1268 Research Article

EphA4 preserves postnatal and adult neural stem

cells in an undifferentiated state in vivo
Konstantin Khodosevich1,2,*, Yasuhito Watanabe1,2 and Hannah Monyer1,2
1
Department of Clinical Neurobiology, Heidelberg University Medical Center, 69120 Heidelberg, Germany
2
Department of Clinical Neurobiology/A230, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
*Author for correspondence (k.khodosevich@dkfz-heidelberg.de)

Accepted 22 November 2010

Journal of Cell Science 124, 1268-1279
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jcs.076059

Summary
In 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 signaling
network that regulates neurogenesis. On the basis of previous data, we have identified Eph receptor A4 (EphA4) as a potential regulator
of 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. In
postnatal and adult brain, Epha4 knockdown caused a decrease in NSCs in the SVZ, eventually resulting in a reduced number of
postnatally generated neuroblasts. Both in vitro and in vivo effects were rescued by co-infection with a modified EphA4 that was
resistant to Epha4 shRNA.
Journal of Cell Science

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

Introduction
Postnatal neurogenesis in mammals persists in two brain regions:
the subgranular zone (SGZ) of the dentate gyrus in the hippocampus
and the subventricular zone (SVZ) of the lateral ventricles (Lledo
et al., 2006; Ninkovic and Gotz, 2007; Zhao et al., 2008). Under
pathological conditions, new neuroblasts also migrate into injured
cortex and striatum (Kreuzberg et al., 2010; Parent et al., 2002).
Neural stem cells (NSCs) in the SGZ and SVZ divide slowly and
are preserved throughout life. Little is known about the cellular
signaling that guarantees that NSCs are maintained in an
undifferentiated state. Postnatal NSCs divide asymmetrically and
differentiate into transit-amplifying precursors, which are fast-
dividing cells that, in turn, give rise to more differentiated
neuroblasts (Abrous et al., 2005; Lledo et al., 2006; Zhao et al.,
2008). Neuroblasts in the hippocampal SGZ migrate into the
granule cell layer of dentate gyrus and integrate into previously
established neural circuits, where they are thought to play a role in
certain forms of hippocampal-dependent learning and memory
(Aimone et al., 2009; Kempermann et al., 2004). Neuroblasts
originating in the SVZ migrate over long distances via the rostral
migratory stream (RMS) to the olfactory bulb, where they mature
into granule or periglomerular neurons and contribute to olfactory
information processing (Alonso et al., 2006; Lledo and Saghatelyan,
2005).
Ephrins and their Eph tyrosine kinase receptors are important
regulators 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 to
EphA 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 regulate
different stages of neurogenesis and are differentially expressed on
distinct 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 et
al., 2006). For instance, ephrins B2 and B3 and their receptor
EphB1 cooperatively regulate proliferation and survival of neural
progenitor cells and migration of neuroblasts in the SVZ/RMS and
SGZ (Chumley et al., 2007; Conover et al., 2000; Ricard et al.,
2006). Two other Eph receptors, namely, EphB2 and EphB3, have
opposite function in the adult SVZ. Whereas EphB2 induces
proliferation in the SVZ (Katakowski et al., 2005), EphB3
suppresses progenitor cell proliferation via a p53-dependent
mechanism (Theus et al., 2010). In the SVZ, the complementary
expression of ephrin A2 in transit-amplifying cells and neuroblasts
and that of EphA7 on ependymal cells and NSCs controls neural
progenitor cell proliferation (Holmberg et al., 2005). In another
study, EphA7 was shown to be involved in the modulation of
apoptosis of neural progenitors during embryonic development
(Depaepe et al., 2005). Also, ephrin B1 has been demonstrated to
contribute to the maintenance of NSCs in an undifferentiated state
(Qiu et al., 2008).
We recently described the generation of transgenic mice in
which EGFP (enhanced green fluorescent protein) is expressed in
the entire RMS (Inta et al., 2008), and optimized a procedure for
RNA isolation from in vivo fluorescent RMS neuroblasts
(Khodosevich et al., 2007). The RMS is a robust and hence an
ideal structure in the postnatal brain that allows the harvesting of
sufficient cells to perform microarray analysis. Using transgenic
5HT3A-EGFP mice (where EGFP is expressed under the control
of the serotonin receptor 3A promoter) with clearly EGFP-labeled
RMS, we isolated neuroblasts from two distinct locations: one in
the immediate vicinity of the SVZ (posterior RMS, pRMS), and
the other more rostral, closer to the bulb (anterior RMS, aRMS)
(Khodosevich et al., 2009). It is safe to assume that upregulated
genes in cells from the aRMS are most probably involved in

migration and differentiation, whereas upregulated genes in cells
from the pRMS that is closer to the SVZ might affect cell
generation.
We identified Epha4, the gene coding for the Eph receptor A4,
as one of the upregulated genes in the pRMS. The upregulation
was comparable with that of other genes known to be expressed in
NSCs (e.g. GFAP, Nr2e1, Nes). Epha4 mRNA expression levels
were below background expression in samples collected from the
aRMS or from the periglomerular region of the olfactory bulb.
EphA4 plays an important role in the developing and postnatal
brain, 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 found
in the adult SVZ (Conover et al., 2000; Liebl et al., 2003). Indeed,
there is an increase in the number of neuroblasts in the SVZ and
RMS in EphA4 knockout animals (Furne et al., 2009). Interestingly
Journal of Cell Science
EphA4 maintains adult neural stem cells 1269
the number of proliferating cells in the SVZ (identified by Ki67
expression) 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 in
the adult neurogenic niches exclusively by NSCs, but not by other
cell types. In neurosphere cultures and in vivo, knockdown of
Epha4 expression caused reduction in cell proliferation and
premature differentiation of NSCs.
Results
Analysis of EphA4 expression in the postnatal brain
To establish the cellular expression of EphA4 in the postnatal
brain, we employed double- and triple-immunohistochemistry in
brain sections from 3-month-old animals. In both adult neurogenic
niches, the SVZ and SGZ, EphA4 co-localizes with GFAP (Fig.
1A–C). Furthermore, GFAP–nestin double-positive cells that are
bona fide NSCs, at least in the SGZ (Lledo et al., 2006; Zhao et
Fig. 1. EphA4 is expressed in
postnatal NSCs of the SVZ and
SGZ, but not in transit-amplifying
progenitors and neuroblasts.
(A)EphA4 is expressed in GFAP-
positive NSCs along the lateral
ventricle. (B)EphA4 is expressed in
nestin and GFAP-double positive
NSCs in the SVZ. Arrowhead labels a
GFAP/nestin/EphA4-triple positive
cell that is magnified on the right side
showing two stacks 2-m apart.
(C,C⬘) EphA4 is expressed in SGZ
radial glia-like cells. (D)EphA4 is
expressed in Sox2-positive cells.
(E,F)EphA4 is expressed in slow-
proliferating cells (F), but is not
expressed in fast-proliferating cells
(E). In E, the arrowhead indicates
BrdU-negative magnified cell shown
on the right. Lv, lateral ventricle. Scale
bars: 20m (A,B,E) and 10m
(C,D,F).
 1270 Journal of Cell Science 124 (8)

