www.elsevier.com/locate/ymcne

Mol. Cell. Neurosci. 31 (2006) 713 – 722

EphrinB3 regulates cell proliferation and survival in

adult neurogenesis

Jerome Ricard, Jessica Salinas, Lissette Garcia, and Daniel J. Liebl*

The Miami Project to Cure Paralysis and Department of Neurosurgery, University of Miami School of Medicine, 1095 NW 14th Terrace,

R-48, Miami, FL 33136, USA

Received 10 August 2005; revised 7 December 2005; accepted 2 January 2006

Available online 17 February 2006

Interactions between ephrins and their receptors have been implicated

in many processes during central nervous system development. In the

adult, ephrins and Eph receptors have been implicated in controlling

cell proliferation and neuroblast migration, although there is no direct

evidence for the role of ephrinB3 in these functions. In addition,

activation of Eph receptors has been shown to regulate transduction

pathways important in cell cycle control as well as cell death. We show

that ephrinB3 contributes to the control of cell proliferation and

survival in the adult subventricular zone (SVZ). EphrinB3�/� mice

exhibit a significant increase in dividing cells along the lateral ventricle,

and altered expression of proteins involved in cell cycle regulation.

Gain-of-function approach by infusing soluble ephrinB3-Fc molecules

in ephrinB3�/� can suppress cell proliferation to wild type levels. At the

same time, ephrinB3 also regulates cell survival as greater numbers of

cells die in the SVZ of ephrinB3�/� mice. Together, our results suggest

that ephrinB3 negatively regulates cell cycle progression and cell

apoptosis in the adult subventricular zone.

D 2006 Elsevier Inc. All rights reserved.

Keywords: Ephrin B3; Eph receptors; Stem cells; Subventricular zone;

Apoptosis; Proliferation

Introduction

The subventricular zone (SVZ) is a principle region of

neurogenesis in the adult rodent brain (Alvarez-Buylla and

Garcia-Verdugo, 2002). In normal physiological conditions, neural

stem/progenitor cells in the SVZ give rise to intermediate

progenitors, also named transit-amplifying precursors, which in

turn differentiate into neuroblasts that are destined for the olfactory

bulb (OB). In the OB, these migrating neuroblasts terminally

differentiate into interneurons (Luskin, 1993; Betarbet et al., 1996).

The mechanisms that govern these events are tightly regulated

1044-7431/$ - see front matter D 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.mcn.2006.01.002

* Corresponding author. Fax: +1 305 243 3914.

E-mail address: dliebl@miami.edu (D.J. Liebl).

Available online on ScienceDirect (www.sciencedirect.com).

during neurogenesis from stem cell to neuron. In particular, to

ensure that there is an adequate pool of cells at each stage of

differentiation, proliferation within these stages requires specific

environmental and intracellular signaling mechanisms. In doing so,

the stem/progenitor cells generate the intermediate progenitors

while maintaining their own pool. The intermediate precursors may

use repeated cell divisions to expand their numbers and thus

greatly amplify the number of neuroblasts produced. Further

divisions within the neuroblast population during migration

contribute to the generation of a large amount of new neurons

(Alvarez-Buylla et al., 2001).

Cell proliferation in the SVZ was shown to be increased by a

variety of growth factors and hormones, including epidermal

growth factor (EGF), fibroblastic growth factor (FGF), vascular

endothelial growth factor (VEGF) and thyroid hormone (Doetsch

et al., 2002a,b; Jin et al., 2002, 2003; Lemkine et al., 2005). Other

molecules involved in the control of neural precursors prolifer-

ation include enhancers such as h1-integrins and retinoic acid

(Leone et al., 2005; Wang et al., 2005) and repressors such as neu-

rofibromin (Dasgupta and Gutmann, 2005). There is a tight link

between cell cycle control and neurogenesis (Ohnuma and Harris,

2003), which is further supported by a reduction in cell proli-

feration and neuroblast generation in mice lacking expression of

the E2F1 transcription factor (Cooper-Kuhn et al., 2002), and by

an increase in the number of proliferating precursors in p107�/�

mice (Vanderluit et al., 2004). An increase in proliferation in the

SVZ is also found in p27Kip1�/� mice and is coupled to a decrease

in neuroblast generation (Doetsch et al., 2002a,b). Similarly, in rats,

a decrease in p27Kip1 expression in the SVZ following a stroke

coincides with an increased cell proliferation (Zhang et al., 2004).

In addition, cell death also controls cell numbers in the SVZ,

probably through the release of yet non-identified diffusible factors

(Agasse et al., 2004). Finally, physiological regulation may also be

provided by sensory signals originating from the OB, where

axotomy-induced deafferentation of the OB leads to an increase

in proliferation in the SVZ (Mandairon et al., 2003). This increase

in proliferation is coupled to an up-regulation of cell death.

Conversely, others have reported decreased proliferation 3 months

J. Ricard et al. / Mol. Cell. Neurosci. 31 (2006) 713–722714

after bulbectomy (Kirschenbaum et al., 1999). It is still notable that

OB inputs do not appear to be essential for neurogenesis to occur in

the SVZ, suggesting that local cues and not diffusible OB factor

may be required to regulate neurogenic events.

