Chemokine CXCL12 and its receptors in the developing central nervous system: Emerging themes and future perspectives

Chemokine CXCL12 and Its Receptors in theDeveloping Central Nervous System: EmergingThemes and Future Perspectives

Yan Zhu, Fujio Murakami

Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, Osaka 565-0871,Japan

Received 8 March 2012; revised 25 May 2012; accepted 1 June 2012

ABSTRACT: Homeostatic chemokine CXCL12

(also known as SDF-1) and its receptor CXCR4 are in-

dispensable for the normal development of the nervous

system. This chemokine system plays a plethora of func-

tions in numerous neural developmental processes, from

which the underlying molecular and cellular mecha-

nisms are beginning to be unravelled. Recent identifica-

tion of CXCR7 as a second receptor for CXCL12 pro-

vides opportunities to gain deeper insights into how

CXCL12 operates in the nervous system. Here, we

review the diverse roles of CXCL12 in the developing

central nervous system, summarize the recent progress

in uncovering CXCR7 functions, and discuss the emerg-

ing common themes from these works and future

perspectives. ' 2012 Wiley Periodicals, Inc. Develop Neurobiol 00:

000–000, 2012

Keywords: chemokine; CXCL12; developing CNS;

neuronal migration; axon guidance

Chemokines are a large family of structurally related,

mostly small secreted polypeptides, whose signals are

transduced by 7-transmembrane G-protein coupled

receptors (GPCRs) (Rossi and Zlotnik, 2000). The

name \chemokine" is coined from Chemotactic Cyto-

kine, because these molecules were originally recog-

nized for their predominant role in controlling leuko-

cyte trafficking during inflammatory response and

immune surveillance (Rot and Von Andrian, 2004;

Moser et al., 2004). However, it has since been real-

ized that chemokines are not merely traffic controllers

in the immune system. They are in fact versatile

intercellular mediators, whose functions include regu-

lating cell migration, proliferation, survival and adhe-

sion, and whose terrain of action extends far beyond

the immune system, reaching the nervous system,

cancer biology, and other developmental and patho-

logical paradigms (Horuk, 2001; Tran and Miller

2003; Li and Ransohoff, 2008; Zlotnik et al., 2011).

The first evidence that chemokines are required for

the proper development of the nervous system

emerged in 1998, when it was found that chemokine

CXCL12 and its receptor CXCR4 regulate cerebellar

granule cell development (Ma et al., 1998; Zou et al.,

1998). In the decade or so that has followed, a large

amount of accumulating evidence points to a wide

involvement of chemokines and their receptors in a

range of processes during the normal development of

the nervous system, with CXCL12/CXCR4 dominat-

ing the scene. The recent identification of CXCR7 as

a second receptor of CXCL12 suggests that some of

the functions of CXCL12 may be mediated or modi-

fied by CXCR7. While the field continues to expand

rapidly, common themes and underlying principles

have begun to emerge. In this review, we will sum-

marize key studies that illustrate the diverse roles

CXCL12 and its two receptors CXCR4, CXCR7 in

Correspondence to: Y. Zhu ([email protected]).Contract grant sponsor: Grant-in-Aid for Scientific Research

from the Ministry of Education, Culture, Sports, Science and Tech-nology, Japan; contract grant number: 23570226 (to Y.Z.), and2222004 (to F.M.).

' 2012 Wiley Periodicals, Inc.Published online in Wiley Online Library (wileyonlinelibrary.com).DOI 10.1002/dneu.22041

1

the development of the central nervous system (CNS)

with focus on three main areas: neuronal migration,

axon guidance, and regulation of neural stem/progen-

itor cells. We will outline the emerging rules and

principles inferred from these studies and discuss

future challenges and perspectives as we go along.

Due to space limitations, this review does not extend

to discuss involvement of CXCL12 and its receptors

in CNS under pathological conditions, which is a vast

and exciting topic in its own right. Readers interested

in this topic may refer to some excellent reviews past

and recent (Tran and Miller, 2003; White et al, 2007;

Li and Ransohoff, 2008; Gross and Meier, 2009; Li

and Ransohoff, 2009; Rostene et al, 2011).

CONTROL OF NEURONAL MIGRATIONAND POSITIONING

Migration of Neuron Precursors

In 1998, two studies showed for the first time that

chemokines could be important for normal brain de-

velopment (Ma et al., 1998; Zou et al., 1998). By gen-

erating and analyzing CXCR4 knockout mice, these

reports showed defective development of the external

granule layer (EGL), which is a secondary prolifera-

tive zone that is positioned beneath the pial meninges

and stretches across the entire surface of the cerebel-

lar primordium. Precursors of cerebellar granule cells

born from the upper rhombic lip first migrate tangen-

tially and superficially into the cerebellar primordium

to form the EGL. These cells then stay and proliferate

within the EGL for a protracted period until an appro-

priate developmental time when they exit the cell

cycle and migrate radially into deep cerebellar cortex

to form the internal granule layer (IGL) (Hatten,

1999; Komuro and Yacubova, 2003, Fig. 1). Retain-

ing granule cell precursors in the EGL serves two

purposes: to ensure sufficient cell proliferation, and

to allow the descending of EGL cells only when the

cerebellar cortex is ready to receive them (Ma et al.,

1998; Zou et al., 1998; Choi et al., 2005). During the

formation of EGL, CXCR4 is expressed in EGL cells,

while CXCL12 is expressed in the overlying pial

meninges (Klein et al., 2001; Reiss et al., 2002; Zhu

et al., 2002). In CXCR4 mutants, granule cell precur-

sors prematurely depart the EGL and descend inter-

nally forming ectopias (Ma et al., 1998; Zou et al.,

1998). In vitro migration assays subsequently demon-

strated that either the pial meninges or recombinant

CXCL12 could chemoattract the cerebellar granule

Figure 1 CXCL12/CXCR4 signaling regulates the migration and positioning of cerebellar gran-