al., 2008), express EphA4 (Fig. 1B for SVZ; Fig. 1C,C⬘ for
SGZ). In SVZ, ependymal cells also exhibit coexpression of
GFAP and nestin (Doetsch et al., 1997). However, EphA4
expression does not co-localize with the ependymal cell marker
S100 (supplementary material Fig. S1). Because the EphA4
antibody almost exclusively labels cell processes, EphA4 co-
labeling with other nuclear or cell body markers was determined
by analyzing series of confocal stacks (supplementary material
Fig. S2A). To avoid cross-reactivity between secondary
antibodies, we used rabbit anti-EphA4, mouse anti-GFAP and
chicken anti-nestin antibodies (see Materials and Methods).
Furthermore, stainings on separate sections with anti-EphA4
and GFAP antibodies resulted in similar labeling patterns
(supplementary material Fig. S2B,C). EphA4-positive cells
coexpress Sox2, another neural stem cell marker (Fig. 1D and
supplementary material Fig. S3A–D).
EphA4 is not present in transit-amplifying precursors and glial
precursors or oligodendrocytes, as revealed by the lack of
coexpression with Mash1 and Olig2, respectively (supplementary
material Fig. S4A,B). EphA4 is also not co-localized with the
neuroblast markers DCX and PSA-NCAM (supplementary material
Fig. S4C,D).
We analyzed the proliferating capacity of EphA4-positive cells
taking recourse to labeling with 5-bromo-2⬘-deoxyuridine (BrdU),
Journal of Cell Science
The phenotype of EphA4-positive cells in the SVZ is
summarized in supplementary material Table S1.
EphA4 is involved in NSC proliferation and differentiation
in vitro
To study the cellular function of EphA4 in NSCs of the postnatal
SVZ, we conducted knockdown experiments using two Epha4
short hairpin RNA (shRNA) constructs (shRNA Epha4-1 and
shRNA Epha2) (see Fig. 2E; supplementary material Fig. S5) and
analyzed NSC proliferation in neurosphere cultures after Epha4
knockdown. We used neurosphere cultures obtained from the SVZ
of P4 mice that were infected with retroviruses expressing red
fluorescent protein and either control shRNA (scrambled) or one
of the two Epha4 shRNAs. After 5 days in culture, BrdU was
added to the culture media and cells were fixed after another 12
hours. In the presence of either of the two Epha4 shRNAs, there
was a significant decrease in the number of infected proliferating
cells in neurospheres (green and red double-positive cells in Fig.
2A,B, quantification in Fig. 2D). Details regarding cell counts in
neurospheres are described in Materials and Methods. Knockdown
of Epha4 in neurospheres was already pronounced at 4 days in
vitro (DIV4) (supplementary material Fig. S6A,B) and by DIV5
was at most 25% of that in control neurospheres (see Fig. 2E for
western-blot analysis and supplementary material Fig. S6C,D for

a marker of dividing cells. After a single injection of BrdU, none immunocytochemistry).

of the EphA4-positive cells retained BrdU (Fig. 1E). However,
EphA4-positive cells were labeled when BrdU was administered
for 1 week followed by 3 weeks with no treatment before sacrificing
the animals (Fig. 1F; supplementary material Fig. S3E–H), which
indicates that EphA4 is expressed in slow-dividing cells, but not in
fast-dividing cells.
The effect of Epha4 knockdown was completely rescued by
coexpression of a modified EphA4, containing three silent
mutations that make it resistant to shRNA Epha4-1 (Fig. 2C, see
double-infected BrdU-positive cells). To visualize the EphA4
mutant it was linked to copGFP via the T2A peptide sequence (for
details 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 retroviruses
expressing tdTomato and control shRNA (scrambled) (A), shRNA Epha4-1 (B) or Epha4-2, and shRNA Epha4-1 together with a modified Epha4 resistant to
Epha4-1 (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 cells
compared with control or rescue conditions (*P<0.01, n6). 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. Both
Epha4 shRNAs knocked down EphA4 expression by at least 75%. Scale bars: 20m.