One group of molecules that has been recently implicated in

proliferation of stem/progenitor cells in the SVZ is the ephrin

family and their Eph receptors. Intraventricular infusions of

ephrinB2-Fc or EphB2-Fc molecules lead to increases in the

number of astrocytes (some of which may be the stem/progenitor

cells) and transit-amplifying precursors, as well as a decrease in the

number of neuroblasts after EphB2-Fc injections. RT-PCR experi-

ments show that ephrinB2 and ephrinB3 ligands, as well as EphB1,

EphB2, EphB3 and EphA4 receptors are expressed in the SVZ

(Conover et al., 2000). Furthermore, EphB2 directly activates the

proliferation of SVZ precursors in vitro (Katakowski et al., 2005).

Recently, the A-class was implicated as well in the control of

neurogenesis: ephrinA2 and EphA7 were identified as negative

regulators of neural precursors proliferation (Holmberg et al.,

2005). However, ephrinA1 does not affect telencephalon embry-

onic progenitors proliferation, but rather directs cells towards a

neuronal fate (Aoki et al., 2004). Ephrins are membrane-bound

ligands and tethered to the cell surface, by either a glycosylphos-

phatidylinositol (GPI)-anchor (A-class) or transmembrane domain

(B-class). Eph receptors, which belong to the largest family of

receptor tyrosine kinases, are also subdivided into two A- and B-

classes, based on their preferential binding for one particular group

of ligands. One exception is EphA4 as it binds ligands from both

subclasses. Therefore, receptor activation by ligand binding is a

direct result of cell–cell contact, and interactions between ephrins

and Eph receptors can result in bidirectional signaling from both

the ligand and receptor (Flanagan and Vanderhaeghen, 1998).

Although the binding between ligands and receptors is considered

very promiscuous, specific ligand–receptor interactions exist

within each subclass (Blits-Huizinga et al., 2004).

Here, we examined a novel role for ephrinB3 in neurogenic

proliferation of the SVZ. Examination of gene-targeted knockout

mice for ephrinB3 revealed a significant increase in the number of

proliferating stem/progenitor cells in the SVZ. We also observed

significant increases in cell cycle activators and decreases in cell

cycle inhibitors in the absence of ephrinB3. An increase in

apoptosis in neurogenic regions is associated with the increase in

proliferation in the absence of ephrinB3. We propose that ephrinB3

functions as a negative regulator of cell proliferation and cell

apoptosis in the SVZ during adult neurogenesis.

Results

Expression of B-class ephrins in the adult SVZ and RMS regions

Neurogenesis continues to persist in the subventricular zone into

adulthood, where stem/progenitor cells give rise to neuroblasts that

migrate through the rostral migratory stream (RMS) to become

interneurons in the olfactory bulb. Previous reports have implicated

Fig. 1. EphrinB ligands are expressed in the adult subventricular zone. EphrinB1

GFAP (green, b, d) but not PSA-NCAM (green, e, g). EphrinB2 (green, h, j, m)

localizes with GFAP (red, i, k) but not PSA-NCAM (red, l, n). Insets: high-mag

staining in ephrinB3hgal mice) is excluded from the RMS (o, sagittal section; inse

devoid of ephrinB3 contains PSA-NCAM-stained neuroblasts (red, r, s), which see

receptors detected by RT-PCR in cultured neurospheres. (u) Expression of Eph re

a role for B-class ephrins in the adult rodent SVZ, however, it is

unclear which ephrin(s) may be involved (Conover et al., 2000). To

further elucidate the role of ephrins in the SVZ, we initially

examined the expression of this family of ligands in the subven-

tricular zone. All three ephrinBs were observed in the SVZ or

surrounding tissues, where ephrinB1 and ephrinB2 were expressed

on cells residing in the SVZwhile ephrinB3 was expressed in tissues

surrounding the SVZ and RMS (Fig. 1). In particular, ephrinB1 was

observed on cells that resided in the SVZ along the striatal, septal,

and cortical sides (Fig. 1a). In addition, ephrinB1 was found to be

co-expressed with GFAP (Figs. 1b–d), an astrocytic marker found

also in stem/progenitor cells in the SVZ, but not PSA-NCAM (Figs.

1e–g). EphrinB2 was expressed in cells that resided in and around

the SVZ and corpus callosum (CC) (Fig. 1h). Like ephrinB1,

ephrinB2 was also co-expressed with GFAP and not PSA-NCAM;

however, some of the GFAP-expressing cells have a ramified

appearance that likely represents mature astrocytes while other

GFAP-expressing cells appeared to be morphologically similar to

stem/progenitor cells (Figs. 1i–n). In addition, we cannot rule out

the possibility that ephrinB1 and ephrinB2may also be expressed on

ependymal cells, since cross-reactivity with anti-GFAP in such cells

antibodies has been shown previously (Doetsch et al., 2002a,b). Co-

expression of ephrinB1 and ephrinB2 with GFAP was confirmed

using confocal microscopy (Figs. 1b–g insets, i–n insets). To

examine ephrinB3 expression, we took advantage of a transgenic

knock-in mouse where h-galactosidase (ephrinB3hgal) replaces thecytoplasmic domain. X-gal staining was observed throughout the

brain, including the cortex, striatum, septum, and CC; however, we

did not observe staining within the SVZ or RMS (Figs. 1o, p). Figs.

