressed) and reverse signaling (in the cellexpressing the ephrin ligand) (Egea and Klein, 2007). The effectsdescribed here following Epha4 knockdown occurred in cells thatwere infected by viruses expressing shRNA Epha4, and it can bethus inferred that EphA4 preserves NSC fate via forward signaling.This is also supported by the results showing that a ‘kinase-dead’

Fig. 8. Epha4 knockdown decreases the number of slow-dividing cells. (A–C) P6 animals were infected with lentiviruses expressing EGFP and control shRNA(scrambled), shRNA Epha4-1 or shRNA Epha4-2. At 40 days post-injection animals received BrdU in the drinking water for a week followed by 3 more weeks withnormal water consumption. Animals were sacrificed and analyzed for BrdU-retaining in infected cells. All images were acquired along the lateral ventricles. There weresignificantly more BrdU-retaining infected cells (arrowheads) around the lateral ventricles relative to the total number of infected SVZ cells in animals infected with thecontrol virus (A) compared with animals expressing Epha4 shRNAs, (B,C) (*P<0.01, n10). Note that magnified cell in A is rotated by 45° in A1. (D–F) Similarresults showing a decrease in the number of BrdU-retaining cells in the SVZ after Epha4 knockdown in adult animals (*P<0.01, n10). Scale bars: 20m.

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mutant of EphA4 was not able to rescue the Epha4 knockdownphenotype. EphA4 function is not restricted to the early postnatalSVZ, but continues to be important in the adult SVZ.

The lack of effect on neurogenesis following Epha4 knockdownusing retroviruses (that generally infect fast-dividing cells)(Rogelius et al., 2005), together with the observed decrease inRMS neuroblasts only as late as 40 days post-injection usinglentiviruses, establish a functional role of EphA4 signaling inNSCs, but not in other proliferative cell types in SVZ. PostnatalNSCs are quiescent for long periods of time and divide rarely,about once in 2 weeks (Doetsch et al., 1999; Morshead et al., 1994;Morshead and van der Kooy, 2004). Thus, a decrease in NSCproliferation will be reflected in the number of their progeny onlyseveral weeks later. Also, immunohistochemical data confirm thepresence of EphA4 in NSC, but not in precursor cells andneuroblasts. The facts that neuroblast morphology in Epha4knockdown mice was comparable with controls and that the numberof neuroblasts was reduced only at 40 days post-injection, togetherwith unaltered neuroblast number after retroviral injections, speakagainst a scenario in which EphA4 is involved in migration.

The acceleration of NSC differentiation can most probablyaccount for the observed decrease in their proliferation. Thisconjecture is supported by several observations. The population ofNSCs, indicated by GFAP-, Sox2- and nestin-positive staining,remained relatively constant in control animals (Fig. 7D,F,I). Theseresults are in line with previous reports that postnatal NSCs divideasymmetrically (Morshead et al., 1998; Suh et al., 2007), eachNSC generating one NSC and one precursor cell, thus explainingthe constancy of the NSC pool. Hence, a decrease in NSCproliferation would not change their number. Therefore, asignificant reduction in NSC population after Epha4 knockdown isthe result of premature differentiation rather than that ofproliferation impairment. Because we did not observe an earlyincrease in the number of transit-amplifying precursors andneuroblasts (as well as an overall proliferation increase), it ispossible that NSCs, upon Epha4 knockdown, are enforced todifferentiate directly to non-dividing cells (e.g. mature neurons)omitting intermediate phenotypes such as transit-amplifyingprecursor cells and neuroblasts. This is in line with the in vitroresults where we observed an increase in the number of matureastrocytes and neurons together with a slight decrease in the totalcell number after Epha4 knockdown. Interestingly, EphA signalingthrough other two members of EphA family, EphA2 and EphA3,was found to be involved in pre- and postnatal neural precursorcell differentiation towards the neuronal lineage (Aoki et al., 2004).

Potential ligands for EphA4 have been identified in other systemsor during embryonic development. Ephrin B1 has been shown tobe required for the maintenance of neural progenitor cell state inthe ventricular zone of the developing embryonic cortex (Qiu etal., 2008). The interaction of EphA4 with ephrin B1 in embryonicNSCs of the ventricular zone in rodents also lends credence to aproposed function for cortical development (North et al., 2009).Another ligand for EphA4 might be ephrin B3, which was shownto interact with Epha4 in HEK293 cells (Furne et al., 2009; Ricardet al., 2006). After interaction with ephrin B3, EphA4 propagatesreverse anti-apoptotic signal to cells expressing ephrin B3, whereasin cells lacking ephrin B3, the reverse signal induces apoptosis(Furne et al., 2009).