Next, we investigated the effect of Epha4 knockdown on
precursor cell maintenance in neurospheres. We cultured infected
neurospheres, and after 6 days we transferred them into media
lacking growth factors. After 2 days following growth factor
removal, Epha4 knockdown caused a prominent decrease in the
percentage of nestin-positive cells relative to the total number of
infected cells (Fig. 3A–C). After 4 days of incubation in media
without growth factors, the percentage of astrocytes (i.e. number
of GFAP-positive infected cells relative to the total number of
infected cells) was much higher in neurospheres expressing Epha4
shRNAs than in control neurospheres (Fig. 3D–F). Whereas, in
vivo, GFAP labels NSCs and astrocytes, in neurospheres GFAP
only labels astrocytes. Nestin- and GFAP-positive cells do not
overlap in neurosphere cultures (supplementary material Fig. S7)
(see also Campos, 2004; Campos et al., 2004). Although GFAP
labels cells in the center of neurospheres (mature astrocytes), nestin
labels precursor cells at the periphery. Using another marker for
mature astrocytes, namely S100, we detected a similar increase
in infected astrocytes (relative to the total number of infected cells)
after Epha4 knockdown as that observed in experiments where
GFAP stainings were performed (Fig. 3J–L). Also, in experiments
in which differentiation was assessed using the neuronal marker
Tuj1, there was an increase in differentiated cells in conditions of
Epha4 knockdown (Fig. 3G–I). The Epha4 knockdown phenotype
Journal of Cell Science
could be rescued when modified EphA4 was coexpressed, whereas
a kinase-dead EphA4 mutant was not able to rescue the Epha4
knockdown (Fig. 3C,F,I,L).
To confirm that knockdown of Epha4 indeed affects NSC
differentiation, we analyzed the formation of secondary
neurospheres derived from primary neurospheres infected by
retroviruses expressing control or Epha4 shRNAs. The formation
of primary neurospheres was not affected by Epha4 knockdown,
except for the slightly smaller average size (data not shown) that
was most probably due to a decrease in the number of infected
proliferating cells (see Fig. 2A–D). However, the formation of
secondary neurospheres was dramatically reduced after Epha4
knockdown in primary neurospheres (Fig. 3M–O). Each secondary
neurosphere is a descendant of a single cell from a primary
neurosphere. Thus, it can be inferred that, after Epha4 knockdown
in primary neurospheres, a decrease in the number of
undifferentiated precursor cells would result in fewer secondary
neurospheres. Conversely, the number of non-infected secondary
neurospheres was similar for control and Epha4 knockdown
conditions (Fig. 3P). Thus, in neurosphere cultures, Epha4
knockdown accelerates NSC differentiation, which might account
at least in part for the observed decrease in proliferation.
EphA4 is involved in NSC proliferation of the postnatal
and adult SVZ in vivo
To extend the results obtained in vitro, we silenced EphA4
expression in the postnatal SVZ of wild-type mice using lentiviruses
containing shRNAs against Epha4. We injected lentiviruses
expressing EGFP and control or Epha4 shRNAs into the SVZ of
postnatal day 6 (P6) mice (for details on viral titers see Materials
and Methods) that were sacrificed at 4, 7, 15, 25, 40, 60, 80 and
100 days post-injection (Fig. 4). Because the number of infected
cells in the SVZ can vary from one injection to another (even when
the viral titer is constant), data were normalized and expressed as
relative to the number of all infected cells in the SVZ (for further
details on the quantification index see Materials and Methods).
There was no difference in the number of migrating RMS
EphA4 maintains adult neural stem cells 1271
neuroblasts when comparing control and Epha4 knockdown mice
at any time point up to 25 days post-injection. However, at 40 days
post-injection and later there was a dramatic decrease of infected
neuroblasts in the RMS (Fig. 4A,B,D) of Epha4 knockdown
animals. Concomitantly, there was a reduction in the number of
infected neurons in the olfactory bulb of Epha4 knockdown animals
at 80 days post-injection and later (Fig. 4E,F).
Interestingly, although the effect of Epha4 knockdown could be
first detected only as late as 40 days post-injection, the decrease of
EphA4 expression levels around the SVZ was already visible 3
days post-injection (supplementary material Fig. S8A,B) and was
more pronounced along the whole SVZ at 6 days post-injection
(supplementary material Fig. S8C–E).
For rescue experiments in vivo, we used lentivirus expressing a
modified EphA4 that contains three silent mutations, making it
resistant to shRNA Epha4-1. Cells infected with the ‘rescue’ virus
can be visualized by copGFP expression whereas infection with
the shRNA Epha4 virus is monitored by EGFP expression. To
obtain a significant number of double-infected cells, we used a
fivefold higher titer for the ‘rescue’ virus. Co-injection into the
SVZ of shRNA Epha4-1 lentivirus (also expressing EGFP, green)
together with lentivirus expressing modified EphA4 (also positive
for copGFP, magenta) rescued the Epha4 knockdown effect (Fig.
4C,D,F). At 40 days post-injection and later, most RMS neuroblasts
expressing shRNA Epha4-1 also contained the ‘rescuing’ modified
EphA4 virus (Fig. 4C). Furthermore, at 80 days post-injection and
later, there was a significant increase in the number of cells double-
infected by Epha4 knockdown and ‘rescue’ lentiviruses in
comparison with the control (i.e. gain-of-function effect) (Fig. 4D).
Cells infected by the virus expressing the modified EphA4 did not
exhibit morphological alterations.
We determined the number of proliferating cells in the SVZ by
administering two BrdU pulses to label dividing cells
(supplementary material Fig. S9A–C). Although at 4 and 25 days
post-injection there was no difference between the numbers of
infected proliferating cells in control and Epha4 knockdown
conditions, at 40 days post-injection there was a 50% reduction in
the number of BrdU-positive infected cells around the SVZ in
Epha4 knockdown animals.
The reduction of infected neuroblasts in the RMS after Epha4
knockdown cannot be explained by a change in the number of
apoptotic cells in the SVZ, as indicated by the unaltered caspase-
3 activation (supplementary material Fig. S9D–F). Thus, both in
vitro and in vivo experiments provided evidence that Epha4
knockdown resulted in a decrease of precursor cell proliferation
that was not a consequence of altered survival. The observed effect
of EphA4 on NSCs is cell autonomous and is mediated by
intracellular signaling of EphA4 because it occurs directly in the
infected cells. Also, a kinase-dead EphA4 mutant (with a lack of
forward signaling) was not able to rescue the Epha4 knockdown
phenotype (Fig. 4D).
The immunohistochemistry results indicated that EphA4 is
expressed only in NSCs, but not in transit-amplifying progenitors
or neuroblasts (Fig. 1). Hence, we expect that the knockdown of
EphA4 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 the
SVZ using retroviruses known to infect only dividing cells. In
comparison with neurosphere cultures where NSCs divide fast
(Campos, 2004; Qian et al., 1998), in postnatal SVZ the NSCs
divide very slowly (Doetsch et al., 1999; Morshead et al., 1994;
 1272 Journal of Cell Science 124 (8)
Journal of Cell Science