1q–s represent an overlay image where PSA-NCAM-expressing

neuroblasts where found to mainly reside along a border of

ephrinB3 expression in the SVZ. To confirm whether ephrinB1

and ephrinB2 but not ephrinB3 are present in stem/progenitor cell

populations, we examined mRNA expression in purified neuro-

spheres. RT-PCR analysis using RNA from cultured neurospheres

generated from adult SVZ cells showed expression of ephrinB1 and

ephrinB2 but not ephrinB3, supporting our immunohistochemical

observations (Fig. 1t). These findings also support the presence of

ephrinB1 and ephrinB2 in the stem/progenitor cell population. We

also found that EphB1, EphB3, and EphA4 receptors were all

expressed in the neurospheres, which specifically contain stem/

progenitor cell populations. Further analysis of carefully dissected

SVZ tissue revealed that in addition to EphB1, EphB3, and EphA4

mRNA expression, EphB2, EphB4, and EphB6 mRNA transcripts

were also localized to tissues in or closely associated to the SVZ

(Fig. 1u). These studies demonstrate that multiple ephrins and Eph

receptors are present in and/or around the SVZ, and may play

important roles in regulating adult neurogenesis.

Deletion of ephrinB3 results in cell proliferation and not migration

abnormalities in the subventricular zone

Previous reports have implicated ephrins in regulating cell

migration and proliferation in the adult SVZ when ephrin-Fc or

(red, a, c, f) is expressed in the walls of the ventricle, and co-localizes with

is expressed along the ventricle and in the corpus callosum, and also co-

nification confocal images. Expression of ephrinB3 (identified using X-gal

t: cross-section of the RMS) and SVZ (p, q). The area lining the ventricle

m to align along the ephrinB3 border (s). (t) Expression of ephrins and Eph

ceptors detected by RT-PCR in carefully dissected SVZ tissues.

J. Ricard et al. / Mol. Cell. Neurosci. 31 (2006) 713–722 715

Eph-Fc molecules were infused into the adult lateral ventricle

(Conover et al., 2000; Holmberg et al., 2005). To examine whether

ephrinB3 is a candidate ephrin for these functions, we examined

cell migration and proliferation in ephrinB3�/� mice. The

expression pattern of ephrinB3 would support a role in maintaining

SVZ and RMS boundaries for migrating stem/progenitor cells or

J. Ricard et al. / Mol. Cell. Neurosci. 31 (2006) 713–722716

neuroblasts. However, analysis of neuroblast migration in the

ephrinB3�/� mice revealed no gross migration abnormalities

where PSA-NCAM-expressing cells were not present outside the

SVZ or RMS boundaries (Figs. 2a, b). In addition, 5-bromodeox-

yuridine (BrdU) pulsing did not reveal any BrdU-positive cells

outside the SVZ or RMS (Figs. 2c, d). To further evaluate potential

migratory defects, we examined the ability of cells to migrate from

the SVZ using the lipophilic tracer, DiO, as well as BrdU

incorporation. DiO was infused into the lateral ventricle to trace

cells that originate in the SVZ and eventually migrate through the

RMS to the OB. DiO is a good marker for migrating cells since it is

maintained in the cell’s bilipidic membrane and does not diffuse

between cells (Salman et al., 2004). Over a 4-day period, we did

not observe a significant difference in the ability of DiO-labeled

wild type or ephrinB3�/� cells to reach the OB (Figs. 2e–j). DiO-

labeled cells co-labeled with PSA-NCAM demonstrating that DiO

labeled cells represent migrating neuroblasts. In addition, BrdU

pulsing followed by a 7-day migration period demonstrated that

daughter cells generated from precursors having incorporated the

BrdU have the ability to migrate to the OB (Figs. 2k, l). We did not

observe BrdU-labeled or Ki67-positive cells outside the RMS zone

in the absence of ephrinB3 (not shown), demonstrating that

ephrinB3 does not function to maintain SVZ and RMS boundaries

by preventing migration into surrounding tissues. However,

stereological counts revealed there were significantly fewer

BrdU-labeled cells in the OB of ephrinB3�/� mice (3635 T 287

mm3) as compared to wild type mice (7765 T 1323; P = 0.038

mm3). Furthermore, this was not a function of proliferation in the

Fig. 2. EphrinB3�/� mice do not show migratory defects in neuroblasts or pro

(red, a, b) or BrdU-labeled cells (green, c, d) showed no expression outside the

the ephrinB3�/� mice (b, d) as compared to wild type (a, c) littermates. (e–

ephrinB3�/� (e–g) and wild type (h– j) mice, and colabel with PSA-NCAM (g,

days after pulsing as compared to wild type OB (k). CC, corpus callosum; St,

OB since Ki67 immunostaining was not observed in the OB (not

shown). These findings demonstrate that ephrinB3 does not

regulate migratory boundaries of the SVZ and RMS, nor their

ability to reach the OB, but may influence the number of cells that

reach the OB.