Involvement of EphA4 in neurogenesis was also indicated in astudy reporting a significant increase in the SVZ and RMS areasin Epha4 knockout animals (Furne et al., 2009). Using knockout

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mice, however, precludes evaluation of the role of EphA4 inneurogenic cells because EphA4 is expressed in many non-neurogenic cells, such as mature astrocytes, neurons and endothelialcells (Deininger et al., 2008; Goldshmit et al., 2004; Goldshmit etal., 2006; Tremblay et al., 2009). The use of low-titer lentivirusesinfecting sparsely distributed cells allows functional studies ofEphA4 forward signaling specifically in infected cells. Furthermore,EphA4 plays an important role during embryonic braindevelopment; thus, prenatally derived changes in brain structureand activity of full Epha4 knockout animals can modify postnatalbrain function, an effect that is avoided when employing postnatalviral injections.

Postnatal neurogenic niches contain several cell types known todifferentially express specific ephrins and/or Eph receptors. Giventhe redundancy of ephrin–receptor interaction (individual ephrinscan interact with several Eph receptors and vice versa), ephrinsignaling in neurogenic niches is mediated by complex intracellularnetworks, most probably involving distinct pathways for certainligand–receptor interactions and common pathways for others. Tobetter understand the role of ephrin signaling for postnatalneurogenesis, it is thus important to identify the expression ofdifferent members in the defined cell types in the neurogenicniches and to link members of the ephrin system with intracellularpathways.

Materials and MethodsAnimalsFor all our experiments, we used wild-type C57Bl/6 mice. All procedures withanimals were performed according to the Heidelberg University Animal CareCommittee.

Materials and reagentsAll chemicals and cell culture reagents were purchased from Sigma-Aldrich(Taufkirchen, Germany) and Invitrogen (Karlsruhe, Germany), respectively, unlessotherwise specified.

The following antibodies were used: polyclonal rabbit anti-EGFP, 1:10000(Invitrogen), chicken anti-EGFP, 1:1000 (Abcam, Cambridge, UK), mouse anti-IIIclass -tubulin, Tuj1, 1:500 (Covance, Princetown, NJ), goat anti-doublecortin,1:500 (Santa Cruz Biotechnology), rabbit anti-EphA4, 1:250 (Abcam), rabbit anti-EphA4, 1:200 (Zymed, San Francisco, CA), rabbit anti-copGFP, 1:3000 (Evrogen,Moscow, Russia), mouse anti-GFAP, 1:4000 (Sigma-Aldrich), rabbit anti-GFAP,1:2000 (Dako, Hamburg, Germany), rabbit anti-nestin, 1:500 (Abcam), mouse anti-nestin, 1:500 (BD Biosciences, Franklin Lakes, NJ), chicken anti-nestin, 1:500(Novus Biologicals, Littleton, CO), rat anti-BrdU, 1:500 (Accurate Chemicals,Westbury, NY), rabbit anti-activated caspase-3, 1:2000 (Chemicon, Temecula, CA),mouse anti-Mash1, 1:500 (BD Biosciences), rat anti-Ki67, 1:100 (Dako), rabbit anti-Sox2 (Santa Cruz Biotechnology), mouse anti-PSA-NCAM, 1:500 (Millipore,Schwalbach/Ts, Germany), goat anti-Olig2, 1:500 (R&D Biosystems), rabbit anti-S100, 1:1000 (Swant, Bellinzona, Switzerland), mouse anti-S100, 1:500(Millipore), Alexa-Fluor-488-conjugated anti-rabbit, anti-mouse, anti-rat, anti-chickenand anti-goat, Alexa-Fluor-350-conjugated anti-rabbit and anti-mouse secondaryantibodies (Invitrogen), anti-mouse, anti-rabbit, anti-rat and anti-goat Cy3 coupled,anti-mouse, anti-goat and anti-rabbit Cy5 coupled secondary antibodies (JacksonImmunoResearch Laboratories, Westgrove, PA), anti-mouse and anti-rabbit HRP-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA).