Fig. 3. EphA4 is involved in NSC differentiation in vitro. (A–L) Neurospheres were prepared from the SVZ of P4 animals and infected by retroviruses
expressing 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 by
shRNA 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 later
for GFAP- (D–F), Tuj1- (G–I) or S100- (J–L) positive infected (double-infected for rescue experiments) cells. Arrowheads indicate infected cells that are positive
for the specific marker; arrows indicate infected cells that are negative for the specific marker. Infection with any of the two Epha4 shRNA-expressing viruses
resulted 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, n6). Kinase-dead EphA4 mutant was not able to rescue the Epha4 knockdown
phenotype. Note that image panels do not necessarily reflect quantitative differences, but rather qualitative differences. (M–P) Primary neurospheres were infected
by retroviruses expressing control or Epha4 shRNAs and after 6 days secondary neurospheres were prepared. In comparison with control condition (M), after
Epha4 knockdown (N) there were fewer secondary neurospheres derived from infected cells in primary neurospheres (O) (*P<0.001, n6). Note that the number of
secondary neurospheres derived from non-infected cells in primary neurospheres was comparable between control and Epha4 knockdown (P). Scale bars: 20m
(A–K) and 100m (M,N).
 EphA4 maintains adult neural stem cells 1273
Journal of Cell Science

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 days
post-injection and later in animals expressing the control and rescue constructs (A and C, respectively), compared with animals expressing shRNA Epha4-1 (B) or
shRNA Epha4-2 (D). Kinase-dead EphA4 mutant was not able to rescue Epha4 knockdown phenotype (D). Neuroblasts in the RMS were visualized by staining
with doublecortin (DCX) antibodies. Note that most shRNA Epha4-1-expressing RMS neuroblasts (green) in C are co-infected with the modified EphA4 resistant
to 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-dividing
cells (*P<0.05, n≥6). Scale bars: 40m (A–C) and 100m (E).

Morshead and van der Kooy, 2004). Indeed it has been established
that the majority of infected cells in the SVZ after retrovirus
injection are fast-dividing transit-amplifying precursors or
neuroblasts, but not slow-dividing NSCs (Rogelius et al., 2005).
Thus, we injected retroviruses expressing tdTomato (a red
fluorescent protein) and either control shRNA or shRNA Epha4-1
into the SVZ of P6 mice, and determined the number of infected
cells in the RMS or olfactory bulb relative to the total number of
infected cells in the SVZ at 5, 8, 12, 18, 25, 40 and 60 days post-
injection (Fig. 5A–C). There was no difference in the number of
infected 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 RMS
per animal at 18 days post-injection and no infected neuroblasts at
40 days post-injection and later (Fig. 5C). The number of BrdU-
positive infected cells in the SVZ, labeled by two short BrdU
pulses, was not different between Epha4 knockdown and control
conditions (Fig. 5D).
 1274 Journal of Cell Science 124 (8)

Fig. 5. Epha4 knockdown in fast-

dividing cells does not affect SVZ
progenitor proliferation in vivo.
(A–C)P6 animals were infected with
retroviruses expressing tdTomato and
control shRNA (scrambled) or shRNA
Epha4-1 and sacrificed 5, 8, 12, 18, 25,
40 and 60 days post-injection.
Arrowheads mark infected cells. Epha4
knockdown in fast-dividing cells
(transit-amplifying progenitors and
neuroblasts) did not result in a decrease
of infected cells in the olfactory bulb
(B) or RMS (C) (n≥6). (D)P6 animals
were infected with retroviruses as
described above and received two BrdU
injections 24 hours prior to perfusion.
There was no difference in BrdU-
positive infected cells in the SVZ
between control and Epha4 knockdown
conditions at any time-point analyzed
(n6). GCL, granule cell layer, GL,
glomerular layer, epl, external
plexiform layer. Scale bars: 100m.

Finally, we extended our in vivo injection experiments to adult neurosphere experiments. When restricting the analysis to the area
Journal of Cell Science

animals and injected the SVZ of 3-month-old mice with lentiviruses
expressing EGFP together with either control shRNA or one of the
Epha4 shRNAs. Animals were sacrificed at 4, 25, 40 and 60 days
post-injection (Fig. 6). Although there was no difference in the
number of infected cells in the RMS of control and Epha4
knockdown animals at 4 and 25 days post-injection, at 40 days
post-injection and later the number of neuroblasts in the RMS was
significantly decreased after Epha4 knockdown (Fig. 6A–C). Thus,
knockdown of Epha4 in adults resulted in the same phenotype as
that observed after injections in P6 animals.
EphA4 is involved in NSC differentiation of the postnatal
and adult SVZ in vivo
We subsequently tested in vivo whether, in addition to proliferation,
EphA4 also affected NCS differentiation, as was suggested by the
around the lateral ventricles, there was a significant decrease in the
number 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 representative
images when using a higher viral titer). In fact, the decrease in NSCs
in the knockdown condition might even be an underestimation if one
considers that some GFAP-positive cells in the area analyzed might
be astrocytes. Similar results were obtained when Sox2 and nestin
were used as NSC markers (Fig. 7E–I; for nestin see also
supplementary material Fig. S10C,D for representative images when
injecting a higher viral titer). Thus, at 4 days post-injection the
number of Sox2-positive infected cells was comparable for all
conditions, whereas after 15 days we observed a significant decrease
in the Sox2-positive cell number in Epha4 knockdown animals in
comparison with control or rescue animals.

Fig. 6. EphA4 affects proliferation of adult SVZ NSCs in vivo. (A–C) Three-month-old animals were infected with lentiviruses expressing EGFP and control
shRNA (scrambled), shRNA Epha4-1 or shRNA Epha4-2. Animals were sacrificed 4, 25, 40 and 60 days post-injection. There were significantly more infected
cells 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, n6). Neuroblasts in the RMS were visualized by staining with doublecortin (DCX) antibodies. Scale bars: 40m (A,B); insets: 20m.
 EphA4 maintains adult neural stem cells 1275
Journal of Cell Science

Fig. 7. Epha4 knockdown contributes to premature differentiation of SVZ NSCs in vivo. (A–O) P6 animals were infected with lentiviruses expressing EGFP
and control shRNA (scrambled), shRNA Epha4-1, shRNA Epha4-2, or shRNA Epha4-1 together with a modified EphA4 resistant to Epha4-1. Animals were
sacrificed 4, 7, 15, 25 and 40 days post-injection. All images were acquired along the lateral ventricles. (A–C) There were significantly more GFAP-positive
infected 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-injection
onwards (*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 observed
starting from 15 days post-injection onwards (*P<0.05, n10). 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) was
significantly decreased 40 days post-injection in comparison with control (G) or rescue animals (I) (*P<0.01, n10). (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, n10). Note that the magnified cell in N is rotated by 45° in N1. Scale bars in all panels except M and N are 20m; scale bars in M,N are 50m.
 1276 Journal of Cell Science 124 (8)

