Download CXCR4 and CXCR7 Have Distinct Functions in Regulating

Neuron
Article
CXCR4 and CXCR7 Have Distinct Functions
in Regulating Interneuron Migration
Yanling Wang,1,* Guangnan Li,2,5 Amelia Stanco,1,5 Jason E. Long,3 Dianna Crawford,4 Gregory B. Potter,1
Samuel J. Pleasure,2 Timothy Behrens,3 and John L.R. Rubenstein1,*
1Department
of Psychiatry, Nina Ireland Laboratory of Developmental Neurobiology
in Neuroscience and Developmental Biology and Department of Neurology
University of California, San Francisco, 1550 4th street, San Francisco, CA 94158, USA
3Genentech Corp., 1 Dna Way, South San Francisco, CA 94080, USA
4Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
5These authors contributed equally to this work
*Correspondence: [email protected] (Y.W.), [email protected] (J.L.R.R.)
DOI 10.1016/j.neuron.2010.12.005
2Programs
SUMMARY
CXCL12/CXCR4 signaling is critical for cortical interneuron migration and their final laminar distribution.
No information is yet available on CXCR7, a newly
defined CXCL12 receptor. Here we demonstrated
that CXCR7 regulated interneuron migration autonomously, as well as nonautonomously through its
expression in immature projection neurons. Migrating cortical interneurons coexpressed Cxcr4
and Cxcr7, and Cxcr7–/– and Cxcr4–/– mutants had
similar defects in interneuron positioning. Ectopic
CXCL12 expression and pharmacological blockade
of CXCR4 in Cxcr7–/– mutants showed that both
receptors were essential for responding to CXCL12
during interneuron migration. Furthermore, live
imaging revealed that Cxcr4–/– and Cxcr7–/– mutants
had opposite defects in interneuron motility and
leading process morphology. In vivo inhibition of Ga
(i/o) signaling in migrating interneurons phenocopied
the interneuron lamination defects of Cxcr4–/–
mutants. On the other hand, CXCL12 stimulation of
CXCR7, but not CXCR4, promoted MAP kinase
signaling. Thus, we suggest that CXCR4 and CXCR7
have distinct roles and signal transduction in regulating interneuron movement and laminar positioning.
INTRODUCTION
Cortical GABAergic interneurons are generated in multiple
progenitor zones of the subpallial (subcortical) telencephalon,
including the lateral, medial, and caudal ganglionic eminences
(LGE, MGE, and CGE) (Anderson et al., 1997a, 2001; Butt
et al., 2005; Fogarty et al., 2007; Marin and Rubenstein, 2003;
Pleasure et al., 2000; Sussel et al., 1999; Wonders and Anderson, 2006). The specification, differentiation, and migration of
these cells are regulated by multiple transcription factors,
including the Dlx1,2,5,6 and Lhx6 homeobox genes. The Dlx
genes are critical for interneuron migration and differentiation.
For example, mice lacking Dlx1/2 show a block in the migration
of most cortical and hippocampal interneurons (Anderson et al.,
1997a; Pleasure et al., 2000). Mice lacking Dlx1 show defects in
dendrite-innervating interneurons (Cobos et al., 2005), whereas
mice lacking either Dlx5 or Dlx5/6 have defects in somal-innervating (parvalbumin+; PV+) interneurons (Wang et al., 2010).
Studies on transcriptional alterations in the Dlx1/2–/– mutants
have begun to elucidate the molecular pathways that regulate
interneuron development and function (Long et al., 2009a,
2009b). We have discovered that the Dlx genes promote the
expression of two chemokine receptors, CXCR4 and CXCR7
(RDC1; CMKOR1) (Long et al., 2009a, 2009b; Wang et al.,
2010). Furthermore, these receptors are also positively regulated
by the Lhx6 transcription factor (Zhao et al., 2008) that is
essential for the differentiation of PV+ and somatostatin+ (SS+)
interneurons (Liodis et al., 2007; Zhao et al., 2008).
CXCR4 and CXCR7 are seven-transmembrane receptors that
bind CXCL12, a chemokine also known as Stromal-derived
factor 1 (SDF1) (Balabanian et al., 2005; Libert et al., 1991).
CXCL12 binding to CXCR4 triggers Gai protein-dependent
signaling, whereas CXCl12 binding to CXCR7 does not activate
Gai signaling (Levoye et al., 2009; Sierro et al., 2007).
On the other hand, many lines of evidence indicate that
CXCR7 has an important role in regulating cell signaling in culture
and in vivo.
In developing zebrafish, CXCR4 and CXCR7 are both implicated in regulating migration of primordial germ cells (PMGs)
and the posterior lateral line primordium, in part through their
differential expression patterns (Boldajipour et al., 2008; Dambly-Chaudiere et al., 2007; Valentin et al., 2007). For instance,
while CXCR4 is expressed in the germ cells, CXCR7 is expressed
in adjacent cells. It has been proposed that CXCR7 functions as
a ‘‘decoy’’ receptor to generate a gradient of CXCL12, through
ligand sequestration; the CXCL12 gradient would impact
CXCR4 signaling through controling ligand availability (Boldajipour et al., 2008).
Another mode for CXCR7 function has been proposed based
on experiments in which transiently transfected cells ectopically
express both CXCR4 and CXCR7 (Levoye et al., 2009; Sierro
et al., 2007). These studies showed that CXCR7 forms
Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc. 61
Neuron
CXCR7 Regulates Cortical Interneuron Migration
heterodimers with CXCR4. In this context, CXCR7 dampened
CXCR4 signaling.
More recently, transient transfection studies have provided
evidence that CXCR7 is a signaling receptor. Unlike traditional
seven-transmembrane receptors, which signal through both G
proteins and b-arrestin, CXCR7 may only signal through b-arrestin (Rajagopal et al., 2010). b-arrestin activation then leads
to stimulation of the MAP kinase casade (Rajagopal et al.,
2010; Xiao et al., 2010).
CXCL12 and CXCR4 cellular functions were first studied in
leukocyte chemotaxis (D’Apuzzo et al., 1997; Valenzuela-Fernandez et al., 2002). However, their wider roles in cell migration
are now appreciated, particularly in CNS development. Mice
deficient in either CXCL12 or CXCR4 exhibit abnormal neuronal
migration in the cerebellum, dentate gyrus, and dorsal root
ganglia (Bagri et al., 2002; Belmadani et al., 2005; Ma et al.,
1998). Meningeal expression of CXCL12 controls positioning
and migration of Cajal-Retzius cells via CXCR4 signaling (Borrell
and Marin, 2006; Paredes et al., 2006). Furthermore, CXCL12/
CXCR4 signaling controls cortical interneuron migration by
focusing the cells within migratory streams and controlling their
position within the cortical plate (Li et al., 2008; Lopez-Bendito
et al., 2008; Stumm et al., 2003; Tiveron et al., 2006).
Analysis of CXCR7 function in mice is limited to studies that
demonstrate its function in heart valve and septum development
(Gerrits et al., 2008; Sierro et al., 2007). Here, using both constitutive and conditional null mouse mutants, we report that Cxcr7
is essential for the migratory properties of mouse cortical
interneurons. We demonstrated that Cxcr4 and Cxcr7 were
coexpressed in migrating cortical interneurons. Each receptor
was essential for interneuron migration based on several lines
of evidence. First, Cxcr7–/– and Cxcr4–/– null mutants had
remarkably similar histological phenotypes. Second, ectopic
expression of CXCL12 in the developing cortex, which ordinarily
attracts interneurons, did not cause interneuron accumulation in
either the Cxcr7–/– or the Cxcr4–/– mutant. Third, pharmacological blockade of CXCR4 in Cxcr7–/– null mutants did not augment
their lamination phenotype. Despite their similar phenotypes on
static histological preparations, live imaging revealed that
migratory Cxcr7–/– and Cxcr4–/– interneurons had opposite
abnormalities in interneuron motility and leading process
morphology. Finally, we demonstrated that in vivo inhibition of
G(i/o) signaling in differentiating interneurons recapitulated the
interneuron positioning defects observed in the cortical plate of
CXCR4 mutants. Using primary MGE cultures, we also demonstrated that CXCR7 but not CXCR4 promotes MAP kinase
signaling in vitro.
RESULTS
Cxcr7 Expression in Mouse Telencephalon
Cxcr7 mRNA is expressed in the prenatal subpallium and pallium
(Long et al., 2009a, 2009b). In the subpallium, Cxcr7 was
primarily expressed in progenitor domains of the septum, LGE,
MGE, and CGE between E12.5 and E15.5 (Figures 1A–1E and
Figures S1A–S1J available online); this expression weakened
at E18.5 (Figures S1K–S1O). In the prenatal pallium, Cxcr7
expression strongly labeled the marginal zone (MZ) and subven62 Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc.
tricular zone/intermediate zone (SVZ/IZ). There were also scattered Cxcr7-expressing cells throughout all layers of the cortical
plate (CP) (Figures 1A–1E).
To identify the molecular features of Cxcr7-expressing cells,
we used Cxcr7-GFP and Lhx6-GFP transgenic mouse lines.