Plasmid cloningThe target sequences for oligos used to construct shRNA Epha4 expression plasmidswere 5�-GCAGCACCATCATCCATTG-3� for Epha4-1 and 5�-ATCCACCTGGAA -GGCGTTG-3� for Epha4-2. Scrambled shRNA sequences were cloned from pSilencervector (Ambion, Austin, TX). Complementary pairs of oligonucleotides were clonedinto pSUPER vector (Oligoengine, Seattle, WA). To make recombinant lentiviralplasmids for in vivo experiments, we re-cloned the shRNA silencing cassettes frompSUPER vector to pFUGW, a lentiviral vector containing EGFP expressed under thecontrol of the ubiquitin promoter (Lois et al., 2002). To generate retroviral plasmidsexpressing shRNAs, we re-cloned tdTomato (pRSET-tdTomato; generous gift ofRoger Tsien, University of California at San Diego, La Jolla, CA) and the CMVpromoter (from pEGFP-C1; Clontech-Takara Bio, Saint-Germain-en-Laye, France)into the pFB retroviral vector (Stratagene, La Jolla, CA), resulting in pFB-CMV-tdTvector. Two shRNA Epha4 cassettes were cloned from the pSUPER shuttle vectorinto the retroviral vector.

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pCMV-SPORT-EphA4 expression plasmid was purchased from Biocat (Heidelberg,Germany). To produce the EphA4 expression plasmid resistant to shRNA Epha4-1,we introduced into the Epha4 open reading frame (ORF) three silent mutations usingQuickchange Mutagenesis Kit (Stratagene), and cloned the modified Epha4 ORFinto the pCDH-EF1-T2A-copGFP (original plasmid from System Bioscience,Mountain View, CA) or pCDH-EF1-T2A-tdT (pCDH-EF1-T2A-copGFP where wesubstituted copGFP for tdTomato) lentiviral vector. To obtain retroviral plasmidcontaining modified EphA4, the whole EF1-modEphA4-T2A-copGFP cassette wasre-cloned into pFB retroviral vector.

CopGFP, a green fluorescent protein, was linked to ORF Epha4 through the T2Apeptide sequence. We were not able to detect the native fluorescent signal of copGFPwhen expressed under the control of the EF1 promoter (elongation factor 1), but therewas a bright copGFP signal in infected cells when stained with copGFP antibodies(supplementary material Fig. S12A–C). The T2A sequence allows the generation oftwo proteins expressed at the same level (Osborn et al., 2005). CopGFP antibodiesdo not cross-react with EGFP (supplementary material Fig. S12C), hence theexpression of both fluorescent proteins in the same cell can be detected unambiguously.

The kinase-dead EphA4 mutant was generated as described previously (Kullanderet al., 2001), exchanging lysine 653 with a methionine residue. The mutation wasintroduced into modified EphA4 resistant to Epha4-1 by Quickchange MutagenesisKit (Stratagene), and the kinase-dead Epha4 ORF was cloned into pCDH-EF1-T2A-copGFP (System Bioscience) lentiviral vector. To obtain retroviral plasmid containingkinase-dead EphA4, the whole EF1-EphA4KD-T2A-copGFP cassette was re-clonedinto pFB retroviral vector.

RNA isolation, cDNA synthesis and quantitative real-time PCRRNA isolation, cDNA synthesis and quantitative real-time (qRT)-PCR was performedas described previously (Khodosevich et al., 2007). mRNA levels detected by qRT-PCR were normalized to mRNA levels for Gapdh.

Analysis of shRNA silencing efficiencyThe efficiency of shRNA silencing was tested using qRT-PCR and western blotusing HEK293 cell culture transfections in triplicates. Two out of five shRNAs thatspecifically knocked down Epha4 gene expression to less than 25% were chosen forfunctional experiments described in the study. The efficiency of shRNA silencingwas also tested by viral infections of neurospheres and in vivo brain infections.

Production of recombinant virusesRecombinant lentiviruses were produced as previously described (Khodosevich etal., 2009). For retrovirus production we used the same conditions as for lentiviruses,except for using GP helper plasmid instead of 8.9 plasmid.

Measurement of viral titerTo measure viral titers, a dilution series across five orders of magnitude of viral stocksolutions were used for HEK293 cell infection. Each sample was analyzed intriplicate. After 4 days of incubation at 37°C, the number of fluorescent cell plaquesat the different viral dilutions was measured and the viral titer was estimated influorescent plaque forming units per milliliter (pfu/ml).

