dlx homeobox genes promote cortical interneuron migration from

Dlx Homeobox Genes Promote Cortical InterneuronMigration from the Basal Forebrain by Direct Repression ofthe Semaphorin Receptor Neuropilin-2*□S

Received for publication, August 7, 2006, and in revised form, January 12, 2007 Published, JBC Papers in Press, January 26, 2007, DOI 10.1074/jbc.M607486200

Trung N. Le‡§, Guoyan Du§¶, Mario Fonseca§¶, Qing-Ping Zhou§¶, Jeffrey T. Wigle‡�, and David D. Eisenstat§¶**1

From the Departments of ‡Biochemistry and Medical Genetics, ¶Pediatrics and Child Health, **Human Anatomy and Cell Science,§Manitoba Institute of Cell Biology, �St. Boniface General Hospital Research Centre, University of Manitoba, Winnipeg,Manitoba R3E 0V9, Canada

Dlx homeobox genes play an important role in vertebrateforebrain development.Dlx1/Dlx2 nullmice die at birth with anabnormal cortical phenotype, including impaired differentia-tion and migration of GABAergic interneurons to the neocor-tex. However, the molecular basis for these defects downstreamof loss ofDlx1/Dlx2 function is unknown.Neuropilin-2 (NRP-2)is a receptor for Class III semaphorins, which inhibit neuronalmigration.Herein, we show thatNeuropilin-2 is a specificDLX1and DLX2 transcriptional target by applying chromatin immu-noprecipitation to embryonic forebrain tissues. Both homeoboxproteins repressNrp-2 expression in vitro, confirming the func-tional significance of DLX binding. Furthermore, the homeodo-main of DLX1 and DLX2 is necessary for DNA binding and thisbinding is essential for Dlx repression of Nrp-2 expression. Ofimportance, there is up-regulated and aberrant expression ofNRP-2 in the forebrains ofDlx1/Dlx2 null mice. This is the firstreport that DLX1 or DLX2 can function as transcriptionalrepressors. Our data show that DLX proteins specifically medi-ate the repression of Neuropilin-2 in the developing forebrain.Aswell, our results support thehypothesis that down-regulationof Neuropilin-2 expression may facilitate tangential interneu-ron migration from the basal forebrain.

Members of theDlx homeobox gene family, orthologs ofDis-tal-less inDrosophila melanogaster, are expressed in the devel-oping brain (1, 2), retina (3), craniofacial structures, and limbs(4). FourDlx genes,Dlx1,Dlx2,Dlx5, andDlx6, are expressed inoverlapping domains in the subcortical telencephalon anddiencephalon, including the ventral thalamus and the gangli-onic eminences. There are distinct boundaries ofDlx1 andDlx2

expression at the pallial-subpallial boundary (1). Insights intothe functional role of Dlx genes in development have been pri-marily gained from analysis of the phenotypes of mice withtargeted deletions ofDlx1/Dlx2 (5–8),Dlx5 (9), andDlx5/Dlx6(10). The single Dlx1 and Dlx2 knockouts have relatively nor-mal forebrain development at birth, which is consistent withfunctional redundancy betweenDlx1 andDlx2 in this anatomicregion (1, 4). However, postnatal Dlx1 mutants show specificdifferentiation defects in interneuron subclasses (11, 12). In theabsence of both Dlx1 and Dlx2 function, there is abnormaldevelopment of the subventricular zone (SVZ)2 of the gangli-onic eminences. There is an almost complete loss of tangentialmigration of GABAergic interneurons from the medial gangli-onic eminence (MGE) to the neocortex and from the lateralganglionic eminence (LGE) to the olfactory bulb (5, 13)3. Thesemigrations comprise the major source of cortical inhibitoryinterneurons in the murine telencephalon (5, 14, 15). Further-more, it is evident that there are both Dlx-dependent and Dlx-independent pathways affecting the differentiation and subse-quent migration of interneurons to the striatum (6), olfactorybulb (13), and hippocampus (16).Our understanding of Dlx gene function is limited by the