The expression pattern of Cxcr7-GFP recapitulated that of
Cxcr7 mRNA in both the ventral and dorsal parts of telencephalon at E15.5 (Figures 1F and 1G and Figures S1P–S1T). We
performed double labeling of GFP+ cells by using GFP immunohistochemical staining in conjunction with fluorescent in situ
RNA hybridization for Cxcl12, Reelin, Cxcr7, Cxcr4, Lhx6, and
Dlx1. None of the Cxcr7-GFP+ cells coexpressed their ligand,
Cxcl12 (Figure 1H), and 5% of the Cxcr7-GFP+ cells coexpressed Reelin (Figure 1I). Furthermore, the vast majority of
Cxcr7-GFP+ cells in the MZ and SVZ coexpressed Cxcr7,
Cxcr4, Lhx6, and Dlx1 (Figures 1J–1R). Next, we investigated
whether Cxcr4 and Cxc7 were expressed in MGE-derived
Lhx6-GFP+ cells by performing GFP immunohistochemical
staining with fluorescent in situ RNA hybridization for Cxcr7
and Cxcr4. We found that 70%–80% of Lhx6-GFP+ cells in the
MZ and SVZ expressed Cxcr4 or Cxcr7 (Figures 1S–1Y). Taken
together, these results indicate that Cxcr7-expressing cells in
the MZ and SVZ of prenatal pallium are primarily immature
interneurons that coexpress Cxcr4 and Cxcr7. Furthermore,
almost identical percentages of Lhx6-GFP+ interneurons
express either Cxcr4 or Cxcr7.
Abnormal Interneuron Distribution in Cxcr7–/– Mutants
To analyze Cxcr7 function, we generated conditional null
mutants in which exon 2 was flanked by LoxP sites; the entire
coding region is included within exon 2 (Cxcr7flox allele). By
breeding these mice to deleter transgenic mice and then outcrossing to wild-type B6 mice, we established a stably transmitting mouse line with deletion of Cxcr7 exon 2 (Figures 2A–2C). To
examine the cellular localization of CXCR7, we performed
CXCR7 antibody staining on the E13.5 MGE cells after 2 DIV.
While Cxcr7/ mutants showed no staining (Figure 2E), wildtype cells showed robust CXCR7 expression that appeared as
intracellular aggregates in the close proximity to the nucleus
(Figure 2D). The majority of Lhx6-GFP+ MGE cells expressed
CXCR7 protein (Figure 2F), consistent with our fluorescent
in situ hybridization results (Figure 1Y).
We began our analysis with the constitutive null Cxcr7/
mutants. To examine whether a loss of Cxcr7 function affected
interneuron migration, we assessed expression of Lhx6-GFP+
cells by immunohistochemical staining. Lhx6-GFP is expressed
in interneurons derived from the medial ganglionic eminence
(Cobos et al., 2007). We followed the progression of phenotype
over different developmental stages. At E13.5, in Cxcr7+/+
brains, Lhx6-GFP+ cells formed the MZ and SVZ migratory
streams; most SVZ cells had elongated processes pointing
toward the dorsal cortex (Figure 2G). In contrast, Lhx6-GFP+
cells in the Cxcr7/ cortex did not advance as far dorsally
(dotted line in Figure 2H). Furthermore, mutant cells in the MZ
and SVZ appeared to be intermingled, and their processes
were less polarized along the dorsal (tangential) dimension (Figure 2H). In the E14.5 Cxcr7+/+ cortex, Lhx6-GFP+ cells were
mainly found in the MZ and SVZ/IZ (Figure 2I), whereas in the
Neuron
CXCR7 Regulates Cortical Interneuron Migration
Figure 1. Expression Pattern of Cxcr7 at E15
(A–E) In situ hybridization of coronal sections show the expression of Cxcr7 in wild-type control at E15.5.
(F) Cxcr7-GFP expression of coronal section through the brain of an E15.5 embryo carrying the Cxcr7-GFP transgene.
(G) High magnification of the boxed area in (F).
(H–Q) Double Cxcr7-GFP immunohistochemical staining and fluorescent in situ hybridization for Cxcl12, Reelin, Cxcr7, Cxcr4, Lhx6, or Dlx1 in the MZ (H–M) and in
the SVZ (N–Q).
(R) Quantification of the percentage of Cxcr7-GFP+ cells expressing Cxcr7, Cxcr4, Lhx6, or Dlx1 in the MZ and in the SVZ. Bars correspond to standard error of the
mean (SEM) (n = 3).
(S–T) Fluorescent in situ hybridization showing expression pattern of Cxcr4 and Cxcr7 through the brain of an E15.5 embryo carrying the Lhx6-GFP transgene.
(U–X) Double Lhx6-GFP immunohistochemical staining and fluorescent in situ for either Cxcr4 or Cxcr7 in the MZ (U–V) and in the SVZ (W–X).
(Y) Quantification of the percentage of Lhx6-GFP+ cells expressing either Cxcr4 or Cxcr7 in the MZ and in the SVZ (n = 3).
CTX, cortex; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; CGE, caudal ganglionic eminence; MZ, marginal zone; CP, cortical plate; IZ,
intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bars represent 500 mm (A–E, F, S, and T), 50 mm (G), and 15 mm (H–Q and U–X). See also
Figure S1.
Cxcr7/ cortex, there were fewer Lhx6-GFP+ cells in the MZ and
SVZ and more cells in the cortical plate (Figure 2J). At stages of
E16.5 and E18.5, the mutants showed further depletion of
Lhx6-GFP+ cells in the cortical MZ and SVZ, and the Lhx6-GFP+
cells continued to accumulate in the CP (Figures 2L, 2N, 2P,
and 2R). The remaining Lhx6-GFP+ cells in the SVZ were chaotically oriented and displayed rudimentary processes (Figures 2N
and 2R). The distribution of interneurons expressing Lhx6 and
Dlx1 RNA showed the same phenotype as the Lhx6-GFP+ cells
in Cxcr7/ mutants (Figure S2).
Because Lhx6-GFP+ cells represent MGE-derived interneurons, but not interneurons derived from the dorsal CGE (dCGE;
Zhao et al., 2008), we investigated whether the Cxcr7 mutation
affected Lhx6-GFP migrating interneurons by using double
immunofluorescence labeling with anti-DLX2 and anti-GFP
antibodies. In the E14.5 control cortex, 55.5% of DLX2+ cells
expressed Lhx6-GFP whereas 99% of Lhx6-GFP+ cells expressed DLX2 (Figures 2S and 2T). Therefore, DLX2+/Lhx6GFP+ cells (yellow) represented MGE-derived interneurons,
whereas DLX2+/Lhx6-GFP cells (red) represented dCGEderived interneurons (Figure 2U). TBR1 antibody staining was
used to mark developing cortical projection neurons. We
analyzed the number of DLX2+/Lhx6-GFP+ cells and DLX2+/
Lhx6-GFP cells within each layer at E14.5 and E18.5. In
Cxcr7/ mutants at E14.5, Lhx6-GFP+ cells were reduced
in the MZ and SVZ and accumulated in the CP; in contrast,
Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc. 63
Neuron
CXCR7 Regulates Cortical Interneuron Migration
64 Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc.
Neuron
CXCR7 Regulates Cortical Interneuron Migration
Lhx6-GFP cells had a normal distribution (Figures 2V–2W
and 2Z). In Cxcr7/ mutants at E18.5, both Lhx6-GFP+ and
Lhx6-GFP cells displayed lamination defects in the MZ and
SVZ and in the CP (Figures 2X–2Y and 2Z). Therefore, our data
indicated that the Cxcr7 mutation preferentially affected Lhx6GFP+ interneurons at early stages, whereas it affected both
Lhx6-GFP+ and Lhx6-GFP interneurons at later stages.
Interneuron Phenotypes of Cxcr7–/– and Cxcr4–/–
Mutants
Next, we asked whether Cxcr4 and Cxcr7 mutations affected
interneuron migration in the same manner. We first studied
Lhx6 mRNA expression in Cxcr4/ and Cxcr7/ mutants at
E15.5 and quantified the total number of Lhx6+ cells in the lateral
neocortex (Figures 3A–3C and 3A0 –3C0 ). We found that both
Cxcr7–/– and Cxcr4/ mutants had 25% fewer Lhx6+ cells in
lateral neocortex as compared to controls (Figure 3D). Next,
we compared the laminar distribution of Lhx6+ cells in both
mutants and we found that they had very similar defects:
reduced numbers of Lhx6+ cells in the MZ and SVZ, and an
increase in the number of Lhx6+ cells in the CP, especially in
the lower CP (Figures 3A00 –3C00 and 3E). Thus, both Cxcr7 and
Cxcr4 had similar functions in maintaining interneurons within
the MZ and SVZ migratory streams and in controlling the timing
for interneuron invasion into the cortical plate.
While Cxcr4/ and Cxcr7/ mutants share very similar interneuron laminar positioning phenotypes in the cortical plate,
these deficits may arise from distinct alterations in migration
dynamics. To further explore the migration behaviors of
Cxcr4/ and Cxcr7/ interneurons in vivo, we performed
real-time imaging of Lhx6-GFP+ cortices from control (Cxcr4+/
or Cxcr7+/), Cxcr4/, and Cxcr7/ embryos at E15.5. We first
examined the transition from tangential to radial migration as
interneurons migrated from either the MZ or the SVZ into the
CP. In the MZ, Cxcr4/ and Cxcr7/ mutants demonstrated
a 3-fold and 2.4-fold increase in the number of Lhx6-GFP+ inter-
neurons switching from tangential to radial migration, respectively (Figures 4A–4C and 4G; Movies S1–S3). In the SVZ,
Cxcr4/ mutants displayed a 2-fold increase in the number of
Lhx6-GFP+ interneurons switching from tangential to radial
migration and no effect was detected in the Cxcr7/ mutants
(Figures 4D–4F and 4H; Movies S4–S6). Therefore, the laminar
deficits from both mutants were mainly due to an increased
number of Lhx6-GFP+ cells moving from either the MZ or the
SVZ into the cortical plate.