Neurosphere culturesSVZ regions were dissected from coronal sections of wild-type P4 mice. All stepsof tissue processing were done in dissection media (10� DM: 100 mM MgCl2, 10mM kynurenic acid, 100 mM HEPES in 1� Hank’s balanced salt solution). DissectedSVZ regions were incubated for 5 minutes with 30 U of papain (Worthington,Lakewood, NJ) and 0.0005% DNase solution, and washed in Neurobasal MediaSupplemented [500 ml of Neurobasal media + 10 ml 50� B27-Supplement + 1.25ml 200 mM L-glutamate + 5 ml penicillin/streptomycin (100 U/ml)] containing alsotrypsin inhibitor (Sigma-Aldrich) and 0.0005% DNAse. Cells were triturated usinga fire-polished Pasteur pipette, counted and plated at a density of 100,000 cells/mlin Neurobasal Media Supplemented containing 20 ng/ml EGF and 20 ng/ml FGF.To obtain secondary neurospheres, the primary neurospheres were dissociated bytrypsin incubation and subsequent trituration using a fire-polished Pasteur pipette.Dissociated cells were counted and plated at a density of 20,000 cells per ml inNeurobasal Media Supplemented containing 20 ng/ml EGF and 20 ng/ml FGF.

Proliferation analysis: at DIV1, cells were infected with shRNA-expressing viruseswith or without the additional presence of rescue virus. At DIV6, BrdU was addedto the culture media to give a final concentration of 10 M. Neurospheres wereincubated with BrdU for 12 hours and subsequently fixed.

Differentiation analysis: at DIV1, cells were infected with shRNA-expressingviruses with or without the additional presence of rescue virus. At DIV7, neurosphereswere transferred to Neurobasal Media Supplemented without growth factors, and atDIV9 (for nestin immunostaining) or DIV11 (for GFAP, S100 and Tuj1immunostainings) cultures were fixed.

Injection of recombinant viruses into mouse brainThe titer of the injected virus was adjusted so as to be equal for all experiments:2�107 units/ml for lentiviruses and 107 units/ml for retroviruses. For rescue studieswe used the same titer for shRNA expressing viruses: 2�107 units/ml, and 108

units/ml of rescue virus. We also confirmed the EphA4 rescue results using a fivefold

higher titer (resulting in a higher number of infected cells) for shRNA and rescueviruses (108 units/ml and 5�108 units/ml, respectively). Throughout the text, ‘low-titer’ for shRNA-expressing viruses indicates 2�107 units/ml, and ‘high-titer’ indicates108 units/ml. An aliquot of 1 l of recombinant retrovirus or lentivirus (or mix ofviruses) was delivered into the SVZ of each hemisphere of P6 C57BL/6 mouse pupswith a Hamilton (Hamilton, Bonaduz, Switzerland) syringe using special needles forprecise animal injections (reduced needle volume, 20 mm length, 26s gauge and 45°tip angle). The animals were sacrificed 5, 8, 12, 18, 25, 40 or 60 days after injectionfor retroviruses and 4, 7, 15, 25, 40, 50, 60, 80 or 100 days after injection forlentiviruses, and fluorescent cells in the olfactory bulb, RMS and SVZ were counted.

Adult (3-month-old) animals were injected as previously described (Celikel et al.,2007). The coordinates that were used for injections into the SVZ were: anterior, 0.9;lateral, 1; ventral, 2.3.

Data analysis and statisticsData were analyzed using t-test or ANOVA by GraphPad Prism version 5.00 for MacOS X (GraphPad Software, San Diego California, www.graphpad.com). All datawere normally distributed according to the d’Agostino and Shapiro-Wilk tests. nalways defines the number of independent experiments. Absolute cell counts, numberof neurospheres and culture coverslips, number of brain sections and animals usedfor all quantifications are shown in supplementary material Table S2.

Co-labeling in neurospheres was quantified using stacks of confocal imagesobtained by using 0.3 m steps acquired with 63� immersion objective (confocalmicroscope LSM 5 Pascal, Zeiss, Germany). For the markers that label only cellprocesses (nestin and GFAP) co-localization was determined only after carefulexamination of tdTomato coexpression in the processes. Low titer retrovirus infectionassured scarcity of infected cells in neurospheres. Quantifications in Figs 2 and 3show mean ± s.d.; number of cells >100, number of neurospheres ≥30, number ofculture coverslips for each condition ≥6.