ule cells, cortical interneurons and hindbrain pontine neurons. Note that migration of pontine neu-

rons is only depicted on the left half of hindbrain. At early stages in development (top panel), the

migrating neurons (red) expressing CXCR4, are compartmentalized within regions expressing high

level of CXCL12 (green). Later in development (bottom panel), CXCL12/CXCR4 signaling is

down-regulated, allowing neurons to decompartmentalize and move into their final positions. The

mechanism underlying this down-regulation is still little understood. CP: cortical plate; EGL: exter-

nal granule layer; IGL: internal granule layer; lRL: lower rhombic lip; MGE: medial ganglionic

eminence; ML: molecular layer; MNG: meninges; PCL: Purkinje cell layer; PMS: pontine migra-

tory stream; PN: pontine nucleus; SVZ/IZ: subventricular zone/intermediate zone.

2 Zhu and Murakami

Developmental Neurobiology

precursors (Klein et al., 2001; Lu et al., 2001; Reiss

et al., 2002; Zhu et al., 2002). Thus, it appears that

CXCL12 derived exclusively from the pial meninges

functions to anchor cerebellar granule precursors

within a favorable proliferative zone via mechanisms

of chemoattraction (Fig 1). Interestingly, CXCL12 in

addition facilitates cell proliferation in the EGL syn-

ergistically with Sonic Hedgehog (Shh), a known

mitogen for the cerebellar precursor cells (Klein et

al., 2001). This dual functionality of CXCL12 in the

developing cerebellum highly resonates with its origi-

nal roles established in the immune system in regulat-

ing the homing of hematopoetic cells to their prolifer-

ative niche, the bone marrow (Nagasawa et al., 1996;

Tachibana et al., 1998; Ma et al., 1998; Zou et al.,

1998) and facilitating their proliferation synergisti-

cally with interleukin-7 (IL-7) (Nagasawa et al.,

1994).

The constitutive expression of CXCR4 in the

developing CNS, and the ubiquitous expression of

CXCL12 in pial meninges overlying the entire neural

tube predicted widespread neural functions of this

chemokine pair (Jazin et al., 1997; McGrath et. al,

1999; Tissir et al., 2004; Stumm et al., 2007). These

predictions were verified by mounting evidence that

followed. For instance, in the hippocampus, defective

CXCL12/CXCR4 signaling causes a malformed den-

tate gyrus (DG) during development (Bagri et al.,

2002; Lu et al., 2002). The defect might be a com-

pound one, involving failed migration of both prolif-

erating progenitors as well as newly differentiated

granule cells. CXCL12 was again proposed to control

cell migration into the DG, as its expression aligns

the migratory path (Bagri et al., 2002; Lu et al.,

2002). More recently, it was shown that CXCL12

helps to relocate the DG neurogenic zone from the

hippocampal ventricular zone to a transient subpial

proliferative zone aligning the DG blades, which

eventually descend into the DG hilus to form the per-

manent subgranular zone (SGZ) as one of two sites of

adult neurogenesis. The role of CXCL12 appears to

get the CXCR4-expressing granule cell progenitors

into the DG and anchoring them beneath the pial

meninges to form the transient subpial proliferative

zone (Li et al., 2009).

Migration of Postmitotic Neurons

It soon became clear that CXCL12 is also a critical

regulator for the tangential migration of postmitotic

neurons. Tangential migration allows new born neu-

rons to be transported over long distances to destina-

tions far from their birthplaces, thus enabling neuro-

nal types from distant origins to interact and connect

efficiently. The invasion of the neocortex by cortical

interneurons born mostly from the medial ganglionic

eminence (MGE) in the subpallium is probably the

most well-known example, owing to the importance

of interneuron positioning for cortical function.

Therefore, that CXCL12 is implicated in this system

attracted much attention in the field. MGE-derived

interneurons enter into the pallium from the lateral to

medial direction following two distinct tangential mi-

gratory streams, namely the marginal zone (MZ) and

the subventricular zone/intermediate zone (SVZ/IZ)

streams, sparing the cortical plate until later stages

when the interneurons switch to migrate radially into

it (Marın and Rubenstein, 2003; Metin et al., 2006;

Tanaka et al., 2009, Fig. 1). In CXCR4 knockout cor-

tex, interneurons enter the pallium diffusely even

within the cortical plate, disrespecting the two migra-

tory streams (Stumm et al., 2003; Tiveron et al.,

2006; Li et al., 2008; Lopez-Bendito et al., 2008).