Next, we studied the tangential migration rate of Lhx6-GFP+
cells that were maintained in the MZ and SVZ during the
20-hour live-imaging session. Compared to controls, Cxcr7/
mutants exhibited a substantial decrease in tangential migration
rate in the MZ and SVZ; and Cxcr4/ mutants displayed a
modest decrease in migration rate in the SVZ (Figure 4I). Thus,
the decreased migration rate may underlie the reduced extent
of interneuron migration into the dorsal cortex observed in
both mutants during early embryonic stages.
Finally, we explored the migration behaviors of Lhx6-GFP+
cells after they entered into the cortical plate. Cxcr4/ and
Cxcr7/ mutants displayed major differences in interneuron
motility and leading process morphology. Cxcr4/ mutants
exhibited a significant increase in the number of motile cells
and in the leading process length of tangentially oriented cells,
while Cxcr7/ mutants showed a significant decrease in the
number of motile cells and in the leading process length of
both radially and tangentially oriented cells (Figure 5 and Movies
S7–S9). Together, these results indicated that CXCR4 and
CXCR7 conferred interneurons with distinctive migratory properties after they entered the cortical plate.
CXCR4 and CXCR7 Are Both Essential in Regulating
Interneuron Migration
Ectopic CXCL12 expression in the lateral prenatal neocortex
causes interneuron accumulation in vivo, which is presumably
mediated by the CXCR4 receptor (Li et al., 2008). However, it
Figure 2. Distribution and Cellular Morphology of Lhx6-GFP+ Cells in Cxcr7–/– Mutants
(A) Schematic representation of the wild-type Cxcr7 locus, the targeted allele, and the deleted locus. Shaded regions represent exon 2 (Ex2). The fragment
includes Ex2 and about 100 bp up- and downstream of Ex2. The 50 homologous arm was cloned from DNA immediately upstream of Ex2 (50 arm), and the 30
homologous arm was cloned from DNA immediately downstream of Ex2 (30 arm). LoxP sites are represented as black triangles and primer sites used for genotyping are represented as small arrowheads. E, EcoRV, neo, neomycin-resistance gene cassette.
(B) Southern-blot analysis of embryonic stem cell clones showing non-targeted (+/+) and targeted (+/f) clones. The 9.4 kb wild-type and 7.1 kb targeted allele
EcoRV-digested fragments were identified by an upstream probe, as shown in (A).
(C) PCR analysis of genomic DNA from E16.5 wild-type (+/+) embryos or embryos heterozygous (+/D) or homozygous (D/D) for Cre-mediated deletion of the targeted
locus. The 3.9 kb wild-type and 1.9 kb deleted products were from a single set of primers outside of Ex2, as shown in (A).
(D and E) Confocal images of Cxcr7/ mutant and control neurons from E13.5 MGE cultures stained with CXCR7 antibody after 2 DIV.
(F) Confocal image of control neurons from E13.5 Lhx6-GFP+ MGE cultures stained with CXCR7 and GFP antibodies after 2 DIV.
(G and H) Lhx6-GFP expression in the lateral cortices of Cxcr7+/+ and Cxcr7–/– embryos carrying Lhx6-GFP transgene at E13.5.
(I and J) Lhx6-GFP expression in the lateral cortices of Cxcr7+/+ and Cxcr7–/– embryos at E14.5. Note that there are fewer Lhx6-GFP+ cells in the MZ and the SVZ
(arrows) and more cells accumulate in the cortical plate in Cxcr7/ brains (asterisks).
(K–R) Lhx6-GFP expression in the lateral cortices of Cxcr7+/+ and Cxcr7–/– embryos at E16.5 and E18.5. Note that interneuron migration defects progress at
these stages.
(S) Confocal image showing normal Lhx6-GFP (green) and DLX2 (red) expression in E14.5 lateral cortex. TBR1 (blue) labels deep cortical plate.
(T) Quantification of the percentage of DLX2+ cells expressing Lhx6-GFP and the percentage of Lhx6-GFP+ cells expressing DLX2 (n = 3).
(U) Graphic illustration of the percentage contribution of Lhx6-GFP+ and Lhx6-GFP cells to DLX2+ cells.
(V–Y) Confocal images showing Lhx6-GFP+ (yellow) and Lhx6-GFP cells (red) from Cxcr7+/+ and Cxcr7/ cortices at E14.5 and E18.5. Number-denoted areas
are shown at higher magnification.
(Z) Quantification of the percentage of Lhx6-GFP+ and Lhx6-GFP cells (n = 5; *p < 0.01, #p < 0.05, x2 test).
MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bars represent 15 mm (D–F), 100 mm
(G–R, V1–Y1, and V2–Y2), and 500 mm (V–Y). See also Figure S2.
Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc. 65
Neuron
CXCR7 Regulates Cortical Interneuron Migration
Figure 3. Lamination Deficits of Cxcr4–/– and Cxcr7–/– Null Mutants
(A–C) In situ hybridization of coronal sections showing expression of Lhx6 in +/+, Cxcr4/, and Cxcr7/ mutants at E15.5. Boxed areas are shown in high
magnification (A00 –C00 ). The reduced extent of migration into the dorsal telencephalon is present in both mutants (arrowheads in A0 –C0 ).
(D) Quantitative analysis of the total number of Lhx6+ cells in the lateral cortices.
(E) Quantitative analysis of the distribution of Lhx6+ cells (n = 4; *p <0.01, #p < 0.05; ANOVA).
MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bars represent 500 mm (A, B, and C), 250 mm
(A0 , B0 , and C0 ), and 50 mm (A00 , B00 , and C00 ).
is possible that CXCR7 also confers interneuron responsiveness
to CXCL12 and that this chemoattraction is mediated by both
CXCR4 and CXCR7. To determine whether cellular responsiveness to CXCL12 is mediated by both CXCR4 and CXCR7, we
used in utero electroporation to ectopically express Cxcl12
and DsRed2 in the ventricular zone of the lateral neocortex of
controls (Cxcr7+/+ and Cxcr4+/+) and Cxcr7–/– and Cxcr4/
E13.5 embryos. Electroporation with DsRed2 alone into wildtype cortices was used as a control (Figure S3). After 48 hours,
we used in situ hybridization to examine the location and extent
of ectopic Cxcl12 expression (Figures 6B, 6D, and 6F) and the
distribution of Lhx6+ interneurons (Figures 6A, 6C and 6E).
Overexpression of Cxcl12 in controls induced an accumulation of Lhx6+ interneurons in the deep cortical plate (dCP) and
intermediate zone (IZ) as well as a depletion of Lhx6+ interneurons in the marginal zone (MZ) above the electroporation site
(Figures 6A–6A2 and 6H; n = 4). In contrast, neither the
Cxcr4/ nor the Cxcr7–/– mutants exhibited a measurable
accumulation of Lhx6+ interneurons around the site of ectopic
Cxcl12 expression (Figures 6C–C2, 6E–6E2, 6G, and 6H; n = 4).
Thus, these results suggested that interneuron attraction to
CXCL12 relied on the integrated function of both CXCR4 and
CXCR7.
To further assess the role of CXCR7 in CXCL12-mediated
chemoattraction, we studied the migration of E13.5 MGE cells
66 Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc.
by using a pharmacological inhibitor of CXCR7 (CCX771) in a
72-hour transwell migration assay. Control experiments showed
a 5-fold stimulation of migration by adding CXCL12 (100 nM) to
the bottom chamber. On the other hand, treatment with the
CXCR7 antagonist CCX771 (1 mM) reduced CXCL12-mediated
cell migration (Figure S4). Similar inhibition was observed with
the CXCR4 antagonist (AMD3100 [5 mM]) (Figure S4). Treatment
with both CCX771 and AMD3100 did not lead to further
reduction in migration (Figure S4).
As Cxcr7 and Cxcr4 were coexpressed within interneurons
(Figure 1K) and were both essential in regulating interneuron
migration, we investigated whether simultaneous disruption of
their function would lead to an enhanced phenotype. Because
these genes are closely linked on mouse chromosome 1
(1.2 Mb apart), we could not generate a double mutant. We
circumvented this problem by pharmacologically inhibiting
CXCR4 with a specific inhibitor, AMD3100 (Donzella et al.,
1998; Lazarini et al., 2000) in Cxcr7–/– mutants. We injected
AMD3100 (1 ml, 12.6 mM in PBS) or PBS (control) into the lateral
ventricle of E14.5 embryos carrying the Lhx6-GFP allele (some of
which were Cxcr7–/– mutants). We analyzed the location of GFP+
cells after 15 hours. As reported previously (Lopez-Bendito et al.,
2008), AMD3100-treated wild-type embryos showed interneuron defects that closely resembled those found in Cxcr4–/–
mutants: fewer cells populated the MZ and SVZ, whereas
Neuron
CXCR7 Regulates Cortical Interneuron Migration
many more cells were located in the CP (Figure 6J). Importantly,
AMD3100 treatment of Cxcr7–/– mutants did not exacerbate their
phenotype (Figures 6K–6M), consistent with the CCX771 and
AMD3100 inhibitor experiments described above (Figure S4).
These data indicated that Cxcr4 did not compensate for the
loss of Cxcr7 function and that CXCR4 and CXCR7 receptors
did not have fully redundant functions in regulating interneuron
migration.