current paucity of established transcriptional targets of Dlxgenes. We have commenced isolating and characterizing can-didateDlx gene targets in the developingmouse, particularly inthe forebrain, using chromatin immunoaffinity purification(ChIP) of embryonic tissues that regionally express Dlx genes.We have optimized the ChIP protocol using embryonic gangli-onic eminence tissues and specific affinity-purified rabbit poly-clonal antibodies to the proteins encoded by theDlx1 andDlx2genes (1) to isolate the Dlx5/Dlx6 intergenic enhancer, a DLXtarget gene promoter sequence (17). In this study, we exploitedthis approach to identify a novel DLX target in the embryonicforebrain.Several factors are important for the regulation of differenti-

ation and/or migration of interneurons from the forebrain.These include transcription factors such as Pax6 (18, 19),Emx1/Emx2 (20), and Tlx (21), Neuregulin-1/Erb-B4 signaling(22, 23), and other subcortical chemorepulsive or cortical che-

* This work was supported by a Canadian Institutes of Health Research (CIHR)strategic training grant and Natural Sciences and Engineering ResearchCouncil graduate studentships (to T. N. L.), a Manitoba Institute of ChildHealth postdoctoral fellowship (to G. D.), a CIHR New Investigator Award(to J. T. W.), March of Dimes Birth Defects Foundation Basil O’ConnorStarter Scholar Award 5-FY00-615 (to D. D. E.), and grants from the Foun-dation Fighting Blindness (Canada), CancerCare Manitoba Foundation,and Manitoba Medical Services Foundation (to D. D. E.). The costs of pub-lication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S7.

1 To whom correspondence should be addressed: Manitoba Inst. of Cell Biol-ogy, 675 McDermot Ave., Winnipeg, Manitoba R3E 0V9, Canada. Tel.: 204-787-1169; Fax: 204-787-2190; E-mail: [email protected].

2 The abbreviations used are: SVZ, subventricular zone; MGE, medial gangli-onic eminence; LGE, lateral ganglionic eminence; ChIP, chromatin immu-noaffinity purification; NRP2, Neuropilin-2; EMSA, electrophoretic gelmobility shift assay; GABA, �-aminobutyric acid.

3 J. E. Long and J. L. Rubenstein, unpublished observation.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 26, pp. 19071–19081, June 29, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

JUNE 29, 2007 • VOLUME 282 • NUMBER 26 JOURNAL OF BIOLOGICAL CHEMISTRY 19071

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

motactic guidance cues (24–26). The semaphorins (Sema) 3Aand 3F, by binding to their receptors Neuropilin-1 and Neuro-pilin-2, respectively, provide strong repulsive guidance cuesand mediate sorting of tangentially migrating interneuronsfrom the ganglionic eminences to the cortex and striatum (15).Different models to assess the function of members of the neu-ropilin family have been generated. A dominant negative formofNrp-1 increases interneuronmigration from theMGE to thestriatum and reduces migration to the neocortex in vitro (15).In Nrp-2 knock-out mice, sympathetic and hippocampal neu-rons have reduced repulsive responses to Sema3F but not toSema3A. There are also axonal pathfinding defects of specificcranial and sensory nerves and hippocampal mossy fiber pro-jections (27–29). However, similar to theNrp-1 dominant neg-ative experiments, there are increased numbers of interneuronsinvading the developing and postnatal striatum in these mice(15). Evidence of ectopic expression of Neuropilin-2 (Nrp-2) inthe Dlx1/Dlx2 mutant forebrain (15) provided a rationale totest whether direct DLX1 and/or DLX2 directly regulate Neu-ropilin-1/2 expression.Herein, using ChIP of mid-gestation ganglionic eminences,