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 with
normal water consumption. Animals were sacrificed and analyzed for BrdU-retaining in infected cells. All images were acquired along the lateral ventricles. There were
Journal of Cell Science

significantly more BrdU-retaining infected cells (arrowheads) around the lateral ventricles relative to the total number of infected SVZ cells in animals infected with the
control virus (A) compared with animals expressing Epha4 shRNAs, (B,C) (*P<0.01, n10). Note that magnified cell in A is rotated by 45° in A1. (D–F) Similar
results showing a decrease in the number of BrdU-retaining cells in the SVZ after Epha4 knockdown in adult animals (*P<0.01, n10). Scale bars: 20m.

A decrease in the number of NSCs would eventually result in a
decreased number of their progeny cells. Indeed, there was a
decrease in the number of transit-amplifying precursors and
neuroblasts around the SVZ, as revealed by immunolabeling with
Mash1 and DCX, respectively. At 40 days post-injection, the
percentage of Mash1-positive infected cells in Epha4 knockdown
animals was half that of controls (Fig. 7J–L). The decrease in
DCX-positive infected cells around the SVZ corresponded to the
decrease in infected neuroblasts in RMS (see Fig. 4D).
If Epha4 knockdown reduces the number of cells expressing
stem cell markers and drives direct differentiation, one would
expect an increase in neuronal markers in the knockdown cells.
Indeed, this was the case. However, to test this, an injection site
located slightly more dorso-medially was used to avoid the infection
of many striatal neurons that occurred when injections were close
to the lateral ventricles. When using these injection coordinates,
Epha4 knockdown resulted in an increase of NeuN-positive infected
cells around the SVZ in comparison with control animals at 40
days post-injection (Fig. 7M–O).
A similar approach was used to analyze cell differentiation in
the SVZ using 3- to 4-month-old mice (supplementary material
Fig. S11). Animals were injected into the SVZ with lentiviruses
expressing EGFP together with either control shRNA or one of the
Epha4 shRNAs and sacrificed at 4, 15 and 40 days post-injection.
Similar to early postnatal injections, Epha4 knockdown decreased
the 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 the
SVZ. Conversely, there was an increase in NeuN-positive cells
(supplementary material Fig. S11J–M).
Finally, we performed BrdU-retaining experiments to directly
demonstrate that Epha4 knockdown indeed reduces the number of
slow-dividing cells. The SVZ of P6 or adult animals was injected
with lentiviruses expressing EGFP together with either control
shRNA or one of the Epha4 shRNAs. To label slow-dividing cells,
at 40 days post-injection BrdU was administered in the drinking
water for 1 week followed by 3 weeks with no treatment before
sacrificing the animals. Both early postnatal (Fig. 8A–C) and adult
(Fig. 8D–F) Epha4 knockdown animals showed a 50% reduction
in the number of infected slow-dividing cells around the SVZ
compared with controls. Thus, several lines of evidence indicate
that Epha4 expression preserves NSCs in an undifferentiated state.
Discussion
Ephrins and their Eph receptors regulate many aspects of neural
system development during embryogenesis (Klein, 2004; Pasquale,
2005). They also participate in the control of neurogenesis in both
postnatal neurogenic regions, the SVZ of the lateral ventricles, and
the hippocampal SGZ (Chumley et al., 2007; Conover et al., 2000;
Holmberg et al., 2005; Jiao et al., 2008; Qiu et al., 2008; Ricard et
al., 2006). In the SVZ, ephrin signaling has been shown to control
progenitor 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 et
al., 2005; Jiao et al., 2008). In this study, we have shown that Eph
receptor A4 is expressed specifically in NSCs, and is necessary to
preserve NSCs in an undifferentiated state. Interaction of Eph
receptors with ephrins activates both forward signaling (in the cell
where the receptor is expressed) and reverse signaling (in the cell
expressing the ephrin ligand) (Egea and Klein, 2007). The effects
described here following Epha4 knockdown occurred in cells that
were infected by viruses expressing shRNA Epha4, and it can be
thus inferred that EphA4 preserves NSC fate via forward signaling.
This is also supported by the results showing that a ‘kinase-dead’