Cxcr7 Was Expressed in Medial Pallial Cortical Plate
Cells, Where It Nonautonomously Regulates
Interneuron Migration
Cxcr7 mRNA and Cxcr7-GFP expression provided evidence that
Cxcr7 was expressed in the immature projection neurons of the
cortical plate (Figure S1). Furthermore, we examined Cxcr7
expression in E15.5 Dlx1/2/ mutants. We found that Cxcr7
expression was greatly reduced in the subpallium and was eliminated in most of the cortex, except for a band of cortical plate
expression extending in a dorsoventral gradient in the dorsomedial pallium (Figures 7B and 7B0 ; Figure S5E). These Cxcr7+
pallial cells are almost certainly not interneurons, as very few
interneurons that reach the cortex in the Dlx1/2/ mutants are
present in a scattered pattern in the SVZ and intermediate
zone (Cobos et al., 2006). Furthermore, E13.5 cortical cultures
(2 DIV) showed that CXCR7 was detectable in 10% of TBR1+
or CTIP2+ cortical projection neurons (Figure 7C and 7D). Therefore, in addition to its expression in migrating interneurons, Cxcr7
is expressed in immature excitatory neurons of the cortical plate,
especially of the dorsomedial cortical plate. Given this, we investigated Cxcr7’s specific functions in immature GABAergic and
glutamatergic neurons by using conditional mutagenesis.
We selectively deleted Cxcr7 gene in telencephalic GABAergic
lineages (including cortical interneurons) and cortical glutamatergic lineages by using the Dlx-I12b-Cre allele (Potter et al.,
2009) and the Emx1-Cre allele (Guo et al., 2000), respectively.
We compared the distribution of Lhx6+ cortical interneurons in
the dorsomedial pallium of Emx1Cre+; Cxcr7f/+, Emx1Cre+;
Cxcr7f/, DlxI12bCre+; Cxcr7f/+, and DlxI12bCre+; Cxcr7f/
embryos at E15.5 and E18.5. At E15.5, both the DlxI12bCre
and Emx1Cre Cxcr7 conditional knockouts had reduced Lhx6+
cells in the MZ and SVZ and excess number of cells in the CP
(Figures 7E–7I and Figures S5F–S5I). These phenotypes persisted at E18.5 (Figures 7J–7M). We assessed whether the
interneuron laminar defects in Emx1Cre+, Cxcr7f/ were due to
the abnormal lamination of Cajal-Retzius cells or cortical projection neurons. We did not detect defects in the laminar organization of Cajal-Retzius cells (based on Reelin and CR expression)
and the cortical plate (based in Cux2, SATB2, CTIP2, and
TBR1 expression) at E15.5 and E18.5 (Figure S6). Thus, we
propose that Cxcr7 functions in the dorsomedial cortical plate
nonautonomously regulate interneuron migration without having
a detectable effect on the position of cortical projection neurons.
To further determine whether the deletion of Cxcr7 in interneurons affects their lamination postnatally, we examined interneuron phenotypes in the adult neocortex of DlxI12bCre+,
Cxcr7f/ mutants by using a Cre-inducible R26R-EYFP reporter
(Srinivas et al., 2001). We found that recombined (EYFP+) interneurons expressing CR, PV, or SS were more frequently present
in the mutant’s deep cortical layers compared to control (Figure S7). Thus, interneuron-specific deletion of Cxcr7 alters the
laminar distribution of cortical interneurons pre- and postnatally.
CXCR4 and CXCR7 Signal through Different Molecular
Mechanisms in Developing Cortical Interneurons
CXCR4 signals through Gai, whereas CXCR7 is not believed to
signal through heterotrimeric G proteins (Boldajipour et al.,
2008; Levoye et al., 2009; Sierro et al., 2007). On the other
hand, recently, CXCR7 has been shown to signal through
b-arrestin and thereby to activate the MAP kinase cascade in
transiently transfected cells (Rajagopal et al., 2010; Regard
et al., 2007; Xiao et al., 2010). To gain insights into the signaling
mechanisms used by CXCR4 and CXCR7 in developing
interneurons, we used in vivo and primary culture assays that
employed CXCR4 and CXCR7 mutant mice, inhibition of Ga(i/o)
with pertussis toxin (PTX), and CXCR4&7 inhibition with small
molecule antagonists. We assessed the effects of these molecular perturbations on interneuron migration and MAP kinase
signaling.
First, we inhibited Ga(i/o) in migrating interneurons in vivo by
using PTX to probe the function of seven-transmembrane receptors (including CXCR4) that couple with this class of heteromeric
G proteins. We used mice carrying an allele in which the S1
subunit of PTX was introduced into the Rosa26 locus (Regard
et al., 2007). Expression of the S1 subunit of PTX in developing
interneurons required removal of a floxed polyadenylation signal
by using Cre recombinase; for this purpose, we used Dlx5/6 Cre
(Kohwi et al., 2007). We analyzed the location of Lhx6+ migrating
interneurons by using in situ hybridization in the Dlx5/6 Cre+;
PTXf/+ mice, compared with PTXf/+ controls at E18.5 (Figures
8A–8C). We found that PTX-expressing embryos phenocopied
the interneuron positioning defects observed in the cortical plate
of Cxcr4/ mutants (compare Figure 8B and Figure S8B).
Therefore, these results show that PTX-sensitive Gai/o signaling
regulates interneuron positioning. Given that CXCR4, and not
CXCR7, is known to signal in this manner, these results provide
strong evidence for the mechanism of CXCR4 signaling in
migrating interneurons. They also suggest that there are no other
receptors signaling through Gai/o that have a major role in
regulating early interneuron development.
We next tested whether CXCL12 promotes MAP kinase
signaling through CXCR4 and/or CXCR7 in MGE primary
cultures. In wild-type cells, CXCL12 increased phospho-p44/
42 MAP Kinase (pErk1/2) expression (Figure 8G). pErk1/2
expression was asymmetrically localized in the cell, typically at
the base of the primary process growing from the cell, and in
that process (arrows in Figure 8G). In addition, pErk1/2 often
colocalized with CXCR7 (arrowheads in Figure 8G0 ).
Finally, we examined whether CXCL12 increased pErk1/2
expression in MGE cells lacking CXCR4 or CXCR7 or exposed
to AMD3100 (CXCR4 antagonist). Cxcr4/ cells or AMD3100treated cells showed no change in the CXCL12-mediated
increase in pErk1/2 (Figures 8H, 8J, and 8L). On the other
hand, Cxcr7/ mutant cells failed to show the CXCL12-mediated increase in pErk1/2 (Figures 8I and 8L). Thus, CXCR7,
and not CXCR4, strongly activates the MAP kinase pathway in
MGE cells.
Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc. 67
Neuron
CXCR7 Regulates Cortical Interneuron Migration
68 Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc.
Neuron
CXCR7 Regulates Cortical Interneuron Migration
DISCUSSION
CXCL12/CXCR4 signaling in interneurons controls their migration and final position in the cortical plate (Li et al., 2008; Lopez-Bendito et al., 2008; Stumm et al., 2003; Tiveron et al.,
2006). Here we demonstrated that CXCR7 regulated these
processes through its distinct roles in immature interneurons
and projection neurons. We showed that migrating immature
cortical interneurons coexpressed Cxcr4 and Cxcr7 and that
Cxcr7–/– and Cxcr4/ single-mutant mice had similar defects
in interneuron position. Both receptors were essential for responding to CXCL12 and for regulating interneuron migration,
based on experiments that studied the effect of (1) ectopic
CXCL12 expression and (2) pharmacological blockade of
CXCR4 in Cxcr7–/– mutants. Furthermore, live imaging revealed
that Cxcr4–/– and Cxcr7/ mutants had opposite defects in
interneuron motility and leading process morphology. We
provided evidence that CXCR4 and CXCR7 signal through
distinct mechanisms in immature interneurons. In vivo inhibition
of Ga(i/o) signaling (the means through which CXCR4 is known to
function) in differentiating interneurons phenocopied the interneuron laminar positioning defects observed in the prenantal
cortex of the Cxcr4/ mutant. Finally, in MGE primary cultures
CXCR7, but not CXCR4, promoted CXCL12-mediated MAP
kinase signaling. As such, we conclude that in migrating interneurons CXCR4 and CXCR7 perform essential but distinct
functions in regulating interneuron movement and laminar
positioning.
Cxcr7 Regulates Interneuron Migration and Laminar
Positioning
We found that Cxcr7/ constitutive null mutants and DlxI12bCre
Cxcr7 conditional mutants (loss of expression in forebrain
GABAergic neurons) had interneuron laminar positioning defects
that were comparable to Cxcr4/ and Cxcl12/ mutants
(Figures 3 and 7; Li et al., 2008; Lopez-Bendito et al., 2008;
Stumm et al., 2003; Tiveron et al., 2006).
Live-imaging analysis provided insights into why Cxcr4/ and
Cxcr7/ mutants had laminar deficits of Lhx6+ interneurons on
fixed brain sections. Both mutants showed an increased number
of tangentially migrating mutant interneurons that switched from
tangential migration (in the MZ and SVZ) to radial migration into
the cortical plate. This is consistent with the idea that mutant
interneurons have reduced responsiveness to CXCL12 in the
SVZ and meninges. Furthermore, live imaging of both mutants
detected a reduced rate of interneuron tangential migration in
the SVZ; this may underlie why the mutant interneurons fail to
advance as far dorsally as wild-type interneurons (arrows in
Figure 3).