To evaluate whether the observed reductions in BrdU cells in

the ephrinB3�/� OB resulted from alterations in proliferation, we

quantitatively examined proliferation in adult SVZ of wild type

and ephrinB3�/� mice using both BrdU incorporation and Ki67

labeling. Quantitative analysis demonstrated that there is a sig-

nificant increase in the number of Ki67-labeled cells in the

ephrinB3�/� mice (17,349 T 3368) as compared to wild type

littermates (12,578 T 2667; P = 0.025) (Fig. 3a). These increases

were not observed in the ephrinB3hgal mice, which retain the

extracellular domain of the ligand and thus the capability to trigger

signaling on respective Eph receptors, suggesting that activation of

the receptors is essential for the proper control of cell proliferation

in the SVZ. Cell counts were also performed on animals lacking

individual receptors in order to uncover the receptor involved in

proliferation control. EphB3, EphA4 and to a lesser degree EphB2

have the ability to bind ephrinB3. However, no other genotype

exhibited the increased proliferation observed in ephrinB3�/�

animals, raising the question of potential compensatory mecha-

nisms due to promiscuous binding properties in the Eph family of

receptors. To provide additional support for the role of ephrinB3 in

regulating cell proliferation in the SVZ, we examined the number

of BrdU-labeled cells in the SVZ following a 1-h pulse. We

observed a significant increase in the number of BrdU-labeled cells

liferating cells residing in the SVZ. Neuroblasts stained for PSA-NCAM

SVZ. Numbers of PSA-NCAM and BrdU-labeled cells were increased in

j) DiO-labeled neuroblasts migrate through the RMS to the OB in both

j). Fewer BrdU-labeled cells (green) in ephrinB3�/� OB (l) are detected 7

striatum; LV, lateral ventricle. Bar: 100 Am.

J. Ricard et al. / Mol. Cell. Neurosci. 31 (2006) 713–722 717

in the ephrinB3�/� mice (7295 T 653) as compared to wild type

mice (3847 T 1238; P = 0.044) (Fig. 3b). This increased

proliferation in the ephrinB3�/� mice correlated with an increase

in the number of neuroblasts present in the SVZ (Fig. 3c). We also

examined vimentin expression (a marker not specific for cell

proliferation but known to be up-regulated in germinal zones

(Alonso, 2001)), and found increased expression of vimentin in the

ephrinB3�/� mice as compared to wild type mice (Figs. 3d, e). In

contradiction to the reduction of BrdU-labeled cells in the OB,

these findings support the role of ephrinB3 in regulating cell

proliferation that results in increased numbers of neuroblasts in the

SVZ of ephrinB3�/� mice.

We also performed a primary neurosphere assay in order to

determine whether the increase in proliferation and neuroblasts

observed in the ephrinB3�/� animals could be linked to the

presence of greater numbers of progenitors. A defined number of

cells were plated from wild type and ephrinB3�/� SVZ and the

Fig. 3. EphrinB3�/� mice have increased numbers of proliferating cells and

neuroblasts in the adult SVZ as compared to wild type mice. (a)

Stereological counts of Ki67-labeled cells in the SVZ of wild type and

ephrinB3�/� mice. Stereological counts of BrdU-labeled cells (b) and PSA-

NCAM-labeled (c) cells in the SVZ of wild type and ephrinB3�/� mice.

Vimentin immunostaining in the SVZ of wild type (d) and ephrinB3�/�

(e) mice.

Fig. 4. EphrinB3�/� SVZ contains more neurosphere-forming cells than

wild type mice. SVZ cells from wild type or ephrinB3�/� mice were put in

culture and neurospheres (inset) formed after 1 week were counted. The

graph shows the mean of six different wells and is representative of three

different experiments. *P < 0.05. Bar: 100 Am.

number of neurospheres present in the culture after 1 week was

counted. The cell suspension obtained from the ephrinB3�/�

mice led to the formation of significantly more neurospheres

(Fig. 4). Although primary neurospheres are not necessarily

uniquely derived from stem cells (Reynolds and Rietze, 2005),

they originate from progenitors able to proliferate in particular

culture conditions. Our results thus indicate that more neuro-

sphere-forming cells, hence more neural progenitors are present

in the SVZ of adult ephrinB3�/� mice as compared to wild type

mice.

To provide additional support for the role of ephrinB3 on the

control of SVZ cells proliferation, we infused pre-clustered

ephrinB3-Fc molecules in the lateral ventricle of wild type and

ephrinB3�/� mice for 1 week using osmotic pumps. Infusion

controls consisted of only the Fc portion of the molecule.

Proliferation was assessed by stereological analysis of BrdU

incorporation, which demonstrated that infusion of ephrinB3-Fc

resulted in a significant decrease in the number of dividing cells

associated with ephrinB3�/� mice (Fig. 5). Interestingly, the

number of proliferating cells in the ephrinB3�/� mice after

exposure to the ligand decreased to levels found in wild type

mice, while wild type mice were not significantly influenced by the

infusion of supplementary ephrinB3 (Fig. 5). Together, these

observations indicate that ephrinB3 exerts an inhibitory role on

proliferation of stem/progenitor cells in the SVZ.

Alterations in cell cycle proteins in ephrinB3�/� mice

To begin examining the role of ephrinB3 on cell cycle, we

initially screened a panel of cell cycle proteins using Western blot

analysis (Pharmingen Powerblot). Briefly, SVZ tissues were

Fig. 5. Infusion of soluble ephrinB3-Fc reduces proliferation in the

ephrinB3�/� mice. BrdU incorporation was assessed after 1 week of

infusion with pre-clustered Fc fragments (for control) or pre-clustered

ephrinB3-Fc molecules. *P < 0.05.