The expression pattern of CXCL12 in the developing

cortex shows stunning alignment with the interneuron

migratory paths in the pial meninges overlying the

MZ and the SVZ/IZ itself (Daniel et al., 2005;

Tiveron et al., 2006, Fig. 1). We and others have

shown that CXCL12 determines the migratory

streams for interneurons, both early in migration by

sorting them into the two pathways at the palium-sub-

palium boundary (Li et al., 2008), and later by keep-

ing the interneurons within the MZ for a substantial

period of time before their diving down into the corti-

cal plate (Tiveron et al., 2006; Li et al., 2008; Lopez-

Bendito et al., 2008; Tanaka et al., 2009). By doing

so, CXCL12 may serve two functions: to channel

interneurons into migratory corridors that are optimal

for their efficient dispersion (for example, MZ is a

relatively cell sparse region), and to prevent inter-

neurons from prematurely contacting their future syn-

aptic partners, pyramidal neurons. If these functions

are truly important, then disrupting them should have

lasting consequences on the final interneuron distri-

bution in the cortex. By generating conditional

CXCR4 knockout mice that survive postnatally (Li et

al., 2008; Tanaka et al., 2010), or using cell transplan-

tation (Lopez-Bendito et al., 2008), we and others

have shown that disrupting the interneuron migratory

paths indeed has long-term consequences, affecting

both the regional and laminar distributions of inter-

neurons. The reduction in the number of interneurons

or interneuron subtypes in certain cortical regions and

the presence of occasional clustered interneurons are

in accordance with the notion that interneurons may

disperse less efficiently outside their migratory paths

(Li et al., 2008; Tanaka et al., 2010). Laminar distri-

bution is also disrupted with defects specific to inter-

CXCL12 in Developing CNS 3


neuron subtypes (Li et al., 2008; Tanaka et al., 2010).

The extent of functional consequences of disrupted

interneuron migratory pathways is still not fully

explored and would be of great interest to revisit once

further knowledge and new tools become available to

analyze in detail interneuron subtypes and their corre-

sponding local circuitries.

A chemoattractive role of CXCL12 has again been

proposed for the migrating cortical interneurons and

gained support from both in vitro and in vivo evi-

dence (Li et al., 2008; Lopez-Bendito et al., 2008;

Liapi et al., 2008; Tanaka et al., 2009). Cortical inter-

neurons in their migratory streams are clearly

attracted to ectopically expressed CXCL12 in vivo or

to CXCL12 in vitro. Both the initial sorting of new-

born interneurons into the MZ and SVZ/IZ streams as

well as the retention of interneurons within migratory

streams could be accounted for by chemoattraction

toward CXCL12. However, an alternative interpreta-

tion has also been suggested to explain the latter

phenomenon (Lysko and Golden, 2011). This study

showed that CXCL12 signaling affects the dynamic

morphology of migrating interneurons in the SVZ/IZ.

Interneurons migrate by frequently branching their

leading processes and subsequently translocating

their soma toward branch points (Bellion et al., 2005;

Martini et al. 2009). These branches may serve as

sensors to detect environmental guidance cues.

CXCL12 signaling appears to reduce branching fre-

quency and increase the speed of migrating interneur-

ons, thereby minimizing the chance of these neurons

to be distracted by cues outside the CXCL12-

enriched migratory path. Interestingly, this function

of CXCL12 depends only on the ability of CXCR4

coupled G protein to inhibit the cAMP pathway,

which alone does not lead to chemotaxis. Thus, the

authors argued that it was not necessary for CXCL12

to act as a chemoattractant to retain interneurons

within their migratory paths. Regardless of the exact

mechanisms, the ability of CXCL12 to direct the

course of migrating interneurons raises the question

of whether CXCL12 may also influence the lateral-

to-medial migratory direction of cortical interneur-

ons. Studies on the role of CXCL12 in the tangential

migration of Cajal-Retzius cells (C-R cells) provide

some interesting insight into this issue. Cortical hem-

derived C-R cells (Takiguchi-Hayashi et al., 2004)

also depend on meningeal CXCL12 for their tangen-

tial migration and dispersion within the MZ stream

(Borrell and Marın, 2006; Paredes et al., 2006), but in

this case from medial-to-lateral direction. This sug-

gests that the meninge-derived CXCL12 is unlikely

to provide cues for the tangential direction of migrat-

ing C-R cells and interneurons. Borrell and Marın

(2006) provide evidence suggesting C-R cells spread

by a mode of contact inhibition, which results in cells

migrating from high to low cell density area. It would

be interesting to see if contact inhibition may also

play a part in controlling the tangential direction of

cortical interneuron migration.

The expression of CXCL12 in the SVZ/IZ is in-

triguing, as it raises the question of which cells

express this chemokine there. Tiveron et al (2006)

showed that CXCL12 is expressed by intermediate

progenitors of future pyramidal neurons that undergo

amplification in the SVZ. An important implication

of this finding is that excitatory components of the

cortex might themselves be involved in the develop-

ment of inhibitory components. A recent study lends

further support to this notion (Sessa et al., 2010),

showing that when Tbr2, a gene expressed in and crit-

ical for the development of intermediate progenitors

in the SVZ, is force-expressed, it leads to the attrac-

tion of cortical interneurons toward the ectopic

expression site. This seems to be achieved by Tbr2-

dependent induction of CXCL12 expression, thus

linking an important intermediate progenitor marker

directly with the chemokine expression profile of

cortical cells.

CXCL12 also controls the tangential migration of

new born neurons in other brain regions. In the hind-

brain, pontine neurons, the most prominent subset of

precerebellar neurons, take a circuitous migratory

path from the lower rhombic lip to the anterior–ven-

tral hindbrain to form the pontine nucleus (Altman

and Bayer, 1997; Kawauchi et al., 2006). These neu-

rons migrate marginally beneath the pial surface and

move anteriorly for a fixed distance before turning

ventrally at a stereotypic position toward the midline

(Fig. 1). We have shown that disrupting CXCL12/

CXCR4 signaling causes a substantial portion of pon-

tine neurons to deviate from their marginal migratory

stream and consequentially turn ventrally without

traveling anteriorly (Zhu et al., 2009). We found that

meninge-derived CXCL12 again functions to retain

migrating pontine neurons within the marginal zone.