Numbers of fluorescent cells in the RMS or olfactory bulb were evaluated relativeto the total number of infected cells around the SVZ. GFAP-, Sox2-, NeuN-,S100-, nestin-, BrdU-, Mash1, Dcx-, activated caspase-3-positive fluorescent cellsin the SVZ were evaluated as percentage of the total number of infected cells aroundthe SVZ. Injections with the same type of virus and titer resulted in approximatelythe same number of infected cells across animals. In rescue experiments, only double-infected cells were calculated. Mis-injected mice were excluded from further analysis.Quantifications in Figs 4–8 and supplementary material Figs S9 and S11 show mean± s.d., number of infected cells >1000, number of independent experiments ≥6.

This way of estimating the quantification index should not be affected by alteredproliferation in the SVZ after Epha4 knockdown for the following reasons. Althoughcell proliferation after Epha4 knockdown decreased by 0.5% at 40 days post-injection(BrdU-labeling, 1 day after BrdU injection), these cells do not enter the equation asnewly born cells in the SVZ and eventually migrate to the olfactory bulb; hence theyare not considered to be ‘infected cells around the SVZ’ once they reach the RMS.Most importantly, all normalized quantifications (except NeuN co-localization) showeda decrease in the number of specifically labeled cells (i.e. GFAP+, Sox2+, BrdU+,Mash1+, nestin+) after Epha4 knockdown. Because the index is obtained by dividingspecifically labeled infected cells by all infected cells in the SVZ, reduced proliferationin the SVZ would result in a decreased number of total infected cells in the SVZ, andhence in a higher index in Epha4 knockdown experiments than controls, which wasnot the case.

Analysis of cell proliferation in vivo by BrdUTo label fast-proliferating cells, mice were injected once with 50 mg BrdU/kg bodyweight. To label slow-proliferating cells, 1 mg/ml BrdU was administered in thedrinking water for 1 week followed by 3 weeks of normal water consumption toobtain BrdU dilution in fast-proliferating cells. For the analysis of dividing cellsafter lentiviral infection, mice were injected with BrdU twice, the two time-pointsbeing 6 hours apart, and sacrificed 24 hours later. Postnatal animals were injectedwith 20 mg BrdU/kg body weight and adult animals with 50 mg/kg.

Immunohistochemistry and immunocytochemistrySagittal brain sections (50–75 m) were cut with a vibratome (Leica VT1000S,Leica, Germany). Immunostainings were carried out on free-floating sections. Sliceswere blocked in 0.2–1% Triton and 2–5% normal goat serum or 5% bovine serumalbumin and incubated overnight with primary antibodies at 4°C followed byincubation with secondary antibodies at room temperature. Primary and secondaryantibodies were listed above. For nuclear staining we used DAPI. For BrdU stainings,slices were preincubated with 1 N HCl at room temperature for 1 hour, thenneutralized with 10 mM Tris (pH 8.5) at room temperature for 15 minutes andfurther processed as for other stainings. Sections were mounted onto slides withMowiol (Carl Roth, Karlsruhe, Germany) or Immu-mount (ThermoScientific, Bonn,Germany) and subsequently analyzed on a fluorescent microscope (Axioplan 2;Zeiss, Germany) or confocal microscope (LSM 5 Pascal; Zeiss, Germany).

Western-blot analysisFor western-blot analysis, protein samples were boiled in SDS gel sample buffer.Denatured proteins were separated by SDS-PAGE, transferred onto PVDF membranesand probed with antibodies. For statistical analysis, antibody signals were quantified

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using NIH ImageJ software and values normalized to the corresponding -actinsignals. Data were analyzed using the Student’s t-test.

We thank Ulla Amtmann, Regina Hinz-Hernkommer and IrmgardPreugschat-Gumbrecht for technical assistance. This work wassupported in part by the Schilling Foundation and DFG (SFB488grant).

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/8/1268/DC1

ReferencesAbrous, D. N., Koehl, M. and Le Moal, M. (2005). Adult neurogenesis: from precursors

to network and physiology. Physiol. Rev. 85, 523-569.Aimone, J. B., Wiles, J. and Gage, F. H. (2009). Computational influence of adult

neurogenesis on memory encoding. Neuron 61, 187-202.Alonso, M., Viollet, C., Gabellec, M. M., Meas-Yedid, V., Olivo-Marin, J. C. and

Lledo, P. M. (2006). Olfactory discrimination learning increases the survival of adult-born neurons in the olfactory bulb. J. Neurosci. 26, 10508-10513.