Fig. 6. Western blot analysis (BD Pharmingen Power blot) of cells cycle

regulating proteins shows a significant increase in cell cycle activators

(p19SKP1 and PCNA) and decrease in cell cycle inhibitors (p27Kip1) in

ephrinB3�/� mice as compared to wild type mice. Increases were also

observed in phosphorylated (p)-Akt as compared to total Akt and h-tubulincontrols. Percent change and significance are shown in Table 1.

J. Ricard et al. / Mol. Cell. Neurosci. 31 (2006) 713–722718

carefully dissected from 5 wild type or ephrinB3�/� mice,

combined, and run on a high-throughput Western blot screen in

triplicate. Table 1 shows the effects of ephrinB3 deficiencies on a

variety of cell cycle genes. We observed several cell cycle markers

or promoters, such as PCNA, p19Skp1 and BM28 (also known as

MCM2), to be up-regulated, while cell cycle inhibitors, such as

p27Kip1, were down-regulated in ephrinB3�/� mice (Fig. 6).

Furthermore, we observed significant alterations in the levels of

Table 1

Cell cycle protein levels are altered in the SVZ between wild type and

ephrinB3�/� mice

Protein identity Changea P value

Cytoskeleton

Beta-tubulin NS N/A

Arp3 NS N/A

p21-Arc NS N/A

Cell cycle

p27Kip1 , 2.4 fold <0.01

CRP2 , 1.7 fold <0.01

Phospho-PKA RIIB j 2.5 fold <0.001

PCNA j 3.2 fold <0.01

Hsp70 j 1.2 fold <0.01

BM28 (MCM2) j 2.3 fold <0.01

Stat2 j 4.7 fold <0.01

p19Skp1 j 2.2 fold <0.05

PP5 j 1.9 fold <0.05

Phospho-AKT j 1.3 fold <0.05

14-3-3e NS N/A

cdc37 NS N/A

Chk2 NS N/A

p21Cip NS N/A

CyclinD3 NS N/A

This table presents observed changes that are significantly different.

Samples were run in triplicate and the mean of the normalized intensities

was used for the statistical analysis. NS, not significant; N/A, not attempted.a Change in ephrinB3�/� compared to wild type. P values obtained using

the student t test.

phosphorylated Akt (p-Akt), Stat2 and Hsp70 among others, while

phosphorylated ERK, CKII, p21Cip, Chk2 and cyclinD3 were

unchanged. In addition, cytoskeletal proteins such as h-tubulin and

Arp3 were unchanged, suggesting that the increases in protein

levels was not simply due to increased numbers of cells associated

with the ephrinB3�/� mice. The cell cycle protein p27Kip1 is a

likely modulator of stem/progenitor cell cycle and has been shown

to be modulated by the Ras-Akt signal cascade, and Eph receptors

signal transduction include Ras pathways (Dodelet et al., 1999;

Zou et al., 1999; Elowe et al., 2001; Miao et al., 2001). Akt is

known to participate in the down-regulation of p27Kip1 (Narita et

al., 2002). The fact that p-Akt was increased in the ephrinB3�/�

mice may suggest that the same mechanism is at play in those

animals to regulate neurogenesis. Together, these results support an

ephrinB3 modulation of cell cycle regulating proteins in the adult

SVZ.

Deletion of ephrinB3 causes an increase in apoptotic cell death in

the subventricular zone

To examine why fewer BrdU labeled cells are present in the OB

of ephrinB3�/� mice when increased proliferation occurs in the

SVZ, we examined whether there were alterations in apoptosis in

the SVZ. Stereological counts of TUNEL-labeled cells in the adult

SVZ revealed a 45% increase in the numbers of TUNEL-positive

cells in the ephrinB3�/� (2336 T 411) mice as compared to wild

type (1329 T 141; P = 0.038) mice (Figs. 7a, b). However, in the

OB there were fewer TUNEL-positive cells in the ephrinB3�/�

mice (824 T 181 mm3) as compared to the wild type mice (2859 T514 mm3; P = 0.02) (Figs. 7c, d), which correlates to the reductions

observed in the number of migrating BrdU-labeled neural

progenitor cells. Many of the cells that were found to be labeled

by the TUNEL procedure were also BrdU-positive (Fig. 7, insets in

c and d), indicating that a fraction of the newly arriving neuroblasts

fail to survive and will not integrate into the bulb circuitry.

Together, these findings suggest that ephrinB3 also functions to

regulate cell apoptosis of neural progenitor cells in the SVZ, where

the excessive numbers of cells associated with the absence of

ephrinB3 could be reduced through apoptosis prior to entering the

Fig. 7. EphrinB3�/� mice have increased numbers of apoptotic cells in the

SVZ but fewer in the OB. Images comparing numbers of TUNEL-labeled

cells in the SVZ and OB of wild type (a, c) and ephrinB3�/� mice (b, d).

Quantification numbers appear in the text. *P < 0.05. Insets in c and d:

some newly generated cells having reached the bulb (labeled with BrdU,

red) were found to be TUNEL-positive (green). Bar: 100 Am.

J. Ricard et al. / Mol. Cell. Neurosci. 31 (2006) 713–722 719

OB. Upon entry into the OB, apoptosis seems to occur at a similar

rate between ephrinB3�/� and wild type mice.