Curiously, a proportion of pontine neurons that

remain to migrate marginally still fail in their anterior

migration as they prematurely turn toward the ventral

midline before their supposed turning point, suggest-

ing that CXCL12 may directly control the anterior

migration of pontine neurons. How can CXCL12

ubiquitously expressed in the pial meninges provide

tangential directions for migrating pontine neurons?

If such guidance is provided by a gradient of extracel-

lular CXCL12 protein along the anteroposterior axis,

then how is such a gradient established in the first

place? To unambiguously visualize and quantify

4 Zhu and Murakami


extracellular distribution of CXCL12 protein, or for

that matter any secreted molecules, is still technically

challenging, yet, will be greatly helpful to address

these questions.

Silencing CXCL12/CXCR4 Signaling

A common theme that has emerged from all these

studies is that CXCL12 functions to compartmental-

ize developmental processes within a specific lamina

zone of highly stratified tissue like neuroepithelium.

Such compartmentalization often is transient with the

subsequent decompartmentalization being carefully

timed to achieve fine coordination with other devel-

opmental processes (Fig. 1). This model begs an im-

portant question as to how the CXCL12/CXCR4 sig-

naling becomes inhibited at specific developmental

times to allow for proper decompartmentalization.

Despite its importance and biological relevance, we

presently know little of its answer. It does not seem

to be a case of simply down-regulating the transcrip-

tion of CXCL12 and CXCR4 as their expression per-

sist even after the presumed inhibition of CXCL12/

CXCR4 signaling (Klein et al., 2001; Reiss et al.,

2002; Stumm et al., 2003). The inhibition might

therefore be achieved by turning off the signaling

pathway downstream of CXCR4. An elegant mecha-

nism of this kind has been proposed for the cerebellar

granule precursors in the EGL, albeit only in vitro(Lu et al., 2001). It has been shown that reverse sig-

naling between EphB and ephrinB can down-regulate

CXCR4 signaling by binding to a PDZ-RGS protein

(RGS stands for Regulator of G protein Signaling),

and presumably bringing RGS close to CXCR4

receptors. Since RGS is essentially a GTPase Activat-

ing Protein (GAP) for the heterotrimeric G protein, it

leads to the hydrolysis of G protein and thus dampens

CXCR4 signaling. Whether such an RGS-based in-

hibitory mechanism operates in vivo and whether

similar or different mechanisms operate in the cases

of cortical interneurons, DG progenitors and pontine

neurons, these are important and imminent questions

awaiting future investigation.

CONTROL OFAXON GUIDANCE:ATTRACTANT OR MODULATOR?

CXCL12 clearly is critical for controlling the migra-

tion of neurons and their precursors by functioning at

least in part as a guidance cue that via CXCR4 signal-

ing leads to biased migration direction. Because the

guidance of migrating neurons and that of growth

cones of extending axons share similar signaling

mechanisms and chemical guidance cues (Guan and

Rao, 2003), it was expected that CXCL12/CXCR4

may also play a role in axon pathfinding. Xiang et al.

(2002) provided the first evidence that growth cones

of rat cerebellar granule neurons, when presented

with a steep gradient of CXCL12, could turn either

away or toward the source depending on the intracel-

lular cyclic GMP levels. A dual functionality of

CXCL12 on axons was also demonstrated by Ara-

kawa et al. (2003). They showed, using a dissociated

mouse cerebellar granule cell culture, that CXCL12

promoted axon elongation at low concentration but

inhibited it at high concentration. The authors further

probed into downstream signaling pathways for these

two opposing responses showing that high CXCL12

concentration selectively activate the Rho-ROCK

pathway which negatively regulates cytoskeleton dy-

namics, while low CXCL12 concentration activates

the Rho-mDia pathway without activating ROCK.

Considering that most reagents controlling axon

extension can cause axon turning when presented as a

point source, one implication from this study is that

CXCL12 can also guide these axons. In line with

these in vitro studies, it was shown in zebrafish that

ectopically expressed CXCL12 appears to aberrantly

attract retinal ganglion cell axons on their way to the

optic stalk (Li et al., 2005).

These initial works, which suggest that CXCL12

is a chemotactic cue for axons, were challenged by

emerging evidence advocating a permissive rather

than chemotropic role of CXCL12 for axons. In

zebrafish, Miyasaka et al. (2007) showed that ubiqui-

tously-expressed CXCL12 works equally effectively

as localized CXCL12 in guiding olfactory sensory

axons. In chick, Chalasani et al. (2003) found, using

collagen explant culture and growth cone collapse

assay, that CXCL12 acts as neither an attractant nor a

repellent on its own, but rather to reduce the repulsive

activity of other chemorepellents when present simul-

taneously. This repellent-reducing activity of

CXCL12 was demonstrated for cultured chicken reti-

nal ganglion cell (RGC) axons, dorsal root ganglion

(DRG) axons, and sympathetic ganglion axons for

their respective repellents, Slit-2, Sema3A, and

Sema3F. Intriguingly, this activity of CXCL12 seems

to solely depend on a CXCR4-mediated elevation of

cAMP levels. The antirepellent effect of CXCL12

has subsequently gained in vivo support in zebrafish

(Chalasani et al., 2007, Xu et al., 2010). Zebrafish

RGC axons frequently make pathfinding errors within

the optic stalk when Slit-Robo signaling is compro-

mised. Chalasani et al. (2007) showed that knocking

down CXCL12 or CXCR4 can partially rescue the

misprojection of RGC axons in a robo2 hypomorph



which retains residual low level robo activities, but

cannot rescue a robo2 null mutant. The authors

argued that the absence of CXCL12/CXCR4

increases the sensitivity of RGC axons to the residual

robo activity in the hypomorph, thus rescuing the

phenotype. In a follow-up study, they showed in the

same system that the antirepellent activity of

CXCL12 is dependent on a calmodulin-activated ade-

nylate cyclase (ADCY8), an enzyme that facilitates

cAMP synthesis, and ADCY8 knockdown rescues the

robo2 hypomorph similarly as CXCL12 knockdown

(Xu et al., 2010).