Cxcr7 expression waned between E15.5 and E18.5 (Figure 1
and Figure S1). This interval correlates with the period when
a large flux of interneurons invade the cortical plate (LopezBendito et al., 2008). Perhaps downregulation of Cxcr7 results
in reduced interneuron responsiveness to CXCL12, thereby
enabling their entry into the cortical plate. This is consistent
with our electroporation experiments showing that interneurons
require both CXCR4 and CXCR7 receptors to respond to
CXCL12-mediated chemoattraction.
CXCR4 and CXCR7 Perform Essential but Distinct
Functions in Regulating Interneuron Migration
Several lines of evidence suggest that both CXCR4 and CXCR7
regulate CXCL12 signaling in migrating interneurons. Cxcr4/
and Cxcr7/ mutants had nearly equivalent interneuron positioning phenotypes. In addition, both Cxcr4 and Cxcr7 were
required for attraction to ectopic CXCL12. Thirdly, the CXCR4
blockage did not exacerbate the phenotype observed in the
Cxcr7/ mutants. These experiments provide evidence that
signaling through both CXCR4 and CXCR7 are essential in
guiding migrating interneurons.
After entering the cortical plate, movements of Lhx6-GFP+
cells from the Cxcr4/ and Cxcr7/ mutants exhibited opposite phenotypes: CXCR4-deficient neurons were highly motile
and elaborated a longer leading process, whereas CXCR7-deficient neurons were much less motile and had a shorter leading
process. These results suggest that signaling downstream of
CXCR4 and CXCR7 have distinct effects on cytoskeletal
organization as previous studies have shown that actin and
microtubule dynamics are the main cytoskeletal contribution
responsible for cell motility (Baudoin et al., 2008; Etienne-Manneville, 2004; Reiner and Sapir, 2009). Our result also implies
that both receptors are required for the interneurons to coordinate their movements in response to guidance cues in the cortex
and eventually situate themselves in their appropriate cortical
positions. Indeed, there are interneuron lamination defects in
the adult cortex of interneuron-specific Cxcr4 and Cxcr7 conditional mutants (Figure S7; Li et al. 2008).
Our data demonstrated that blocking Ga(i/o) signaling in vivo,
using PTX, phenocopied the CXCR4 mutant (Figure 8B and
Figure S8B), consistent with CXCR4’s known ability to signal
through Ga(i/o) (Albert and Robillard, 2002; Hamm, 1998; Hesselgesser et al., 1998; Ma et al., 1998; Patrussi and Baldari,
2008; Teicher and Fricker, 2010). On the other hand, because
CXCR7 does not appear to signal through heteromeric G
Figure 4. Real-Time Assessment of Interneuron Migration in the Cortical Wall Reveals Abnormal Migration in Cxcr4–/– and Cxcr7–/– Mutant
Embryos
(A–C) Live imaging of E15.5 Lhx6-GFP+ cortices showing interneurons transitioning from tangential to radial migration along the MZ in Cxcr4+/ or Cxcr7+/
controls (A, cells in red), Cxcr4/ (B, cells in blue), and Cxcr7/ (C, cells in purple) mutants.
(D–F) Interneurons transitioning from tangential to radial migration along the SVZ in Cxcr4+/ or Cxcr7+/ controls (D, cells in red), Cxcr4/ (E, cells in blue), and
Cxcr7/ (F, cells in purple) mutants.
(G–H) Quantification of the percentage of interneurons transitioning from tangential to radial migration along the MZ and SVZ.
(I) Quantification of rates of tangential migration through the MZ and SVZ. Time elapsed since the onset of observations is indicated in minutes. n = 5; *p < 0.01
one-way ANOVA and Tukey-Kramer post-hoc test. MZ, marginal zone; CP, cortical plate; SVZ, subventricular zone. Scale bar represents 50 mm. See also
Movies S1–S6.
Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc. 69
Neuron
CXCR7 Regulates Cortical Interneuron Migration
Figure 5. Cxcr4–/– and Cxcr7–/– Mutant Interneurons Display Opposite Morphological and Motility
Deficits within the Cortical Plate at E15.5
(A–C) Confocal image showing Lhx6-GFP expression in
controls (+/+ or +/) and Cxcr4/ and Cxcr7/ mutants.
(D) Quantification of the number of motile cells (i.e., cells
migrating at a rate of > 5 mm/hr) within the CP of Cxcr4/,
Cxcr7/, and control cortices.
(E–K) Interneurons from the CP, oriented in a radial (E–G)
or tangential (I–K) direction are aligned by their cell soma
(yellow arrowhead). The leading process length is assessed from the base of the soma to the tip of the leading
process (red arrowhead).
(H and L) Quantification of leading process length of
interneurons oriented in either radial or tangential direction
(n = 5; *p < 0.01 one-way ANOVA and Tukey-Kramer
post-hoc test).
MZ, marginal zone; CP, cortical plate; SVZ, subventricular
zone. Scale bars represent 110 mm (A–C), 19 mm (D–F), and
20 mm (G–I). See also Movies S7–S9.
proteins, the mechanism underlying its function has been
controversial.
CXCR7 has been reported to regulate responses to CXCL12 in
tissue culture cells, where CXCR4 and CXCR7 form heterodimers (Levoye et al., 2009; Sierro et al., 2007). CXCL12 stimulation of HEK293 cells results in stronger calcium flux and more
robust phosphorylation of MAPKp42/44 in cells that coexpress
CXCR4 and CXCR7 than in cells that express only CXCR4 (Sierro
et al., 2007). This opens the possibility that CXCR4 and CXCR7
form heterodimers in migrating interneurons and that the
balance of CXCR dimers and monomers modulates how the
interneurons respond to CXCL12.
70 Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc.
Recently, CXCR7 has been shown to signal
through b-arrestin to activate MAP kinases in
transiently transfected cells (Rajagopal et al.,
2010; Regard et al., 2007; Xiao et al., 2010).
Our data using cultures of MGE cells support
these findings. We found that Cxcr7–/– mutant
cells failed to show a CXCL12-mediated
increase in pErk1/2, whereas loss of CXCR4
function did not alter this process (Figures 8H–
8L). Thus, in immature MGE interneurons
CXCR7, but not CXCR4, strongly promotes
MAP kinase signaling.
Therefore, our data provide evidence that
CXCR4 and CXCR7 signal through different
pathways in the developing interneurons. Future
studies are needed to test whether these
signaling differences underlie the opposite
defects in interneuron motility and leading
process morphology of Cxcr4–/– and Cxcr7–/–
mutants (Figure 5).
In the cultured MGE cells and cortical cells,
CXCR7 is predominantly expressed inside the
cell in perinuclear aggregates that may be
internal membranous vesicles (Figures 2D,
2F, 7C, and 7D). CXCL12-induced pErk1/2
partially colocalized with CXCR7 (Figure 8G0 ). There is evidence that internalized seven-transmembrane receptors continue to signal by G-protein-independent mechanisms, such
as through b-arrestins to activate the MAP kinase cascade in
signalsomes (Cottrell et al., 2009; Luttrell et al., 1999; Tohgo
et al., 2002). Thus, perhaps CXCR7 activates pErk in endosomes, through b-arrestins. Currently we are uncertain of the
location of the CXCR4, as all of the antibodies we tested
continue to stain the surface of Cxcr4–/– cells. Thus, future
studies are needed to fully elucidate how CXCR4 and CXCR7
differentially regulate signaling, morphology, and motility of
developing interneurons.
Neuron
CXCR7 Regulates Cortical Interneuron Migration
Figure 6. Ectopic Cxcl12 Expression in the Lateral Cortices of Cxcr4–/– and Cxcr7–/– Mutants and Pharmacological Blockade of CXCR4 in
Cxcr7–/– Mutants Show that Both Receptors Are Essential in Mediating Interneuron Migration In Vivo
(A, C, and E) In situ hybridization showing expression of Lhx6 in +/+ (A), Cxcr4/ (C), and Cxcr7–/– (E) brain sections. Boxed areas are shown in higher magnification (A1–E1 and A2–E2).
(B, D, and F) Cxcl12 in situ reveals the site of ectopic Cxcl12 expression. (G) Quantitative analysis of the Lhx6+ cell accumulation in electroporated lateral cortices.
The ratios were generated as cell number on electroporated side/cell number on contralateral side.
(H) Quantitative analysis of the Lhx6+ cell accumulation in each layer. (n = 4; #p < 0.05, *p <0.01, ANOVA).
(I) Confocal image showing the normal Lhx6-GFP expression in the lateral cortex of Cxcr7+/+ embryos injected with PBS.
(J–L) Lhx6-GFP expression in the lateral cortices of Cxcr7+/+ embryos injected with AMD3100 (J), Cxcr7–/– embryos injected with PBS (K), and Cxcr7–/– embryos
injected with AMD3100 (L).
(M) Quantitative analysis of the distribution of Lhx6-GFP+ cells in each layer. n = 5; *p < 0.01, #p < 0.05; one-way ANOVA, Tukey-Kramer post-hoc test.
MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bars represent 500 mm (A–F), 100 mm (A1, A2,
C1, C2, E1, and E2), and 200 mm (I–L). See also Figures S3 and S4.
CXCR7 Function in the Cortical Plate Regulates
Interneuron Positioning
We showed that Cxcr7 mRNA and Cxcr7-GFP were expressed in
the immature projection neurons of the cortical plate (Figure S1).