Aoki, M., Yamashita, T. and Tohyama, M. (2004). EphA receptors direct thedifferentiation of mammalian neural precursor cells through a mitogen-activated proteinkinase-dependent pathway. J. Biol. Chem. 279, 32643-32650.

Bourgin, C., Murai, K. K., Richter, M. and Pasquale, E. B. (2007). The EphA4 receptorregulates dendritic spine remodeling by affecting beta1-integrin signaling pathways. J.Cell Biol. 178, 1295-1307.

Campos, L. S. (2004). Neurospheres: insights into neural stem cell biology. J. Neurosci.Res. 78, 761-769.

Campos, L. S., Leone, D. P., Relvas, J. B., Brakebusch, C., Fassler, R., Suter, U. andffrench-Constant, C. (2004). Beta1 integrins activate a MAPK signalling pathway inneural stem cells that contributes to their maintenance. Development 131, 3433-3444.

Celikel, T., Marx, V., Freudenberg, F., Zivkovic, A., Resnik, E., Hasan, M. T.,Licznerski, P., Osten, P., Rozov, A., Seeburg, P. H. et al. (2007). Select overexpressionof homer1a in dorsal hippocampus impairs spatial working memory. Front. Neurosci.1, 97-110.

Chumley, M. J., Catchpole, T., Silvany, R. E., Kernie, S. G. and Henkemeyer, M.(2007). EphB receptors regulate stem/progenitor cell proliferation, migration, andpolarity during hippocampal neurogenesis. J. Neurosci. 27, 13481-13490.

Conover, J. C., Doetsch, F., Garcia-Verdugo, J. M., Gale, N. W., Yancopoulos, G. D.and Alvarez-Buylla, A. (2000). Disruption of Eph/ephrin signaling affects migrationand proliferation in the adult subventricular zone. Nat. Neurosci. 3, 1091-1097.

Deininger, K., Eder, M., Kramer, E. R., Zieglgansberger, W., Dodt, H. U., Dornmair,K., Colicelli, J. and Klein, R. (2008). The Rab5 guanylate exchange factor Rin1regulates endocytosis of the EphA4 receptor in mature excitatory neurons. Proc. Natl.Acad. Sci. USA 105, 12539-12544.

Depaepe, V., Suarez-Gonzalez, N., Dufour, A., Passante, L., Gorski, J. A., Jones, K.R., Ledent, C. and Vanderhaeghen, P. (2005). Ephrin signalling controls brain sizeby regulating apoptosis of neural progenitors. Nature 435, 1244-1250.

Doetsch, F., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (1997). Cellular compositionand three-dimensional organization of the subventricular germinal zone in the adultmammalian brain. J. Neurosci. 17, 5046-5061.

Doetsch, F., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (1999). Regeneration of agerminal layer in the adult mammalian brain. Proc. Natl. Acad. Sci. USA 96, 11619-11624.

Egea, J. and Klein, R. (2007). Bidirectional Eph-ephrin signaling during axon guidance.Trends Cell Biol. 17, 230-238.

Egea, J., Nissen, U. V., Dufour, A., Sahin, M., Greer, P., Kullander, K., Mrsic-Flogel,T. D., Greenberg, M. E., Kiehn, O., Vanderhaeghen, P. et al. (2005). Regulation ofEphA 4 kinase activity is required for a subset of axon guidance decisions suggestinga key role for receptor clustering in Eph function. Neuron 47, 515-528.

Fu, W. Y., Chen, Y., Sahin, M., Zhao, X. S., Shi, L., Bikoff, J. B., Lai, K. O., Yung, W.H., Fu, A. K., Greenberg, M. E. et al. (2007). Cdk5 regulates EphA4-mediateddendritic spine retraction through an ephexin1-dependent mechanism. Nat. Neurosci.10, 67-76.

Furne, C., Ricard, J., Cabrera, J. R., Pays, L., Bethea, J. R., Mehlen, P. and Liebl, D.J. (2009). EphrinB3 is an anti-apoptotic ligand that inhibits the dependence receptorfunctions of EphA4 receptors during adult neurogenesis. Biochim. Biophys. Acta 1793,231-238.