Discussion

There are a number of mitogenic extracellular cues that can

activate stem/progenitor cells proliferation; however, less is known

about cues that suppress proliferation. This article describes

ephrinB3 as a new negative regulator of stem/progenitor cell

proliferation in the adult SVZ. We show that ephrinB1, ephrinB2,

and Eph receptors are expressed in stem/progenitor cells, while

ephrinB3 is expressed in regions that border the SVZ and RMS.

Examination of ephrinB3�/� mice revealed that ephrinB3 func-

tions to inhibit cell proliferation in the SVZ, and that increased

proliferation in the knockout animals can be blocked by infusion of

soluble ephrinB3. We also demonstrate that ephrinB3 may function

through Eph receptor signaling to up-regulate cell cycle activators

and down-regulate cell cycle inhibitors. Finally, we show that the

number of newly generated neurons can be counterbalanced by an

ability of ephrinB3 to also regulate cell apoptosis in neurogenic

regions. Together, these findings demonstrate a role for ephrinB3

in its ability to regulate cell turn over and cell death to maintain

proper neural progenitor numbers during adult neurogenesis.

EphrinB3 was not found to be expressed in the SVZ per se and

is excluded from the first few layers of cells lining the lumen of the

ventricle. Rather, it is expressed in the striatum, bordering the

germinal zone. These findings differ from previous reports

(Conover et al., 2000), but are the first to study histochemical

localization and expression in neurospheres. The lack of ephrinB3

expression in the progenitors cultures supports this observation.

However, it is unlikely that ephrinB3 functions to prevent stem/

progenitor cell or neuroblasts migration into the surrounding

striatum, since in its absence or excess, no aberrant migration is

observed. Infusion of soluble ephrinB2-Fc and EphB2-Fc resulted

in neuroblast migration defects (Conover et al., 2000), suggesting

that either ephrinB3 and ephrinB2 have different migratory

functions or that infusion of high concentrations of ephrinB2-Fc

or EphB2-Fc could result in activation or inhibition of multiple

family members. Compensation between ephrins or Eph receptors

is well documented in the literature (Blits-Huizinga et al., 2004).

The expression of ephrinB3 in bordering regions would suggest

that precursors and/or neuroblasts must be in intermittent contact

with surrounding tissues to regulate cellular functions. For the

precursors, this would represent a short period of time prior to their

differentiation into neuroblasts, whereas neuroblasts would need to

contact ephrinB3 expression tissues throughout their migration to

the OB. This may represent a mechanism by which neural

progenitor numbers are regulated, simply by the fact that there

are a defined number of cells that can be in contact with the

peripheral regions of the RMS conduit. Furthermore, it should be

pointed out that ephrinB3 is not the only ephrin expressed in

regions bordering the SVZ, in particular, ephrinB2 is largely

expressed on both stem/progenitor cells and mature astrocytes that

are closely associated with the SVZ border and may compensate

for ephrinB3 deficiencies.

EphrinB3 is thought to mainly interact with EphB3 and EphA4

receptors, which are both expressed on stem/progenitor cells in the

adult SVZ. This could lead to the conclusion that ephrinB3/Eph

receptor interactions occur when stem/progenitor cells are closely

associated with the bordering striatum. We could also conclude

that Eph receptor signaling is required and not ephrinB3 or

bidirectional signaling, since we did not observe any increase in

proliferation in mice expressing only the extracellular/transmem-

brane domains of the ligand (ephrinB3hgal mice). However, while

unlikely, we cannot rule out potential signaling functions associ-

ated with the transmembrane and extracellular domains of

ephrinB3. Thus, the activation of ephrinB3 receptors appears

critical to exert a regulatory control over the proliferation of SVZ

cells.

Ephrins and their Eph receptors have recently gained a lot of

attention in regulating proliferation in neurogenic regions (Conover

et al., 2000; Aoki et al., 2004; Holmberg et al., 2005; Katakowski

et al., 2005). Both B-class (ephrinB2, EphB2) and A-class

(ephrinA2, EphA7) members of this family increase proliferation

when infused into the lateral ventricle (Conover et al., 2000;

Holmberg et al., 2005) and EphB2 increases neural precursor

proliferation in vitro (Katakowski et al., 2005). These findings

suggested that ephrins and Eph receptors may function as positive

regulators of stem/progenitor cell proliferation. However, exami-

nation of ephrinA2�/� or EphA7�/� mice showed an opposite

effect where increased proliferation was observed in the absence of

these molecules (Holmberg et al., 2005). It was concluded by

Holmberg and colleagues that in both studies, soluble ephrins and

Eph receptors function as antagonists of cell proliferation;

however, studies are pending analysis of B-class ephrin knockout

mice. In addition, the promiscuous binding between family

members makes it difficult to evaluate the specificity of infusion

J. Ricard et al. / Mol. Cell. Neurosci. 31 (2006) 713–722720

experiments. Our studies address both these points through

analysis of cell proliferation in the SVZ of ephrinB3�/� mice.