In addition, the remaining Cxcr7 expression in the dorsomedial
pallium of Dlx1/2/ mutants also supports this idea, as
Dlx1/2/ mutants have very few cortical interneurons. On the
other hand, we did not detect Cxcr4 expression in the neocortical
plate (Figure S5B). Thus, we hypothesize that CXCR7 functions
as homodimers in immature cortical projection neurons. Deletion
of Cxcr7 in cortical plate cells with Emx1-Cre revealed that Cxcr7
non-cell-autonomously regulates interneuron migration, especially in the dorsomedial pallium. Furthermore, this regulation
was not secondary to changes in the laminar position of CajalRetzius cells and projection neurons (Figure S6).
Cxcr7 cortical plate expression followed a dorsoventral
gradient (Figure S5E), which was opposite to the gradient of
Cxcl12 SVZ expression (Figure S5A). The inverse gradients of
the ligand and its receptor in the developing cortex are intriguing,
because CXCR7 has been reported to act as a scavenger
receptor that can reduce the concentration of CXCL12 available
for signaling (Boldajipour et al., 2008). Possibly, CXCR7 in the
cortical plate may lower the concentration of CXCL12 and
generate a gradient from either MZ or SVZ to the cortical plate,
thereby regulating the cortical invasion of migrating interneurons. In our study, elimination of Cxcr7 in the cortical plate may
Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc. 71
Neuron
CXCR7 Regulates Cortical Interneuron Migration
Figure 7. Cxcr7 Has Cell-Autonomous and Non-Cell-Autonomous Functions in Regulating Interneuron Laminar Distribution
(A and B) In situ hybridization showing expression of Cxcr7 in Dlx1/2+/+ and Dlx1/2/ brain sections. Boxed areas in (A) and (B) are shown in higher magnification.
(C and D) Confocal images of wild-type neurons from E13.5 cortical cultures stained with CXCR7 and TBR1 or CTIP2 antibodies after 2 DIV.
(E–H) Coronal sections through the telencephalons of Emx1cre+; Cxcr7f/+ (E) and Emx1cre+; Cxcr7f/- (F), DlxI12bCre+; Cxcr7f/+(G), and DlxI12bCre+; Cxcr7f/ (H)
showing Lhx6 expression at E15.5. Note that both conditional mutants exhibit laminar distribution defects of Lhx6+ interneurons.
(I) Quantitative analysis of the distribution of Lhx6+ cells/each layer at E15.5 (n = 3; *p < 0.01, ANOVA).
(J–M) E18.5 Lhx6 expression in the dorsomedial pallium of Emx1cre+; Cxcr7f/+ (J) and Emx1cre+; Cxcr7f/ (K), DlxI12bCre+, Cxcr7f/+(L), and DlxI12bCre+, Cxcr7f/
embryos (M).
MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bars represent 1 mm (A and B), 500 mm (A0 –B0
and E0 –H0 ), 200 mm (E–H and J–M), and 15 mm (C and D). See also Figures S5–S7.
72 Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc.
Neuron
CXCR7 Regulates Cortical Interneuron Migration
Figure 8. CXCR4 and CXCR7 Function through Different Molecular Pathways
(A and B) In situ hybridization showing expression of Lhx6 on Dlx5/6 Cre; PTXf/+(A) and Dlx5/6 Cre+; PTXf/+ (B) brain sections at E18.5.
(A0 and B0 ) Boxed areas in (A) and (B) are shown in higher magnification.
(C) Quantitative analysis of the distribution of Lhx6+ cells in each layer (n = 3; *p < 0.01, ANOVA).
(D) Wild-type MGE cells stained with CXCR7.
(E) Wild-type cells treated with 100 nM CXCL12-1a showing no effect on CXCR7 staining.
(F) Wild-type cells stained with pErk1/2.
(G and G0 ) Wild-type cells treated with 100 nM CXCL12-1a showing increased pErk1/2 staining; some of the activated pErk1/2 expression colocalized with
CXCR7 staining (arrowheads).
(H) pErk1/2 expression from Cxcr4/ MGE cells treated with CXCL12-1a.
(I) pErk1/2 expression from Cxcr7/ MGE cells treated with CXCL12-1a.
(J and K) pErk1/2 expression from wild-type and Cxcr7/ MGE cells treated with CXCL12-1a and AMD3100.
(L) Quantification of relative pErk1/2 staining intensity (n = 3; *p < 0.01, ANOVA).
MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bars represent 800 mm (A and B), 200 mm
(A0 and B0 ), and 15 mm (D–K). See also Figure S8.
disrupt this gradient and thus results in a premature interneuron
entry.
Transcription Factor Hierarchy Regulating Interneuron
Development
Several transcription factors have been demonstrated to regulate the migration of interneurons generated from the ganglionic
eminences; these include Arx, Dlx1, Dlx2, Dlx5, Lhx6, Nkx2.1,
and Sox6 (Alifragis et al., 2004; Anderson et al., 1997a, 1997b;
Azim et al., 2009; Batista-Brito et al., 2009; Casarosa et al.,
1999; Colasante et al., 2008; Liodis et al., 2007; Marin et al.,
2000; Nobrega-Pereira et al., 2008; Pleasure et al., 2000; Sussel
et al., 1999; Zhao et al., 2008). Effector downstream genes that
are implicated in regulating interneuron migration such as
Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc. 73
Neuron
CXCR7 Regulates Cortical Interneuron Migration
Cxcr4 and Cxcr7 are beginning to be identified (Long et al., 2007,
2009a, 2009b; Zhao et al., 2008).
In Dlx1/2/ double mutants, expression of Cxcr4 and Cxcr7 is
greatly reduced in cortical interneurons and the ganglionic
eminences (Figure S5; Long et al., 2009a, 2009b). Our data
provide evidence that CXCR4 and CXCR7 function is not
required for interneurons to migrate from the ganglionic
eminences to the cortex (Figure 3), whereas in Dlx1/2/ mutants
the migration block is nearly complete (Anderson et al., 1997a;
Long et al., 2007). Thus, other factors must contribute to the
failure of interneuron migration in the Dlx1/2/ mutants such
as overexpression of Pak3 (Cobos et al., 2007).
On the other hand, Lhx6, Cxcr4, and Cxcr7 mutants share
similar defects in interneuron migration rate and loss from the
cortical MZ (Zhao et al., 2008). Lhx6 mutants have greatly
reduced Cxcr4 and Cxcr7 expression (Zhao et al., 2008). Thus,
it is appealing to conclude that failure to express these receptors
is a principle mechanism contributing to the interneuron
migration defects in Lhx6 mutants.
EXPERIMENTAL PROCEDURES
Mice
All experimental procedures were approved by the Committees on Animal
Health and Care at the University of California, San Francisco (UCSF). Mouse
colonies were maintained at UCSF in accordance with National Institutes of
Health and UCSF guidelines. Cxcr4 null mice were previously described
(Ma et al., 1998; Stumm et al., 2003). The Dlx5/6Cre and DlxI12bCre lines
were previously described (Kohwi et al., 2007; Potter et al., 2009), and the
Lhx6-GFP Cxcr7-GFP BAC lines were purchased from The Gene Expression
Nervous System Atlas Project (GENSAT) at The Rockefeller University
(New York, NY). The Rosa26-pertussis toxin (PTX) mice were obtained from
Shaun Coughlin (Regard et al., 2007). See the Supplemental Data for details
on the generation of Cxcr7flox/+ mice.
Histology
See the Supplemental Data for details on immunohistochemistry and in situ
hybridization.
In Utero Electroporation and Drug Administration
In utero electroporation with pCAGGS-Cxcl12 and pCAGGS-DsRed2 or
pCAGGS alone was performed as described (Li et al., 2008). For blocking of
CXCR4 receptors, 1 ml of AMD3100 solution (12.6 mM; Sigma) or PBS was
injected into the lateral ventricle at E14.5.
Real-Time Imaging
Coronal slices (200 mm) of E15.5 Cxcr4+/ or Cxcr7+/ control and littermate
Cxcr4/ or Cxcr7/ mutant brains were placed onto nucleopore membrane
filters over 35 mm glass-bottom dishes containing Minimum Essential Medium
(GIBCO) and 10% FBS. Mounted slices were immediately transferred to
a 37 C/5% CO2 live tissue incubation chamber attached to a Zeiss inverted
microscope and a PASCAL confocal laser scanning system and imaged
repeatedly every 12 min for up to 20 hr. Real-time interneuronal migration
patterns were quantified by using Zeiss LSM Image Browser software.
Primary Cell Cultures and CXCR7 Immunohistochemistry
See the Supplemental Data for details on the primary cultures and the transwell
assay. For CXCR7 staining, the cells were fixed in 2% formaldehyde for 30 min
at RT and then permeabilized with permeabilization solution (PBS/0.1% BSA/
0.2% saponin) for 10 min at RT. Cells were incubated in blocking solution
(PBS/1% BSA/0.1% saponin/1% goat serum) for 30 min at RT. Cells were
then incubated with CXCR7 antibody (11G8, 1:400; ChemoCentryx) in blocking solution for 30 min on ice. For double labeling, cells were first incubated
74 Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc.
with CXCR7 antibody and then incubated with the other primary antibodies
for 2 hr at RT.
Statistical Analysis
Statistical analysis was performed with Student’s t test, x2 test, or the one-way
ANOVA and Tukey-Kramer post-hoc test using Prism4 (GraphPad Software)
and XLSTAT (Addinsoft) software. p <0.05 was considered statistically
significant.