A critical and rather puzzling question arose from

these studies. CXCR4 is traditionally thought to cou-

ple with the Gai/o subunit of the heterotrimeric G

protein that decreases cAMP levels upon CXCL12

binding. How then can CXCL12 stimulation in axons

lead to an increase in cAMP levels? This question is

particularly pertinent since elevation of cAMP upon

CXCL12 stimulation has been directly demonstrated

in cultured chicken RGC using a FRET-based cAMP

sensor (Xu et al., 2010). A recent study has provided

some clues as to how this might happen (Twery and

Raper, 2011). By selectively blocking various Gasubunits as well as Gbc in cultured embryonic

chicken DRG neurons, the authors showed that Gai,

Gaq, Gbc, and phospholipase C (PLC) all seem to

mediate the antirepellent activity of CXCL12. Taken

previous studies together, they proposed a model in

which downstream of CXCR4, Gai, Gaq, and Gbccooperatively activate PLC, which in turn activates

calcium-calmodulin activated ADCY8, thus resulting

in an increase of cAMP. This model, particularly its

in vivo relevance, awaits future validation. It should

be noted that the Twery and Raper report demon-

strates just how complex and poorly understood are

the downstream signaling pathways that GPCR recep-

tors like CXCR4 could evoke in different cellular

contexts. Much is to be explored on this front particu-

larly in in vivo systems.

Curiously, studies reporting axon guidance defects

in CXCL12- or CXCR4-deficient mice are few. Chala-

sani et al. (2003) analyzed the overall axon profiles in

the spinal cord of CXCR4 mutant mice and reported

a general hyperfasciculation of axon tracts and mis-

projected sensory afferents within the spinal cord.

Lieberam et al. (2005) demonstrated an involvement

of CXCL12 in guiding motor axons ventrally out of

the mouse spinal cord and hindbrain. They showed

that CXCR4 is transiently expressed in ventrally-pro-

jecting motor neurons, while CXCL12 is expressed in

the mesenchyme surrounding the ventral motor exit

point. Disruption of CXCL12/CXCR4 signaling

resulted in a failure of many motor axons to exit the

neural tube. The underlying mechanism of this phe-

nomenon is still unclear. Interestingly, the authors

noted that they could not detect any chemoattractive

activity of CXCL12 in motor axons growing out of

spinal cord explants, raising an alternative possibility

that CXCL12/CXCR4 signaling in these motor axons

may be to antagonize repulsion from repellents

located near the ventral motor exit points. However, a

recent study also focusing on motor axons but in ros-

tral hindbrains showed that CXCL12 could chemoat-

tract chicken oculomotor axons in a collagen explant

assay and that oculomotor axons exiting hindbrain

are reduced in CXCR4 mutant mice (Lerner et al.,

2010). Whether CXCL12 acts as an antirepellent

modulator or a chemotactic guidance cue for develop-

ing axons in vivo is still an unsettled issue. The differ-

ences in existing reports may partly reflect differen-

ces in the types of in vitro chemotaxis assays being

used and partly owe to different cellular contexts.

Nevertheless, these attempts to clarify the role of

CXCL12 in axon guidance highlight two important

issues: (1) the complexity and context-dependency of

downstream signaling of CXCL12/CXCR4; and (2)

the possibility of cross-talks between signaling path-

ways of CXCL12 and other axon guidance cues.

CXCR7, ANOTHER RECEPTOR OFCXCL12 THAT HAS MULTIFACETEDROLES

The near identical phenotypes between CXCR4- and

CXCL12-deficient mice have been interpreted to

mean a one-to-one relationship between the receptor

and the ligand, making them an exception in the gen-

erally promiscuous chemokine family. However, this

monogamous relationship was refuted following the

recent discovery of a second receptor for CXCL12,

namely CXCR7 (also known as RDC1) (Balabanian

et al., 2005; Burns et al., 2006). CXCR7, a prior

orphan GPCR, binds to CXCL12 with higher affinity

than CXCR4 and has a second ligand CXCL11 (also

known as ITAC). The addition of a third member to

CXCL12 chemokine system predicted a higher level

of complexity in the mechanisms underlying

CXCL12 functions and urged us to review existing

work in this new context.

Early assessments of CXCR7 already have

revealed signs that it may not be a typical chemokine

receptor. Although CXCR7 shares sequence homol-

ogy with 7-transmembrane GPCRs, the \DRYLAIV"amino acid motif in the second intracellular loop,

which is highly conserved among chemokine recep-

tors and considered to be necessary for G protein

6 Zhu and Murakami


coupling (Colvin et al., 2004; Lagane et al., 2005), is

altered to \DRYLSIT" in CXCR7 (Sierro et al.,

2007; Thelen and Thelen, 2008). In line with this

sequence alternation, typical chemokine responses

such as calcium mobilization, chemotaxis, and activa-

tion of PI3K/AKT pathways, often fail to be detected

upon ligand stimulation of CXCR7 (Burns et al.,

2006; Sierro et al., 2007; Mazzinghi et al., 2008; Bol-

dajipour et al., 2008). These \oddities" set CXCR7

apart from classical chemokine receptors like

CXCR4, suggesting that the two are unlikely to be

functionally redundant.