we demonstrate that DLX1 andDLX2 directly bind to a specificregion of the Nrp-2 but not to the Nrp-1 promoter in vivo.Furthermore, both DLX1 and DLX2 inhibit transcription ofNrp-2 in vitro. Moreover, loss of Dlx1 and Dlx2 function leadsto the increased and ectopic expression of Neuropilin-2 as sub-ventricular ectopias in the medial and lateral ganglionic emi-nences. These results support our hypothesis that a subpopula-tion of late-born Dlx-expressing interneurons (Neuropilin-2low/non-expressing) successfully bypass semaphorin repulsivecues to migrate to the neocortex. Conversely, loss of Dlx1 andDlx2 function results in increased and aberrant Neuropilin-2expression in this population, and their resultant responsive-ness to semaphorin signaling may contribute to block tangen-tial interneuronmigration from the basal telencephalon. Thesedata delineate part of the molecular mechanism by whichmigrating interneurons may bypass inhibitory cues to reachtheir correct destinations in the forebrain.

MATERIALS AND METHODS

Animals—Mice with null mutations ofDlx1 andDlx2 (a kindgift from Dr. J. Rubenstein, University of California, San Fran-cisco)weremaintained in aCD1background atManitoba Insti-tute of Cell Biology following protocols approved by the Uni-versity ofManitoba under the auspices of theCanadianCouncilon Animal Care. Dlx1/Dlx2 mutants were genotyped usingpublished protocols (7, 27). Embryonic age was determined bythe day of appearance of the vaginal plug (E0.5), and tissueswere processed as previously described (3). For comparativestudies all mutant embryos were paired with wild-type litter-mate controls.Chromatin Immunoprecipitation Assays—ChIP was per-

formed under the following conditions as described (17). 1–2�107 E13.5 ganglionic eminence cells were fixed with 1%paraformaldehyde for 90 min at room temperature. Sonicationof cells (Sonifier cell disruptor 350) in SDS lysis buffer (1% SDS,50mMTris-HCl, pH8.1, 10mMEDTA)on ice generated solublechromatin complexes with DNA fragment lengths ranging

between 100 and 300 bp. Specific polyclonal high affinity DLXantibodies were used to immunoprecipitate genomic DNA tar-gets cross-linked to DLX1 or DLX2 homeoproteins. GenomicDNA from an E13.5 mouse embryo was used as a positive con-trol. DNA derived from E13.5 hindbrain was used as a negativecontrol because this tissue does not express any Dlx familymembers.Thermal Cycling/Polymerase Chain Reaction—We gener-

ated oligonucleotide primer pairs to two regions of the mouseNeuropilin-1 (Nrp-1) sequence (GenBankTM AF482432), des-ignated Nrp-1i and Nrp-1ii. For Nrp-1i, the sense primer was5�-GGAACCGGACTACATGG-3� and the antisense primerwas 5�-AAGACTGGCAACGACCC-3�. For Nrp-1ii, the senseprimer was 5�-AACCTCAGGCTGACACC-3� and the anti-sense primer was 5�-ACGAGATCTCTGCACCC-3�. We alsodesigned oligonucleotide primer pairs to two regions of themouse Neuropilin-2 promoter (Nrp-2) sequence (GenBankTMAF022854) that were designated Nrp-2i and Nrp-2ii. For Nrp-2i, the sense primer was 5�-GAGATCACACAGCTGCC-3�and the antisense primer was 5�-CCTACAACATCACGAGG-3�. For Nrp-2ii, the sense primer was 5�-CGTTGATCGTTA-GAGACC-3� and the antisense primer was 5�-GACAGAGAG-GCTCTCTC-3�.Electrophoretic Mobility Shift Assays—We used a region