SUPPLEMENTAL INFORMATION
Supplemental Information includes eight figures, nine movies, and Supplemental Experimental Procedures and can be found with this article online at
doi:10.1016/j.neuron.2010.12.005.
ACKNOWLEDGMENTS
We thank Marc von Zastrow and Mark Penfold for helpful discussions and
reagents. This work was supported by the research grants to J.L.R.R. from
Citizens United for Research in Epilepsy (C.U.R.E.), Nina Ireland, Larry
L. Hillblom Foundation, Weston Havens Foundation, and NIMH R37
MH049428; to Y.W. from C.U.R.E Rhode Island Award from the Epilepsy
Foundation; and to S.J.P. from K02 MH074985 and R01 MH077694. Timothy
W. Behrens and Jason E. Long are full-time employees of Genentech, Inc.
Dianna Crawford is a full-time employee of Amgen, Inc.
Accepted: November 4, 2010
Published: January 12, 2011
REFERENCES
Albert, P.R., and Robillard, L. (2002). G protein specificity: Traffic direction
required. Cell. Signal. 14, 407–418.
Alifragis, P., Liapi, A., and Parnavelas, J.G. (2004). Lhx6 regulates the migration of cortical interneurons from the ventral telencephalon but does not
specify their GABA phenotype. J. Neurosci. 24, 5643–5648.
Anderson, S.A., Eisenstat, D.D., Shi, L., and Rubenstein, J.L. (1997a).
Interneuron migration from basal forebrain to neocortex: Dependence on Dlx
genes. Science 278, 474–476.
Anderson, S.A., Qiu, M., Bulfone, A., Eisenstat, D.D., Meneses, J., Pedersen,
R., and Rubenstein, J.L. (1997b). Mutations of the homeobox genes Dlx-1 and
Dlx-2 disrupt the striatal subventricular zone and differentiation of late born
striatal neurons. Neuron 19, 27–37.
Anderson, S.A., Marin, O., Horn, C., Jennings, K., and Rubenstein, J.L. (2001).
Distinct cortical migrations from the medial and lateral ganglionic eminences.
Development 128, 353–363.
Azim, E., Jabaudon, D., Fame, R.M., and Macklis, J.D. (2009). SOX6 controls
dorsal progenitor identity and interneuron diversity during neocortical development. Nat. Neurosci. 12, 1238–1247.
Bagri, A., Gurney, T., He, X., Zou, Y.R., Littman, D.R., Tessier-Lavigne, M., and
Pleasure, S.J. (2002). The chemokine SDF1 regulates migration of dentate
granule cells. Development 129, 4249–4260.
Balabanian, K., Lagane, B., Infantino, S., Chow, K.Y., Harriague, J., Moepps,
B., Arenzana-Seisdedos, F., Thelen, M., and Bachelerie, F. (2005). The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in
T lymphocytes. J. Biol. Chem. 280, 35760–35766.
Batista-Brito, R., Rossignol, E., Hjerling-Leffler, J., Denaxa, M., Wegner, M.,
Lefebvre, V., Pachnis, V., and Fishell, G. (2009). The cell-intrinsic requirement
of Sox6 for cortical interneuron development. Neuron 63, 466–481.
Baudoin, J.P., Alvarez, C., Gaspar, P., and Metin, C. (2008). Nocodazoleinduced changes in microtubule dynamics impair the morphology and
directionality of migrating medial ganglionic eminence cells. Dev. Neurosci.
30, 132–143.
Neuron
CXCR7 Regulates Cortical Interneuron Migration
Belmadani, A., Tran, P.B., Ren, D., Assimacopoulos, S., Grove, E.A., and
Miller, R.J. (2005). The chemokine stromal cell-derived factor-1 regulates the
migration of sensory neuron progenitors. J. Neurosci. 25, 3995–4003.
Boldajipour, B., Mahabaleshwar, H., Kardash, E., Reichman-Fried, M., Blaser,
H., Minina, S., Wilson, D., Xu, Q., and Raz, E. (2008). Control of chemokineguided cell migration by ligand sequestration. Cell 132, 463–473.
Borrell, V., and Marin, O. (2006). Meninges control tangential migration of
hem-derived Cajal-Retzius cells via CXCL12/CXCR4 signaling. Nat.
Neurosci. 9, 1284–1293.
Butt, S.J., Fuccillo, M., Nery, S., Noctor, S., Kriegstein, A., Corbin, J.G., and
Fishell, G. (2005). The temporal and spatial origins of cortical interneurons
predict their physiological subtype. Neuron 48, 591–604.
Casarosa, S., Fode, C., and Guillemot, F. (1999). Mash1 regulates neurogenesis in the ventral telencephalon. Development 126, 525–534.
Cobos, I., Calcagnotto, M.E., Vilaythong, A.J., Thwin, M.T., Noebels, J.L.,
Baraban, S.C., and Rubenstein, J.L. (2005). Mice lacking Dlx1 show
subtype-specific loss of interneurons, reduced inhibition and epilepsy. Nat.
Neurosci. 8, 1059–1068.
Kohwi, M., Petryniak, M.A., Long, J.E., Ekker, M., Obata, K., Yanagawa, Y.,
Rubenstein, J.L., and Alvarez-Buylla, A. (2007). A subpopulation of olfactory
bulb GABAergic interneurons is derived from Emx1- and Dlx5/6-expressing
progenitors. J. Neurosci. 27, 6878–6891.
Lazarini, F., Casanova, P., Tham, T.N., De Clercq, E., Arenzana-Seisdedos, F.,
Baleux, F., and Dubois-Dalcq, M. (2000). Differential signalling of the chemokine receptor CXCR4 by stromal cell-derived factor 1 and the HIV glycoprotein
in rat neurons and astrocytes. Eur. J. Neurosci. 12, 117–125.
Levoye, A., Balabanian, K., Baleux, F., Bachelerie, F., and Lagane, B. (2009).
CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G
protein signaling. Blood 113, 6085–6093.
Li, G., Adesnik, H., Li, J., Long, J., Nicoll, R.A., Rubenstein, J.L., and Pleasure,
S.J. (2008). Regional distribution of cortical interneurons and development of
inhibitory tone are regulated by Cxcl12/Cxcr4 signaling. J. Neurosci. 28,
1085–1098.
Libert, F., Passage, E., Parmentier, M., Simons, M.J., Vassart, G., and Mattei,
M.G. (1991). Chromosomal mapping of A1 and A2 adenosine receptors, VIP
receptor, and a new subtype of serotonin receptor. Genomics 11, 225–227.
Cobos, I., Long, J.E., Thwin, M.T., and Rubenstein, J.L. (2006). Cellular
patterns of transcription factor expression in developing cortical interneurons.
Cereb. Cortex 16 (Suppl 1 ), i82–i88.
Liodis, P., Denaxa, M., Grigoriou, M., Akufo-Addo, C., Yanagawa, Y., and
Pachnis, V. (2007). Lhx6 activity is required for the normal migration and specification of cortical interneuron subtypes. J. Neurosci. 27, 3078–3089.
Cobos, I., Borello, U., and Rubenstein, J.L. (2007). Dlx transcription factors
promote migration through repression of axon and dendrite growth. Neuron
54, 873–888.
Long, J.E., Garel, S., Alvarez-Dolado, M., Yoshikawa, K., Osumi, N., AlvarezBuylla, A., and Rubenstein, J.L. (2007). Dlx-dependent and -independent
regulation of olfactory bulb interneuron differentiation. J. Neurosci. 27,
3230–3243.
Colasante, G., Collombat, P., Raimondi, V., Bonanomi, D., Ferrai, C., Maira,
M., Yoshikawa, K., Mansouri, A., Valtorta, F., Rubenstein, J.L., and Broccoli,
V. (2008). Arx is a direct target of Dlx2 and thereby contributes to the tangential
migration of GABAergic interneurons. J. Neurosci. 28, 10674–10686.
Long, J.E., Cobos, I., Potter, G.B., and Rubenstein, J.L. (2009a). Dlx1 and
Mash1 transcription factors control MGE and CGE patterning and differentiation through parallel and overlapping pathways. Cereb. Cortex 19, i96–i106.
Cottrell, G.S., Padilla, B.E., Amadesi, S., Poole, D.P., Murphy, J.E., Hardt, M.,
Roosterman, D., Steinhoff, M., and Bunnett, N.W. (2009). Endosomal endothelin-converting enzyme-1: A regulator of beta-arrestin-dependent ERK
signaling. J. Biol. Chem. 284, 22411–22425.
Long, J.E., Swan, C., Liang, W.S., Cobos, I., Potter, G.B., and Rubenstein, J.L.
(2009b). Dlx1&2 and Mash1 transcription factors control striatal patterning and
differentiation through parallel and overlapping pathways. J. Comp. Neurol.
512, 556–572.
Dambly-Chaudiere, C., Cubedo, N., and Ghysen, A. (2007). Control of cell
migration in the development of the posterior lateral line: Antagonistic interactions between the chemokine receptors CXCR4 and CXCR7/RDC1. BMC Dev.
Biol. 7, 23.
Lopez-Bendito, G., Sanchez-Alcaniz, J.A., Pla, R., Borrell, V., Pico, E.,
Valdeolmillos, M., and Marin, O. (2008). Chemokine signaling controls intracortical migration and final distribution of GABAergic interneurons.