Cellular and Molecular Functions

The cellular and molecular function of CXCR7 has

been the subject of heated pursuits ever since its dis-

covery, and mounting evidence emerged from which,

mostly biochemical but also in vivo in zebrafish, gave

rise to three major models (Fig. 2). (1) The first

model suggests that CXCR7 functions as a scavenger

receptor that predominantly mediates CXCL12 inter-

nalization and subjects it to degradation in lysosomes

(Dambly-Chaudiere et al., 2007; Boldajipour et al.,

2008; Zabel et al., 2009; Luker et al., 2010; Naumann

et al., 2010). Like other scavenger chemokine recep-

tors (Haraldsen and Rot, 2006), the capability of

CXCR7 to clear excess CXCL12 from the extracellu-

lar space may help keep the concentration of bioac-

tive CXCL12 within an optimal range, and/or shape

the distribution of CXCL12 protein at tissue levels.

Strong in vivo support of this model comes from an

elegant study in zebrafish on the migration of primor-

dial germ cells (PGCs) (Boldajipour et al.., 2008).

PGCs generated in the head region express CXCR4

and migrate posteriorly following an attractive signal

associated with a CXCL12 expression domain that

itself dynamically shifts toward the future gonad pos-

teriorly. Boldajipour et al. (2008) provided several

lines of complementary evidence by showing that

CXCR7 expressed in the somatic tissue mediates

CXCL12 endocytosis, whereby clearing CXCL12

protein from nontarget areas and sharpening the

localized CXCL12 protein distribution necessary for

precise PGCs guidance. (2) The second model sug-

gests that CXCR7 can form heterodimers with

CXCR4 when copresent in the same cell and as a

result modify CXCR4 downstream signaling (Sierro

et al., 2007; Levoye et al.., 2009; Decaillot et al.,

2011). While some reports have shown that the coex-

pression of CXCR4 and CXCR7 in cell lines enhan-

ces calcium mobilization (Sierro et al., 2007) and

chemotaxis (Decaillot et al., 2011), others have dem-

onstrated compromised CXCR4 signaling (Levoye et

al., 2009). (3) The third model suggests that CXCR7

can mediate signaling independently from CXCR4 in

response to CXCL12 (Balabanian et al., 2005; Valen-

tin et al., 2007; Zabel et al., 2009; Odemis et al.,

2010, 2012; Rajagopal et al., 2010). Most GPCRs can

signal via both G proteins and b-arrestins in a rather

balanced manner. Coupling with b-arrestins triggers

receptor desensitization and endocytosis, and also

activates downstream ERK1/2 pathway (Luttrell and

Lefkowitz, 2002). Whereas CXCR7 is considered

incapable of signaling via G proteins, a couple of

studies recently have provided direct evidence that

CXCR7 can recruit b-arrestin 2 and activate ERK1/2

pathway (Zabel et al., 2009; Rajagopal et al., 2010),

Figure 2 Three major models that have been proposed for the cellular and molecular functions

of CXCR7. Green: CXCL12; orange: CXCR7; blue: CXCR4.



putting CXCR7 into the category of b-arrestin-biased

GPCRs. More recently, one report showed quite sur-

prisingly that CXCR7 could even signal via G protein

and trigger calcium mobilization in CXCR4-deficient

primary asctrocytes upon CXCL12 binding (Odemis

et al., 2012). However, caution should be taken as it

is still unclear in this case whether CXCR7 signals by

itself or by heterodimerizing with another as-of-yet

unknown receptor. One implication from models (2)

and (3) is that CXCR7 alone or the CXCR7/CXCR4

heterodimer can mediate different downstream sig-

naling and thus lead to different cellular responses

compared with CXCR4 alone. Indeed, several lines

of evidence suggest that while CXCR4 predomi-

nantly controls chemotaxis, CXCR7 is more impor-

tant for regulating cell-cell adhesion (Burns et al.,

2006; Hartmann et al, 2008; Mazzinghi et al., 2008;

Zabel et al., 2009). In summary, each of the above

three models has gathered its fair share of experimen-

tal evidence and none are mutually exclusive. For

example, coupling to b-arrestin 2 would enable

CXCR7 to trigger CXCL12 internalization as well as

b-arrestin 2-mediated ERK1/2 activation simultane-

ously in the same cell. The differential expression

patterns of CXCR7 and CXCR4 showing coexpres-

sion in some cells and nonoverlapping in others, and

the fact that CXCR7 and CXCL12 are often coex-

pressed in somatic tissues (Schonemeier et al., 2008;

Tiveron et al., 2010; Zhu et al., unpublished observa-

tion) suggest that all three models might apply in vivodependent on the cellular and tissue context.