mutant of EphA4 was not able to rescue the Epha4 knockdown
phenotype. EphA4 function is not restricted to the early postnatal
SVZ, but continues to be important in the adult SVZ.
The lack of effect on neurogenesis following Epha4 knockdown
using retroviruses (that generally infect fast-dividing cells)
(Rogelius et al., 2005), together with the observed decrease in
RMS neuroblasts only as late as 40 days post-injection using
lentiviruses, establish a functional role of EphA4 signaling in
NSCs, but not in other proliferative cell types in SVZ. Postnatal
NSCs 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 NSC
proliferation will be reflected in the number of their progeny only
several weeks later. Also, immunohistochemical data confirm the
presence of EphA4 in NSC, but not in precursor cells and
neuroblasts. The facts that neuroblast morphology in Epha4
knockdown mice was comparable with controls and that the number
of neuroblasts was reduced only at 40 days post-injection, together
with unaltered neuroblast number after retroviral injections, speak
against a scenario in which EphA4 is involved in migration.
The acceleration of NSC differentiation can most probably
account for the observed decrease in their proliferation. This
conjecture is supported by several observations. The population of
NSCs, indicated by GFAP-, Sox2- and nestin-positive staining,
Journal of Cell Science
remained relatively constant in control animals (Fig. 7D,F,I). These
results are in line with previous reports that postnatal NSCs divide
asymmetrically (Morshead et al., 1998; Suh et al., 2007), each
NSC generating one NSC and one precursor cell, thus explaining
the constancy of the NSC pool. Hence, a decrease in NSC
proliferation would not change their number. Therefore, a
significant reduction in NSC population after Epha4 knockdown is
the result of premature differentiation rather than that of
proliferation impairment. Because we did not observe an early
increase in the number of transit-amplifying precursors and
neuroblasts (as well as an overall proliferation increase), it is
possible that NSCs, upon Epha4 knockdown, are enforced to
differentiate directly to non-dividing cells (e.g. mature neurons)
omitting intermediate phenotypes such as transit-amplifying
precursor cells and neuroblasts. This is in line with the in vitro
results where we observed an increase in the number of mature
astrocytes and neurons together with a slight decrease in the total
cell number after Epha4 knockdown. Interestingly, EphA signaling
through other two members of EphA family, EphA2 and EphA3,
was found to be involved in pre- and postnatal neural precursor
cell differentiation towards the neuronal lineage (Aoki et al., 2004).
Potential ligands for EphA4 have been identified in other systems
or during embryonic development. Ephrin B1 has been shown to
be required for the maintenance of neural progenitor cell state in
the ventricular zone of the developing embryonic cortex (Qiu et
al., 2008). The interaction of EphA4 with ephrin B1 in embryonic
NSCs of the ventricular zone in rodents also lends credence to a
proposed function for cortical development (North et al., 2009).
Another ligand for EphA4 might be ephrin B3, which was shown
to interact with Epha4 in HEK293 cells (Furne et al., 2009; Ricard
et al., 2006). After interaction with ephrin B3, EphA4 propagates
reverse anti-apoptotic signal to cells expressing ephrin B3, whereas
in cells lacking ephrin B3, the reverse signal induces apoptosis
(Furne et al., 2009).
Involvement of EphA4 in neurogenesis was also indicated in a
study reporting a significant increase in the SVZ and RMS areas
in Epha4 knockout animals (Furne et al., 2009). Using knockout
EphA4 maintains adult neural stem cells 1277
mice, however, precludes evaluation of the role of EphA4 in
neurogenic cells because EphA4 is expressed in many non-
neurogenic cells, such as mature astrocytes, neurons and endothelial
cells (Deininger et al., 2008; Goldshmit et al., 2004; Goldshmit et
al., 2006; Tremblay et al., 2009). The use of low-titer lentiviruses
infecting sparsely distributed cells allows functional studies of
EphA4 forward signaling specifically in infected cells. Furthermore,
EphA4 plays an important role during embryonic brain
development; thus, prenatally derived changes in brain structure
and activity of full Epha4 knockout animals can modify postnatal
brain function, an effect that is avoided when employing postnatal
viral injections.
Postnatal neurogenic niches contain several cell types known to
differentially express specific ephrins and/or Eph receptors. Given
the redundancy of ephrin–receptor interaction (individual ephrins
can interact with several Eph receptors and vice versa), ephrin
signaling in neurogenic niches is mediated by complex intracellular
networks, most probably involving distinct pathways for certain
ligand–receptor interactions and common pathways for others. To
better understand the role of ephrin signaling for postnatal
neurogenesis, it is thus important to identify the expression of
different members in the defined cell types in the neurogenic
niches and to link members of the ephrin system with intracellular
pathways.
Materials and Methods
Animals
For all our experiments, we used wild-type C57Bl/6 mice. All procedures with
animals were performed according to the Heidelberg University Animal Care
Committee.
Materials and reagents
All chemicals and cell culture reagents were purchased from Sigma-Aldrich
(Taufkirchen, Germany) and Invitrogen (Karlsruhe, Germany), respectively, unless
otherwise specified.
The following antibodies were used: polyclonal rabbit anti-EGFP, 1:10000
(Invitrogen), chicken anti-EGFP, 1:1000 (Abcam, Cambridge, UK), mouse anti-III
class -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-chicken
and anti-goat, Alexa-Fluor-350-conjugated anti-rabbit and anti-mouse secondary
antibodies (Invitrogen), anti-mouse, anti-rabbit, anti-rat and anti-goat Cy3 coupled,
anti-mouse, anti-goat and anti-rabbit Cy5 coupled secondary antibodies (Jackson
ImmunoResearch Laboratories, Westgrove, PA), anti-mouse and anti-rabbit HRP-
conjugated secondary antibodies (Vector Laboratories, Burlingame, CA).
Plasmid cloning
The target sequences for oligos used to construct shRNA Epha4 expression plasmids
were 5⬘-GCAGCACCATCATCCATTG-3⬘ for Epha4-1 and 5⬘-ATCCACCTGGAA-
GGCGTTG-3⬘ for Epha4-2. Scrambled shRNA sequences were cloned from pSilencer
vector (Ambion, Austin, TX). Complementary pairs of oligonucleotides were cloned
into pSUPER vector (Oligoengine, Seattle, WA). To make recombinant lentiviral
plasmids for in vivo experiments, we re-cloned the shRNA silencing cassettes from
pSUPER vector to pFUGW, a lentiviral vector containing EGFP expressed under the
control of the ubiquitin promoter (Lois et al., 2002). To generate retroviral plasmids
expressing shRNAs, we re-cloned tdTomato (pRSET-tdTomato; generous gift of
Roger Tsien, University of California at San Diego, La Jolla, CA) and the CMV
promoter (from pEGFP-C1; Clontech-Takara Bio, Saint-Germain-en-Laye, France)
into the pFB retroviral vector (Stratagene, La Jolla, CA), resulting in pFB-CMV-tdT
vector. Two shRNA Epha4 cassettes were cloned from the pSUPER shuttle vector
into the retroviral vector.
 1278 Journal of Cell Science 124 (8)
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 using
Quickchange Mutagenesis Kit (Stratagene), and cloned the modified Epha4 ORF
into the pCDH-EF1-T2A-copGFP (original plasmid from System Bioscience,
Mountain View, CA) or pCDH-EF1-T2A-tdT (pCDH-EF1-T2A-copGFP where we
substituted copGFP for tdTomato) lentiviral vector. To obtain retroviral plasmid
containing modified EphA4, the whole EF1-modEphA4-T2A-copGFP cassette was
re-cloned into pFB retroviral vector.
CopGFP, a green fluorescent protein, was linked to ORF Epha4 through the T2A
peptide sequence. We were not able to detect the native fluorescent signal of copGFP
when expressed under the control of the EF1 promoter (elongation factor 1), but there
was a bright copGFP signal in infected cells when stained with copGFP antibodies
(supplementary material Fig. S12A–C). The T2A sequence allows the generation of
two proteins expressed at the same level (Osborn et al., 2005). CopGFP antibodies