J. Neurosci. 28, 1613–1624.
D’Apuzzo, M., Rolink, A., Loetscher, M., Hoxie, J.A., Clark-Lewis, I., Melchers,
F., Baggiolini, M., and Moser, B. (1997). The chemokine SDF-1, stromal cellderived factor 1, attracts early stage B cell precursors via the chemokine
receptor CXCR4. Eur. J. Immunol. 27, 1788–1793.
Luttrell, L.M., Ferguson, S.S., Daaka, Y., Miller, W.E., Maudsley, S., Della
Rocca, G.J., Lin, F., Kawakatsu, H., Owada, K., Luttrell, D.K., et al. (1999).
Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein
kinase complexes. Science 283, 655–661.
Donzella, G.A., Schols, D., Lin, S.W., Este, J.A., Nagashima, K.A., Maddon,
P.J., Allaway, G.P., Sakmar, T.P., Henson, G., De Clercq, E., and Moore,
J.P. (1998). AMD3100, a small molecule inhibitor of HIV-1 entry via the
CXCR4 co-receptor. Nat. Med. 4, 72–77.
Etienne-Manneville, S. (2004). Actin and microtubules in cell motility: Which
one is in control? Traffic 5, 470–477.
Fogarty, M., Grist, M., Gelman, D., Marin, O., Pachnis, V., and Kessaris, N.
(2007). Spatial genetic patterning of the embryonic neuroepithelium generates
GABAergic interneuron diversity in the adult cortex. J. Neurosci. 27, 10935–
10946.
Gerrits, H., van Ingen Schenau, D.S., Bakker, N.E., van Disseldorp, A.J., Strik,
A., Hermens, L.S., Koenen, T.B., Krajnc-Franken, M.A., and Gossen, J.A.
(2008). Early postnatal lethality and cardiovascular defects in CXCR7-deficient
mice. Genesis 46, 235–245.
Guo, H., Hong, S., Jin, X.L., Chen, R.S., Avasthi, P.P., Tu, Y.T., Ivanco, T.L.,
and Li, Y. (2000). Specificity and efficiency of Cre-mediated recombination
in Emx1-Cre knock-in mice. Biochem. Biophys. Res. Commun. 273, 661–665.
Hamm, H.E. (1998). The many faces of G protein signaling. J. Biol. Chem. 273,
669–672.
Hesselgesser, J., Liang, M., Hoxie, J., Greenberg, M., Brass, L.F., Orsini, M.J.,
Taub, D., and Horuk, R. (1998). Identification and characterization of the
CXCR4 chemokine receptor in human T cell lines: Ligand binding, biological
activity, and HIV-1 infectivity. J. Immunol. 160, 877–883.
Ma, Q., Jones, D., Borghesani, P.R., Segal, R.A., Nagasawa, T., Kishimoto, T.,
Bronson, R.T., and Springer, T.A. (1998). Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc. Natl. Acad. Sci. USA 95, 9448–9453.
Marin, O., and Rubenstein, J.L. (2003). Cell migration in the forebrain. Annu.
Rev. Neurosci. 26, 441–483.
Marin, O., Anderson, S.A., and Rubenstein, J.L. (2000). Origin and molecular
specification of striatal interneurons. J. Neurosci. 20, 6063–6076.
Nobrega-Pereira, S., Kessaris, N., Du, T., Kimura, S., Anderson, S.A., and
Marin, O. (2008). Postmitotic Nkx2-1 controls the migration of telencephalic
interneurons by direct repression of guidance receptors. Neuron 59, 733–745.
Paredes, M.F., Li, G., Berger, O., Baraban, S.C., and Pleasure, S.J. (2006).
Stromal-derived factor-1 (CXCL12) regulates laminar position of CajalRetzius cells in normal and dysplastic brains. J. Neurosci. 26, 9404–9412.
Patrussi, L., and Baldari, C.T. (2008). Intracellular mediators of CXCR4-dependent signaling in T cells. Immunol. Lett. 115, 75–82.
Pleasure, S.J., Anderson, S., Hevner, R., Bagri, A., Marin, O., Lowenstein,
D.H., and Rubenstein, J.L. (2000). Cell migration from the ganglionic
eminences is required for the development of hippocampal GABAergic
interneurons. Neuron 28, 727–740.
Potter, G.B., Petryniak, M.A., Shevchenko, E., McKinsey, G.L., Ekker, M., and
Rubenstein, J.L. (2009). Generation of Cre-transgenic mice using Dlx1/Dlx2
Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc. 75
Neuron
CXCR7 Regulates Cortical Interneuron Migration
enhancers and their characterization in GABAergic interneurons. Mol. Cell.
Neurosci. 40, 167–186.
Rajagopal, S., Kim, J., Ahn, S., Craig, S., Lam, C.M., Gerard, N.P., Gerard, C.,
and Lefkowitz, R.J. (2010). Beta-arrestin- but not G protein-mediated signaling
by the "decoy" receptor CXCR7. Proc. Natl. Acad. Sci. USA 107, 628–632.
Regard, J.B., Kataoka, H., Cano, D.A., Camerer, E., Yin, L., Zheng, Y.W.,
Scanlan, T.S., Hebrok, M., and Coughlin, S.R. (2007). Probing cell typespecific functions of Gi in vivo identifies GPCR regulators of insulin secretion.
J. Clin. Invest. 117, 4034–4043.
Reiner, O., and Sapir, T. (2009). Polarity regulation in migrating neurons in the
cortex. Mol. Neurobiol. 40, 1–14.
Sierro, F., Biben, C., Martinez-Munoz, L., Mellado, M., Ransohoff, R.M., Li, M.,
Woehl, B., Leung, H., Groom, J., Batten, M., et al. (2007). Disrupted cardiac
development but normal hematopoiesis in mice deficient in the second
CXCL12/SDF-1 receptor, CXCR7. Proc. Natl. Acad. Sci. USA 104, 14759–
14764.
Srinivas, S., Watanabe, T., Lin, C.S., William, C.M., Tanabe, Y., Jessell, T.M.,
and Costantini, F. (2001). Cre reporter strains produced by targeted insertion
of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4.
Stumm, R.K., Zhou, C., Ara, T., Lazarini, F., Dubois-Dalcq, M., Nagasawa, T.,
Hollt, V., and Schulz, S. (2003). CXCR4 regulates interneuron migration in the
developing neocortex. J. Neurosci. 23, 5123–5130.
neuron precursors and invading interneurons via stromal-derived factor 1
(CXCL12)/CXCR4 signaling in the cortical subventricular zone/intermediate
zone. J. Neurosci. 26, 13273–13278.
Tohgo, A., Pierce, K.L., Choy, E.W., Lefkowitz, R.J., and Luttrell, L.M. (2002).
beta-Arrestin scaffolding of the ERK cascade enhances cytosolic ERK activity
but inhibits ERK-mediated transcription following angiotensin AT1a receptor
stimulation. J. Biol. Chem. 277, 9429–9436.
Valentin, G., Haas, P., and Gilmour, D. (2007). The chemokine SDF1a coordinates tissue migration through the spatially restricted activation of Cxcr7 and
Cxcr4b. Curr. Biol. 17, 1026–1031.
Valenzuela-Fernandez, A., Planchenault, T., Baleux, F., Staropoli, I.,
Le-Barillec, K., Leduc, D., Delaunay, T., Lazarini, F., Virelizier, J.L., Chignard,
M., et al. (2002). Leukocyte elastase negatively regulates Stromal cell-derived
factor-1 (SDF-1)/CXCR4 binding and functions by amino-terminal processing
of SDF-1 and CXCR4. J. Biol. Chem. 277, 15677–15689.
Wang, Y., Dye, C., Sohal, V., Long, J., Estrada, R., Roztocil, T., Lufkin, T.,
Deisseroth, K., Baraban, S., and Rubenstein, J.L. (2010). Dlx5 and Dlx6
regulate the development of parvalbumin-expressing cortical interneurons.
J. Neurosci. 30, 5334–5345.
Wonders, C.P., and Anderson, S.A. (2006). The origin and specification of
cortical interneurons. Nat. Rev. Neurosci. 7, 687–696.
Sussel, L., Marin, O., Kimura, S., and Rubenstein, J.L. (1999). Loss of Nkx2.1
homeobox gene function results in a ventral to dorsal molecular respecification
within the basal telencephalon: Evidence for a transformation of the pallidum
into the striatum. Development 126, 3359–3370.
Xiao, K., Sun, J., Kim, J., Rajagopal, S., Zhai, B., Villen, J., Haas, W., Kovacs,
J.J., Shukla, A.K., Hara, M.R., et al. (2010). Global phosphorylation analysis of
{beta}-arrestin-mediated signaling downstream of a seven transmembrane
receptor (7TMR). Proc. Natl. Acad. Sci. USA 107, 15299–15304.
Teicher, B.A., and Fricker, S.P. (2010). CXCL12 (SDF-1)/CXCR4 pathway in
cancer. Clin. Cancer Res. 16, 2927–2931.
Zhao, Y., Flandin, P., Long, J.E., Cuesta, M.D., Westphal, H., and Rubenstein,
J.L. (2008). Distinct molecular pathways for development of telencephalic
interneuron subtypes revealed through analysis of Lhx6 mutants. J. Comp.
Neurol. 510, 79–99.
Tiveron, M.C., Rossel, M., Moepps, B., Zhang, Y.L., Seidenfaden, R., Favor, J.,
Konig, N., and Cremer, H. (2006). Molecular interaction between projection
76 Neuron 69, 61–76, January 13, 2011 ª2011 Elsevier Inc.