THE ROLE OF CXCR7 FUNCTION IN THEDEVELOPING CNS

The high sequence conservation of CXCR7 across

mammalian species implies its functional importance,

and indeed two independent CXCR7-deficient mice

lines that have been generated showed defects in

heart development and lethality soon after birth

(Sierro et al., 2007; Gerrits et al., 2008). However, an

initial analysis of the developing nervous system

including the cerebellum, dentate gyrus, and spinal

cord, all of which are noted for showing defects in

CXCR4- or CXCL12-deficient mice, did not show

obvious defects in CXCR7 mutant (Sierro et al.,

2007). Nevertheless, recent in-depth analyzes of

CXCR7 mutant cortex uncovered its importance in

regulating the intracortical tangential migration of

interneurons (Sanchez-Alcaniz et al., 2011; Wang et

al.., 2011). Both studies showed that interneurons in

CXCR7-deficient mice failed to respect the stereo-

typic MZ and SVZ/IZ migratory streams and prema-

turely entered the cortical plate, a defect highly iden-

tical to that in CXCR4-deficient mice (see an earlier

section). CXCR7 and CXCR4 are coexpressed in the

majority of migrating cortical interneurons, and fur-

thermore interneuron specific conditional deletion of

CXCR7 causes similar phenotype as constitutive null

mutant, suggesting a cell-autonomous requirement.

So what role does CXCR7 play in cortical interneur-

ons? This is where the two reports begin to diverge.

Sanchez-Alcaniz et al. (2011) proposed a scavenger

role for CXCR7. They have shown an abnormally

high level of CXCL12 protein and a loss of CXCR4

protein in CXCR7 deficient cortex. They have further

suggested that the former causes the latter since

excess CXCL12 has previously been shown to over-

trigger desensitization and degradation of CXCR4

(Marchese and Benovic, 2001; Alsayed et al., 2007;

Kolodziej et al.., 2008). Thus, the authors conclude

that CXCR7, even when copresent with CXCR4 in

cortical interneurons, functions as a scavenger recep-

tor to prevent over-accumulation of CXCL12 in the

cortex, thereby ensuring a sufficient amount of

CXCR4 protein to be present to mediate chemokine

responses. Wang et al. (2011), on the other hand,

although not dismissing the scavenger role for

CXCR7, suggested that CXCR4 and CXCR7 each

plays distinct functions in regulating interneuron

migration with CXCR4 via G proteins and CXCR7

via the ERK1/2 pathway. How these distinct signal-

ing profiles relate to their in vivo functions is still

unclear, but the authors showed by time-lapse imag-

ing that CXCR7-deficient interneurons displayed

lower motility with shorter leading processes in con-

trast to higher motility with longer leading processes

of CXCR4-deficient interneurons. These two models

being proposed are not necessarily mutually exclu-

sive. For example, the reduced motility and shorter

leading processes seen in CXCR7-deficient interneur-

ons could be a consequence of an abnormally high

level of CXCL12 in the environment. This possibility

is supported by two previous observations. First,

migrating PGCs in zebrafish display reduced motility

when either CXCL12 is overexpressed or CXCR7 is

depleted (presumably via an increasing ambient

CXCL12 level), in contrast to normal motility when

CXCL12 is simply depleted (Boldajipour et al.,

2008). Second, in the context of axon growth, high

levels of CXCL12 was found to activate the Rho/

Rock pathway which negatively regulates cytoskele-

ton as discussed above (Arakawa et al., 2003). To

build a more coherent model in the future will require

better understanding of how signaling properties and

cellular responses are affected by varying chemokine

concentrations and receptor combinations. Neverthe-

8 Zhu and Murakami


less, the two in vivo studies on migrating cortical

interneurons offer important insights, (1) CXCR7

may comprise a general mechanism employed to

keep CXCL12 at modest levels so as to prevent the

undesirable negative effect that occurs with excess

CXCL12; (2) CXCR7 may independently signal and

cause distinct cellular effects in vivo.

The role of CXCR7 in the developing nervous sys-

tem is bound to extend beyond the migrating cortical

interneurons. We have found that precerebellar neu-

ronal migration is affected in CXCR7-deficient mice

and the defect differs from that in CXCR4 mutant

(Zhu et al., unpublished observation). It is almost cer-

tain that the list will grow and new roles will be

assigned to this second receptor of CXCL12. The key

issue, however, is to have a clear and thorough under-

standing of the intricate mechanisms that have been

carried out through the interplay between CXCL12

and its two receptors in a complex and dynamic tissue

environment such as the developing brain.

Role of CXCL12 in Neural Stem andNeural Progenitor Cells

In the developing CNS, CXCR4 is expressed predom-

inantly in neural stem and neural progenitor cells

residing in the primary (e.g. ventricular zone) and

secondary (e.g. cerebellar EGL) proliferative zones

from the spinal cord to the forebrain (McGrath et al.,

1999; Tissir et al., 2004; Corti et al., 2005; Stumm et

al., 2007; Diotel et al., 2010). In postnatal brains,

CXCR4 expression continues in neural progenitor

cells in limited locations where adult neurogenesis

persists, such as the SGZ in dentate gyrus and the

SVZ of the lateral ventricle (Tissir et al., 2004; Tran

et al., 2007). As for the ligand, CXCL12 expression

in embryos is mainly confined to the pial meninges

(McGrath et al., 1999; Tissir et al., 2004), with which

neural progenitor cells usually taking on the form of

radial glia cells have contact via their basal endfeet.