Gallarda, B. W., Bonanomi, D., Muller, D., Brown, A., Alaynick, W. A., Andrews, S.E., Lemke, G., Pfaff, S. L. and Marquardt, T. (2008). Segregation of axial motor andsensory pathways via heterotypic trans-axonal signaling. Science 320, 233-236.

Goldshmit, Y., Galea, M. P., Wise, G., Bartlett, P. F. and Turnley, A. M. (2004). Axonalregeneration and lack of astrocytic gliosis in EphA4-deficient mice. J. Neurosci. 24,10064-10073.

Goldshmit, Y., Galea, M. P., Bartlett, P. F. and Turnley, A. M. (2006). EphA4 regulatescentral nervous system vascular formation. J. Comp. Neurol. 497, 864-875.

Holmberg, J., Armulik, A., Senti, K. A., Edoff, K., Spalding, K., Momma, S., Cassidy,R., Flanagan, J. G. and Frisen, J. (2005). Ephrin-A2 reverse signaling negativelyregulates neural progenitor proliferation and neurogenesis. Genes Dev. 19, 462-471.

Inoue, E., Deguchi-Tawarada, M., Togawa, A., Matsui, C., Arita, K., Katahira-Tayama, S., Sato, T., Yamauchi, E., Oda, Y. and Takai, Y. (2009). Synaptic activityprompts gamma-secretase-mediated cleavage of EphA4 and dendritic spine formation.J. Cell Biol. 185, 551-564.

Inta, D., Alfonso, J., von Engelhardt, J., Kreuzberg, M. M., Meyer, A. H., van Hooft,J. A. and Monyer, H. (2008). Neurogenesis and widespread forebrain migration ofdistinct GABAergic neurons from the postnatal subventricular zone. Proc. Natl. Acad.Sci. USA 105, 20994-20999.

Jiao, J. W., Feldheim, D. A. and Chen, D. F. (2008). Ephrins as negative regulators ofadult neurogenesis in diverse regions of the central nervous system. Proc. Natl. Acad.Sci. USA 105, 8778-8783.

Katakowski, M., Zhang, Z., deCarvalho, A. C. and Chopp, M. (2005). EphB2 inducesproliferation and promotes a neuronal fate in adult subventricular neural precursor cells.Neurosci. Lett. 385, 204-209.

Kempermann, G., Wiskott, L. and Gage, F. H. (2004). Functional significance of adultneurogenesis. Curr. Opin. Neurobiol. 14, 186-191.

Khodosevich, K., Inta, D., Seeburg, P. H. and Monyer, H. (2007). Gene expressionanalysis of in vivo fluorescent cells. PLoS One 2, e1151.

Khodosevich, K., Seeburg, P. H. and Monyer, H. (2009). Major signaling pathways inmigrating neuroblasts. Front. Mol. Neurosci. 2, 7.

Klein, R. (2004). Eph/ephrin signaling in morphogenesis, neural development and plasticity.Curr. Opin. Cell Biol. 16, 580-589.

Kreuzberg, M., Kanov, E., Timofeev, O., Schwaninger, M., Monyer, H. andKhodosevich, K. (2010). Increased subventricular zone-derived cortical neurogenesisafter ischemic lesion. Exp. Neurol. 226, 90-99.

Kullander, K., Mather, N. K., Diella, F., Dottori, M., Boyd, A. W. and Klein, R. (2001).Kinase-dependent and kinase-independent functions of EphA4 receptors in major axontract formation in vivo. Neuron 29, 73-84.

Liebl, D. J., Morris, C. J., Henkemeyer, M. and Parada, L. F. (2003). mRNA expressionof ephrins and Eph receptor tyrosine kinases in the neonatal and adult mouse centralnervous system. J. Neurosci. Res. 71, 7-22.

Lledo, P. M. and Saghatelyan, A. (2005). Integrating new neurons into the adult olfactorybulb: joining the network, life-death decisions, and the effects of sensory experience.Trends Neurosci. 28, 248-254.

Lledo, P. M., Alonso, M. and Grubb, M. S. (2006). Adult neurogenesis and functionalplasticity in neuronal circuits. Nat. Rev. Neurosci. 7, 179-193.

Lois, C., Hong, E. J., Pease, S., Brown, E. J. and Baltimore, D. (2002). Germlinetransmission and tissue-specific expression of transgenes delivered by lentiviral vectors.Science 295, 868-872.