do not cross-react with EGFP (supplementary material Fig. S12C), hence the
expression of both fluorescent proteins in the same cell can be detected unambiguously.
The kinase-dead EphA4 mutant was generated as described previously (Kullander
et al., 2001), exchanging lysine 653 with a methionine residue. The mutation was
introduced into modified EphA4 resistant to Epha4-1 by Quickchange Mutagenesis
Kit (Stratagene), and the kinase-dead Epha4 ORF was cloned into pCDH-EF1-T2A-
copGFP (System Bioscience) lentiviral vector. To obtain retroviral plasmid containing
kinase-dead EphA4, the whole EF1-EphA4KD-T2A-copGFP cassette was re-cloned
into pFB retroviral vector.
RNA isolation, cDNA synthesis and quantitative real-time PCR
RNA isolation, cDNA synthesis and quantitative real-time (qRT)-PCR was performed
as described previously (Khodosevich et al., 2007). mRNA levels detected by qRT-
PCR were normalized to mRNA levels for Gapdh.
Analysis of shRNA silencing efficiency
Journal of Cell Science
The efficiency of shRNA silencing was tested using qRT-PCR and western blot
using HEK293 cell culture transfections in triplicates. Two out of five shRNAs that
specifically knocked down Epha4 gene expression to less than 25% were chosen for
functional experiments described in the study. The efficiency of shRNA silencing
was also tested by viral infections of neurospheres and in vivo brain infections.
Production of recombinant viruses
Recombinant lentiviruses were produced as previously described (Khodosevich et
al., 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 titer
To measure viral titers, a dilution series across five orders of magnitude of viral stock
solutions were used for HEK293 cell infection. Each sample was analyzed in
triplicate. After 4 days of incubation at 37°C, the number of fluorescent cell plaques
at the different viral dilutions was measured and the viral titer was estimated in
fluorescent plaque forming units per milliliter (pfu/ml).
Neurosphere cultures
SVZ regions were dissected from coronal sections of wild-type P4 mice. All steps
of tissue processing were done in dissection media (10⫻ DM: 100 mM MgCl2, 10
mM kynurenic acid, 100 mM HEPES in 1⫻ Hank’s balanced salt solution). Dissected
SVZ regions were incubated for 5 minutes with 30 U of papain (Worthington,
Lakewood, NJ) and 0.0005% DNase solution, and washed in Neurobasal Media
Supplemented [500 ml of Neurobasal media + 10 ml 50⫻ B27-Supplement + 1.25
ml 200 mM L-glutamate + 5 ml penicillin/streptomycin (100 U/ml)] containing also
trypsin inhibitor (Sigma-Aldrich) and 0.0005% DNAse. Cells were triturated using
a fire-polished Pasteur pipette, counted and plated at a density of 100,000 cells/ml
in Neurobasal Media Supplemented containing 20 ng/ml EGF and 20 ng/ml FGF.
To obtain secondary neurospheres, the primary neurospheres were dissociated by
trypsin 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 in
Neurobasal Media Supplemented containing 20 ng/ml EGF and 20 ng/ml FGF.
Proliferation analysis: at DIV1, cells were infected with shRNA-expressing viruses
with or without the additional presence of rescue virus. At DIV6, BrdU was added
to the culture media to give a final concentration of 10 M. Neurospheres were
incubated with BrdU for 12 hours and subsequently fixed.
Differentiation analysis: at DIV1, cells were infected with shRNA-expressing
viruses with or without the additional presence of rescue virus. At DIV7, neurospheres
were transferred to Neurobasal Media Supplemented without growth factors, and at
DIV9 (for nestin immunostaining) or DIV11 (for GFAP, S100 and Tuj1
immunostainings) cultures were fixed.
Injection of recombinant viruses into mouse brain
The 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 studies
we 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 rescue
viruses (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’ indicates
108 units/ml. An aliquot of 1 l of recombinant retrovirus or lentivirus (or mix of
viruses) was delivered into the SVZ of each hemisphere of P6 C57BL/6 mouse pups
with a Hamilton (Hamilton, Bonaduz, Switzerland) syringe using special needles for
precise 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 injection
for retroviruses and 4, 7, 15, 25, 40, 50, 60, 80 or 100 days after injection for
lentiviruses, 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 statistics
Data were analyzed using t-test or ANOVA by GraphPad Prism version 5.00 for Mac
OS X (GraphPad Software, San Diego California, www.graphpad.com). All data
were normally distributed according to the d’Agostino and Shapiro-Wilk tests. n
always defines the number of independent experiments. Absolute cell counts, number
of neurospheres and culture coverslips, number of brain sections and animals used
for all quantifications are shown in supplementary material Table S2.
Co-labeling in neurospheres was quantified using stacks of confocal images
obtained by using 0.3 m steps acquired with 63⫻ immersion objective (confocal
microscope LSM 5 Pascal, Zeiss, Germany). For the markers that label only cell
processes (nestin and GFAP) co-localization was determined only after careful
examination of tdTomato coexpression in the processes. Low titer retrovirus infection
assured scarcity of infected cells in neurospheres. Quantifications in Figs 2 and 3
show mean ± s.d.; number of cells >100, number of neurospheres ≥30, number of
culture coverslips for each condition ≥6.
Numbers of fluorescent cells in the RMS or olfactory bulb were evaluated relative
to the total number of infected cells around the SVZ. GFAP-, Sox2-, NeuN-,
S100-, nestin-, BrdU-, Mash1, Dcx-, activated caspase-3-positive fluorescent cells
in the SVZ were evaluated as percentage of the total number of infected cells around
the SVZ. Injections with the same type of virus and titer resulted in approximately
the 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 altered
proliferation in the SVZ after Epha4 knockdown for the following reasons. Although
cell 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 as
newly born cells in the SVZ and eventually migrate to the olfactory bulb; hence they
are not considered to be ‘infected cells around the SVZ’ once they reach the RMS.
Most importantly, all normalized quantifications (except NeuN co-localization) showed
a decrease in the number of specifically labeled cells (i.e. GFAP+, Sox2+, BrdU+,
Mash1+, nestin+) after Epha4 knockdown. Because the index is obtained by dividing
specifically labeled infected cells by all infected cells in the SVZ, reduced proliferation
in the SVZ would result in a decreased number of total infected cells in the SVZ, and
hence in a higher index in Epha4 knockdown experiments than controls, which was
not the case.
Analysis of cell proliferation in vivo by BrdU
To label fast-proliferating cells, mice were injected once with 50 mg BrdU/kg body
weight. To label slow-proliferating cells, 1 mg/ml BrdU was administered in the
drinking water for 1 week followed by 3 weeks of normal water consumption to
obtain BrdU dilution in fast-proliferating cells. For the analysis of dividing cells
after lentiviral infection, mice were injected with BrdU twice, the two time-points
being 6 hours apart, and sacrificed 24 hours later. Postnatal animals were injected
with 20 mg BrdU/kg body weight and adult animals with 50 mg/kg.
Immunohistochemistry and immunocytochemistry
Sagittal brain sections (50–75 m) were cut with a vibratome (Leica VT1000S,
Leica, Germany). Immunostainings were carried out on free-floating sections. Slices
were blocked in 0.2–1% Triton and 2–5% normal goat serum or 5% bovine serum
albumin and incubated overnight with primary antibodies at 4°C followed by
incubation with secondary antibodies at room temperature. Primary and secondary
antibodies 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, then
neutralized with 10 mM Tris (pH 8.5) at room temperature for 15 minutes and
further processed as for other stainings. Sections were mounted onto slides with
Mowiol (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 analysis
For western-blot analysis, protein samples were boiled in SDS gel sample buffer.
Denatured proteins were separated by SDS-PAGE, transferred onto PVDF membranes
and probed with antibodies. For statistical analysis, antibody signals were quantified