In adult neurogenic regions, CXCL12 is found in

cells adjacent to the proliferating neural progenitors

(Banisadr et al., 2003; Kokovay et al., 2010). These

expression patterns suggest that neural stem/progeni-

tor cells may develop and function under the influ-

ence of CXCL12. In vitro, CXCR4 expression is

almost a hallmark of neural progenitor cells or neuro-

spheres isolated and grown from embryonic or adult

brains (Tran et al., 2004; Peng et al., 2004; Dziem-

bowska et al., 2005). What then are the effects of

CXCL12 on neural progenitor cells? There are sev-

eral occasions where neural progenitor cells need to

be relocated from their primary sites of residence to

secondary proliferative zones within which neurogen-

esis takes place. CXCL12 seems critically involved

in this process by mobilizing neural progenitor cells

and then retaining them in their second niches, as

exemplified by the cases of cerebellar EGL and den-

tate gyrus SGZ (discussed in an earlier section). A

similar scenario was recently described in adult SVZ

where CXCL12 regulates the lineage progression of

SVZ progenitors by transporting them from their rela-

tively quiescent ependymal niche to a basal vascula-

ture niche where these cells undergo active amplifica-

tion (Kokovay et al., 2010). In all these scenarios, it

is thought that CXCL12 fulfils its function by regulat-

ing neural progenitor cell migration as a chemoattrac-

tant. Indeed, CXCL12 can attract and enhance migra-

tion of in vitro cultured neural progenitor cells pre-

pared from a variety of regions in the CNS

(Dziembowska et al., 2005; Imitola et al., 2004; Car-

bajal et al., 2010, Luo et al., 2005). Perhaps the most

important and relevant implication of this observation

is that CXCL12 may be a key chemoattractant capa-

ble of directing either endogenous or transplanted

neural progenitor cells to sites of CNS injury and

engaging them for tissue repair, a possibility that has

already gained support from several mouse models of

brain pathology (Imitola et al., 2004; Corti et al.,

2005; Carbajal et al., 2010).

It should be noted, however, that most CXCR4-

expressing neural progenitor cells reside in the ven-

tricular zone where neurogenesis takes place on site,

and thus do not undergo migration during embryonic

development. This suggests that CXCL12 may regu-

late some other aspects of these cells: conceivably,

proliferation and survival. In fact, CXCL12 was orig-

inally cloned owing to its ability to stimulate prolifer-

ation of pre-B cells synergistically with IL-7 (Naga-

sawa et al., 1994). So far, several studies have

reported that CXCL12 can stimulate proliferation of

in vitro cultured neural progenitor cells from both

rodent and human origins (Imitola et al., 2004; Pritch-

ett et al., 2007; Wu et al., 2009; Li et al., 2011),

although contradictory results show that CXCL12

induces human neural progenitor cells to enter quies-

cence (Krathwohl and Kaiser, 2004). What accounts

for the discrepancy is still unclear. Similarly,

CXCL12 also appears to promote survival of neural

progenitor cells cultured in vitro (Dziembowska et

al., 2005; Prichett et al., 2007). However, beyond

these in vitro studies, an effect of CXCL12 on prolif-

eration and survival of neural progenitor cells in the

ventricular zones of CXCR4- or CXCL12-deficient

mice has not yet been reported. One possibility could

be redundancy resulting from the presence of a num-

ber of growth promoting factors operating in this



system. Li et al. (2011) recently suggested that

CXCL12 may only stimulate neural progenitor prolif-

eration in synergy with epidermal growth factor

(EGF) and fibroblast growth factor (FGF). Further-

more, neural progenitor cells appear to express sev-

eral other chemokine receptors besides CXCR4

(Peng et al., 2004; Tran et al., 2007), raising the pos-

sibility of involvement of other chemokine molecules

in regulating the proliferation and survival of neural

progenitor cells. An alternative possibility that should

be considered is that CXCL12/CXCR4 may regulate

some aspects of neural progenitor cells in vivo other

than migration, proliferation, or survival. Indeed, we

recently found that CXCL12 signaling is required to

maintain the integrity of radial glial processes of neu-

ral progenitor cells in the caudal spinal cord (Zhu et

al., unpublished observation).

CONCLUDING REMARKS

Among the large chemokine family, the CXCL12

chemokine receptor system is best known for its

involvement in the normal development of CNS. This

may partly be due to the vast amount of studies focus-

ing on this chemokine system in comparison with the

others. A few other chemokine receptors have also

been found to be expressed in the developing CNS

and may have roles in glial cell development (Bajetto

et al., 2001; Tsai et al., 2002; Tran and Miller, 2003;

Ambrosini and Aloisi, 2004; Tran et al., 2007),

although little in vivo function has so far been attrib-

uted to them. Future effort may uncover new players

from the chemokine family for the normal brain de-

velopment. On the other hand, the predominant role

of CXCL12 and its receptors in CNS development

may have its roots in the fact that CXCL12 and

CXCR4 are evolutionarily more ancient than most

other chemokine members, and that their ancestral

role could very well be in the development of the

CNS which predates the emergence of the adaptive

immune system in vertebrates (Huising et al., 2003).

The plethora of functions CXCL12 and its receptors

play during the development of CNS is impressive

and the list will continue to grow. Yet we still do not

have a coherent view of how this chemokine system

operates mechanistically in the complex and dynamic

tissue environment like the developing brain. Impor-

tant questions remain unanswered. How CXCL12

signaling is turned off for the developmental process

to move onto the next step? What different down-

stream effectors are activated in different cell types

or even different compartment of one cell? How

CXCL12 is distributed and how this distribution is

maintained or reshaped as tissue morphogenesis takes

place? How CXCL12 signaling interacts with other

signaling pathways to influence cell behaviors? To

answer these questions, particularly in vivo, poses

future challenges for us developmental neurobiolo-

gists who work on chemokines in the nervous system.

The authors thank Dr. Peter Karagiannis for editing the

manuscript.

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Chemokine CXCL12 and its receptors in the developing central nervous system: Emerging themes and future perspectives