We found that ephrinB3 functions as a negative regulator of stem/

progenitor cell proliferation in the SVZ, and unlike the previous

studies we have found that infusion of clustered ephrinB3 func-

tions to suppress proliferation, hence restoring proliferation to

levels similar to the ones found in wild type animals. The fact that

ephrinB3 infusion does not have a significant effect on prolifer-

ation in wild type mice supports the specificity of the function of

ephrinB3 in regulating cell proliferation and suggests that ephrinB3

may only partially regulate cell proliferation. This is confirmed by

the alterations in cell cycle proteins in SVZ tissues, where cell

cycle activators (e.g., PCNA) are up-regulated while cell cycle

inhibitors (e.g., cyclin-dependent kinase inhibitor p27Kip1) are

down-regulated. One could argue that the increased numbers of

proliferating cells in the SVZ are due to an impairment of

neuroblast chain migration in the RMS, since fewer cells reach

the OB in the ephrinB3�/� mice. While we cannot completely

exclude this possibility, the increased expression of vimentin in the

ephrinB3�/� SVZ, an intermediate filament associated with

precursor and glial cells but not PSA-NCAM-positive neuroblasts

(Doetsch et al., 1997), supports the role of ephrinB3 in stem/

progenitor cell proliferation. Moreover, the changes in cell cycle

proteins detected by Western blot analysis also support a

proliferative function for ephrinB3.

One perplexing observation from our studies is the lack of

effect on proliferation when the receptors of ephrinB3 are knocked

out. Neither EphB3 nor EphA4 deletion resulted in the similar

increases in proliferation. One would expect that if the interaction

between an ephrin and its receptor(s) triggers a signal to regulate

cell proliferation, the lack of either signaling partner would result

in the same effect. This was the case for ephrinA2 and EphA7, as

mice lacking the expression of either one similarly exhibited more

proliferation in the SVZ (Holmberg et al., 2005). The absence of

proliferation increase in mice lacking EphA4 or EphB3 may

suggest that the two receptors could compensate for each other, and

that the deletion of both of them would be required to observe any

changes in proliferation. This hypothesis is supported by the fact

that both receptors are expressed on stem/progenitors cell in the

adult SVZ.

An increase in cell death was observed in the ephrinB3�/� mice

in parallel to increases in cell proliferation. This has been observed

in several cases when proliferation was up-regulated in the SVZ

(Belvindrah et al., 2002; Doetsch et al., 2002a,b; Vanderluit et al.,

2004). Conversely, in mice lacking E2F1 expression, a decrease in

the proliferation level in the SVZ occurs simultaneously with a

decrease in TUNEL-positive cells (Cooper-Kuhn et al., 2002).

However, some studies have shown opposite trends between

proliferation and cell death in the case of PTEN (Groszer et al.,

2001), or no effect in apoptosis as in the case of VEGF (Jin et al.,

2002). More likely, fluctuations in proliferation precede alterations

in cell death, since apoptosis is known to be one of the regulatory

components that control stem cell numbers and progeny output

(Sommer and Rao, 2002). This would ensure constant levels of

new neurons to meet the specific needs of the OB. Recently, A-

class ephrins and their receptors have been implicated in apoptotic

events (Dohn et al., 2001; Depaepe et al., 2005). In particular,

EphA7 and ephrinA5 have been shown to control neural progenitor

cell death, where EphA7�/� mice exhibit decreased cell death

(Depaepe et al., 2005). This would suggest that ephrinB3 function

in an opposite manner to suppress the apoptotic response.

Together, ephrinB3 may be a key regulator of regulating of both

cellular proliferation and apoptosis needed to maintain proper

neuronal numbers in the OB.

Here, we have identified ephrinB3 as a new negative regulator

of cell proliferation and positive regulator of cell survival within

the adult SVZ, where stem/progenitor cells expressing Eph

receptors interact with bordering tissues expressing ephrinB3 in

the adult SVZ. These findings demonstrate a novel function for

ephrinB3 and reinforce the importance of the ephrin/Eph families

as endogenous modulators of neurogenesis.

Experimental methods

Animals and tissue preparation

The generation of the mutant CD1 mice has been described

(Henkemeyer et al., 1996; Orioli et al., 1996; Cowan et al., 2000;

Yokoyama et al., 2001). Genotyping was performed by PCR analysis. Mice

were perfused intracardially with 4% paraformaldehyde. Brains were

removed and post-fixed for 2 h (for X-gal or Ki67 staining) or overnight

in the same solution, cryopreserved in 25% sucrose, and frozen in OCT

(Tissue Tek).

Staining procedures

For immunostaining, sections were washed with PBS, permeabilized for

10 min in 0.2% Triton-X100 and blocked in 5% BSA for 30 min. Primary

antibodies were applied for 1 h at room temperature (polyclonal anti-GFAP:

1/1000, Dako; monoclonal anti-GFAP: 1/1000, Pharmingen; anti-PSA-

NCAM: 1/1000, Chemicon) or overnight at 4-C (anti-Ki67: 1/25, Dako).