Morshead, C. M. and van der Kooy, D. (2004). Disguising adult neural stem cells. Curr.Opin. Neurobiol. 14, 125-131.

Morshead, C. M., Reynolds, B. A., Craig, C. G., McBurney, M. W., Staines, W. A.,Morassutti, D., Weiss, S. and van der Kooy, D. (1994). Neural stem cells in the adultmammalian forebrain: a relatively quiescent subpopulation of subependymal cells.Neuron 13, 1071-1082.

Morshead, C. M., Craig, C. G. and van der Kooy, D. (1998). In vivo clonal analysesreveal the properties of endogenous neural stem cell proliferation in the adult mammalianforebrain. Development 125, 2251-2261.

Ninkovic, J. and Gotz, M. (2007). Signaling in adult neurogenesis: from stem cell nicheto neuronal networks. Curr. Opin. Neurobiol. 17, 338-344.

North, H. A., Zhao, X., Kolk, S. M., Clifford, M. A., Ziskind, D. M. and Donoghue,M. J. (2009). Promotion of proliferation in the developing cerebral cortex by EphA4forward signaling. Development 136, 2467-2476.

Osborn, M. J., Panoskaltsis-Mortari, A., McElmurry, R. T., Bell, S. K., Vignali, D. A.,Ryan, M. D., Wilber, A. C., McIvor, R. S., Tolar, J. and Blazar, B. R. (2005). Apicornaviral 2A-like sequence-based tricistronic vector allowing for high-leveltherapeutic gene expression coupled to a dual-reporter system. Mol. Ther. 12, 569-574.

Parent, J. M., Vexler, Z. S., Gong, C., Derugin, N. and Ferriero, D. M. (2002). Ratforebrain neurogenesis and striatal neuron replacement after focal stroke. Ann. Neurol.52, 802-813.

Pasquale, E. B. (2004). Eph-ephrin promiscuity is now crystal clear. Nat. Neurosci. 7,417-418.

Pasquale, E. B. (2005). Eph receptor signalling casts a wide net on cell behaviour. Nat.Rev. Mol. Cell Biol. 6, 462-475.

Qian, X., Goderie, S. K., Shen, Q., Stern, J. H. and Temple, S. (1998). Intrinsicprograms of patterned cell lineages in isolated vertebrate CNS ventricular zone cells.Development 125, 3143-3152.

Qiu, R., Wang, X., Davy, A., Wu, C., Murai, K., Zhang, H., Flanagan, J. G., Soriano,P. and Lu, Q. (2008). Regulation of neural progenitor cell state by ephrin-B. J. CellBiol. 181, 973-983.

Ricard, J., Salinas, J., Garcia, L. and Liebl, D. J. (2006). EphrinB3 regulates cellproliferation and survival in adult neurogenesis. Mol. Cell. Neurosci. 31, 713-722.

Rogelius, N., Ericson, C. and Lundberg, C. (2005). In vivo labeling of neuroblasts inthe subventricular zone of rats. J. Neurosci. Methods 142, 285-293.

Suh, H., Consiglio, A., Ray, J., Sawai, T., D’Amour, K. A. and Gage, F. H. (2007). Invivo fate analysis reveals the multipotent and self-renewal capacities of Sox2+ neuralstem cells in the adult hippocampus. Cell Stem Cell 1, 515-528.

Theus, M. H., Ricard, J., Bethea, J. R. and Liebl, D. J. (2010). EphB3 limits theexpansion of neural progenitor cells in the subventricular zone by regulating p53 duringhomeostasis and following traumatic brain injury. Stem Cells 28, 1231-1242.

Tremblay, M. E., Riad, M., Chierzi, S., Murai, K. K., Pasquale, E. B. and Doucet, G.(2009). Developmental course of EphA4 cellular and subcellular localization in thepostnatal rat hippocampus. J. Comp. Neurol. 512, 798-813.

Wegmeyer, H., Egea, J., Rabe, N., Gezelius, H., Filosa, A., Enjin, A., Varoqueaux, F.,Deininger, K., Schnutgen, F., Brose, N. et al. (2007). EphA4-dependent axon guidanceis mediated by the RacGAP alpha2-chimaerin. Neuron 55, 756-767.

Zhao, C., Deng, W. and Gage, F. H. (2008). Mechanisms and functional implications ofadult neurogenesis. Cell 132, 645-660.

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