using NIH ImageJ software and values normalized to the corresponding -actin
signals. Data were analyzed using the Student’s t-test.
We thank Ulla Amtmann, Regina Hinz-Hernkommer and Irmgard
Preugschat-Gumbrecht for technical assistance. This work was
supported in part by the Schilling Foundation and DFG (SFB488
grant).
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/124/8/1268/DC1
References
Abrous, 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 the
differentiation of mammalian neural precursor cells through a mitogen-activated protein
kinase-dependent pathway. J. Biol. Chem. 279, 32643-32650.
Bourgin, C., Murai, K. K., Richter, M. and Pasquale, E. B. (2007). The EphA4 receptor
regulates 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. and
ffrench-Constant, C. (2004). Beta1 integrins activate a MAPK signalling pathway in
neural stem cells that contributes to their maintenance. Development 131, 3433-3444.
Celikel, T., Marx, V., Freudenberg, F., Zivkovic, A., Resnik, E., Hasan, M. T.,
Journal of Cell Science
Licznerski, P., Osten, P., Rozov, A., Seeburg, P. H. et al. (2007). Select overexpression
of 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, and
polarity 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 migration
and 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 Rin1
regulates 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 size
by regulating apoptosis of neural progenitors. Nature 435, 1244-1250.
Doetsch, F., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (1997). Cellular composition
and three-dimensional organization of the subventricular germinal zone in the adult
mammalian brain. J. Neurosci. 17, 5046-5061.
Doetsch, F., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (1999). Regeneration of a
germinal 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 of
EphA 4 kinase activity is required for a subset of axon guidance decisions suggesting
a 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-mediated
dendritic 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 receptor
functions 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 and
sensory 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). Axonal
regeneration 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 regulates
central 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 negatively
regulates 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 activity
prompts gamma-secretase-mediated cleavage of EphA4 and dendritic spine formation.
J. Cell Biol. 185, 551-564.
EphA4 maintains adult neural stem cells 1279
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 of
distinct 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 of
adult 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 induces
proliferation 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 adult
neurogenesis. Curr. Opin. Neurobiol. 14, 186-191.
Khodosevich, K., Inta, D., Seeburg, P. H. and Monyer, H. (2007). Gene expression
analysis of in vivo fluorescent cells. PLoS One 2, e1151.
Khodosevich, K., Seeburg, P. H. and Monyer, H. (2009). Major signaling pathways in
migrating 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. and
Khodosevich, K. (2010). Increased subventricular zone-derived cortical neurogenesis
after 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 axon
tract formation in vivo. Neuron 29, 73-84.
Liebl, D. J., Morris, C. J., Henkemeyer, M. and Parada, L. F. (2003). mRNA expression
of ephrins and Eph receptor tyrosine kinases in the neonatal and adult mouse central
nervous system. J. Neurosci. Res. 71, 7-22.
Lledo, P. M. and Saghatelyan, A. (2005). Integrating new neurons into the adult olfactory
bulb: 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 functional
plasticity in neuronal circuits. Nat. Rev. Neurosci. 7, 179-193.
Lois, C., Hong, E. J., Pease, S., Brown, E. J. and Baltimore, D. (2002). Germline
transmission 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 adult
mammalian 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 analyses
reveal the properties of endogenous neural stem cell proliferation in the adult mammalian
forebrain. Development 125, 2251-2261.
Ninkovic, J. and Gotz, M. (2007). Signaling in adult neurogenesis: from stem cell niche
to 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 EphA4
forward 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). A
picornaviral 2A-like sequence-based tricistronic vector allowing for high-level
therapeutic 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). Rat
forebrain 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). Intrinsic
programs 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. Cell
Biol. 181, 973-983.
Ricard, J., Salinas, J., Garcia, L. and Liebl, D. J. (2006). EphrinB3 regulates cell
proliferation and survival in adult neurogenesis. Mol. Cell. Neurosci. 31, 713-722.
Rogelius, N., Ericson, C. and Lundberg, C. (2005). In vivo labeling of neuroblasts in
the 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). In
vivo fate analysis reveals the multipotent and self-renewal capacities of Sox2+ neural
stem 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 the
expansion of neural progenitor cells in the subventricular zone by regulating p53 during
homeostasis 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 the
postnatal 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 guidance
is mediated by the RacGAP alpha2-chimaerin. Neuron 55, 756-767.
Zhao, C., Deng, W. and Gage, F. H. (2008). Mechanisms and functional implications of
adult neurogenesis. Cell 132, 645-660.