We observed no significant difference in the immunostaining pattern in the

SVZ between the monoclonal and polyclonal GFAP antibodies (not

shown). The detection of Ki67 required antigen retrieval prior to the

staining (sections placed in 10 mM citrate pH 6.0 and heated in a

microwave oven twice for 4 min). Staining for ephrinB1 (1/100; Santa

Cruz) and ephrinB2 (1/100; Santa Cruz) was performed on fresh frozen

tissue (isopentane at dry ice temperature). The specificity of the antibodies

was first assessed on recombinant ligands (100 ng/lane ephrinB1-Fc,

ephrinB2-Fc, and ephrinB3-Fc molecules; R&D Systems) by Western blot,

where both anti-ephrinB1 and anti-ephrinB2 were found to be specific for

their respective antigens (not shown). Alexa Fluor 488- or Alexa Fluor 594-

conjugated secondary antibodies (Molecular Probes) were used for 30 min

at room temperature.

EphrinB3hgal mice (expressing fusion proteins where h-galactosidasereplaces the intracytoplasmic domain of the ephrin) were used to visualize

the areas of expression of ephrinB3. X-gal staining (5 mM potassium ferro/

ferricyanide, 2 mM MgCl2, 1 mg/ml X-gal in PBS) was performed at room

temperature until a strong blue color was visible.

TUNEL staining was performed on PFA-fixed tissue using the ApopTag

Plus Fluorescein In Situ detection kit (Chemicon) according to the

manufacturer’s instructions.

BrdU injection and detection

Mice were injected intraperitoneally with 50 Ag BrdU per gram of body

weight once for 1 h for stereological counts or three times (2 h apart) before

waiting for 7 days for migration studies. Prior to the immunostaining (anti-

BrdU: 1/25, Roche), sections were incubated in 2N HCl for 30 min at 37-Cand neutralized in 0.1 M borate pH 8.5.

Stereological procedures

Cell counts were performed on 30 Am-thick cryostat brain sections (the

first section was collected when the rostral part of the lateral ventricle was

reached). Staining was performed on 10 sections every fifth section (total

J. Ricard et al. / Mol. Cell. Neurosci. 31 (2006) 713–722 721

thickness of the examined tissue was 1500 Am). For neuroblasts counts,

PSA-NCAM was revealed with an Alexa Fluor 594 secondary antibody, the

nuclei were counterstained using Sytox Green (1/100,000, Molecular

Probes), and the tissue was analyzed under a dual FITC/Rhodamine filter

on a Zeiss Axiophot microscope equipped with a CCD camera. The

analysis was conducted using the StereoInvestigator software (Micro-

BrightField) to estimate the total number of cells in the volume examined.

Western blot analysis

Western blot analysis was performed by BD Pharmingen using the

Power blot system. Protein samples were prepared according to Pharmingen

instructions. Briefly, proteins were separated on 4–15% gradient SDS-

polyacrylamide gels. After transfer to Immobilon-P membrane (Millipore),

the membrane was blocked for 1 h with blocking buffer (LI-COR). Primary

antibodies were applied to the membrane using a manifold that isolates 40

different channels. Primary antibodies were allowed to bind for 1 h at 37-C.The secondary antibodies were Alexa 680-conjugated goat anti-mouse

(Molecular Probes) and IRDye 800-conjugated goat anti-rabbit (Rockland).

The membrane was scanned on an Odyssey Infrared Imaging System (LI-

COR Biosciences) and the raw measured intensities were normalized

against intensities obtained from a standardization cocktail of proteins.

Samples were run in triplicate. Antibodies from the Cell Cycle and

Phosphoproteins sets (BD Pharmingen) were chosen for the study.

Neurosphere cultures

Adult neural progenitor cells were isolated from the lateral ventricle

wall of 2 month old CD1 mice. 1 mm coronal slices corresponding to

anteroposterior coordinates from 1 mm to 0 relative to bregma were cut.

The ventricle wall on the striatal side was dissected out and the tissue was

dissociated in 1.33 mg/ml trypsin, 0.7 mg/ml hyaluronic acid and 0.2 mg/ml

kynurenic acid in HBSS, and cells were grown in DMEM-F12 medium

supplemented with B27 (Life Technologies) in the presence of 20 ng/ml

EGF (Life Technologies) and 10 ng/ml bFGF (Chemicon). Half of the

medium was replaced every 4 days with fresh mitogens. For the primary

neurosphere assay, 2000 cells (from the SVZ tissue of 3 pooled mice) were

plated in 6-well plates and cultured for 7 days, after which the numbers of

neurospheres formed were counted.

Intracerebral infusions

EphrinB3-Fc ligands (R&D Systems; 140 Ag/ml in PBS) were pre-

clustered for 2 h at room temperature (anti-human-Fc ratio 10:1) and were

infused over a 7-day period using osmotic pumps (100 Al volume, rate 0.5

Al/h, connected to brain infusion devices (Alzet) at the following

coordinates (from bregma: lateral 0.5 mm; posterior �0.7 mm). Clustered

Fc fragments were used as controls. For tracing, 1 Al of a 2 mg/ml solution

of DiO (3,3V-dihexadecyloxacarbocyanine perchlorate; Molecular Probes)

was injected in the ventricle (coordinates as above) using a Hamilton

syringe. Animals were analyzed 4 days after injection.

Acknowledgments

We thank Dr. Mark Henkemeyer for the gracious gift of the

ephrinB3 knockout mice. We also thank Dr. Steven Kernie for

critical reading of the manuscript. This work was supported by The

Miami Project to Cure Paralysis, Ralph C. Wilson Sr./Ralph C.

Wilson Jr. Medical Research Foundation, and the National

Institutes of Health, NINDS (NS049545/NS30291).

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