(444–562 nt) of the Neuropilin-2 promoter (Nrp-2ii) (Gen-BankTM AF022854) and labeled double-stranded oligonucleo-tides with [�-P32]dATP using the Klenow large fragment ofDNA Polymerase I (Promega). The binding reaction mixture(20 �l total volume) contained labeled probes (80,000 cpm) inbinding buffer (20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5mMdithiothreitol, 250mMNaCl, 50mMTris-HCl, pH 7.5), 1�gof poly(dI-dC), and purified recombinant proteins or nuclearextracts derived from E13.5 ganglionic eminences. The bindingreactions were incubated for 30 min at room temperature. Weadded unlabeled probes for “cold competition” assays and spe-cific polyclonal DLX1 or DLX2 antibodies for “supershift”assays and polyclonal Secretory Component antibody (Dako,Mississauga, Ontario) to control for nonspecific binding (17).Reporter Gene Assays—Weused the Lipofectamine 2000 rea-

gent (Invitrogen) to transiently co-transfect luciferase genereporters (1 �g) into 106 human embryonic kidney 293 cellswith pRSV-� galactosidase (Promega) (0.4 �g) as an internalcontrol andharvested cell lysates 36 h later. Subsequently, lucif-erase activities were measured with the Luciferase ReporterAssay System (Promega), normalizing luciferase activity with�-galactosidase activity.The Nrp-2 luciferase reporter containsthe Nrp-2 promoter (444–562 nt) (GenBankTM AF022854)directing expression of the luciferase gene.We expressedDLX1and DLX2 (Dlx1 and Dlx2 cDNAs were a kind gift from Dr. J.Rubenstein, UCSF) and their derivatives from the pcDNA3expression plasmid (Invitrogen). We generated Q50E variantsusing the QuikChange site-directed mutagenesis kit (Strat-agene, La Jolla, CA). DLX-VP16 and DLX-Engrailed fusionconstructs were generated as follows: we excised the N-termi-nal domain of eitherDlx1 orDlx2 by HindIII/PpuMI or EcoRI/BsmBI (New England Biolabs) restriction enzyme digestions,respectively, and maintained the nuclear localization signal,homeodomain, and C-terminal domains. Engrailed (888-bp)

Transcriptional Control of Neuropilin-2 by Dlx Genes

19072 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 26 • JUNE 29, 2007


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

and VP16 (255-bp) domains (30) were ligated to the 5�-end ofthese N-terminal-modified Dlx constructs.Tissue Preparations and in SituHybridization—Tissueswere

prepared as previously described (1, 17). E13.5 tissues (wholeembryos) were fixed using 4% paraformaldehyde and cryopre-served using sucrose gradients followed by embedding in OCTmedium (Tissue Tek), whereas central nervous system tissuesfrom E16.5 and E18.5 embryos were prepared following dissec-tion. Tissue samples were also sectioned coronally at a thick-ness of 15 �m using a cryostat (Cryotome, ThermoShandon,Cheshire, UK). Digoxigenin in situ hybridization was carriedout as described (1, 3, 17) using cRNA riboprobes forNrp-1 andNrp-2 (a kind gift from Dr. M. Tessier-Lavigne, Genentech,South San Francisco, CA).Immunohistochemistry, Immunofluorescence, and Immuno-

blotting—Immunohistochemistry and single and doubleimmunofluorescence experiments were performed asdescribed (1, 3, 17). For immunohistochemistry and immuno-fluorescence we used the following primary antibodies: rabbitanti-DLX1 (1:100, N114 affinity-purified), rabbit anti-DLX2(1:300, C199 affinity-purified) (DLX1 and DLX2 polyclonalantisera were kind gifts fromDr. J. Rubenstein, UCSF, andwereaffinity-purified (1)), rabbit anti-GABA (1:8000; Sigma), andrabbit anti-Neuropilin-2 (1:500) (a kind gift fromDr. A. Kolod-kin and Dr. D. Ginty, Johns Hopkins University). Secondaryantibodies were fluorescein-conjugated goat anti-rabbit (1:100;Sigma), biotin-SP-conjugated goat anti-rabbit (1:200; JacksonImmunoResearch, West Grove, PA), streptavidin-conjugatedOregonGreen-488 (1:200;Molecular Probes, Eugene, OR), andstreptavidin-conjugated Texas Red (1:200; Vector Laborato-ries, Burlingame, CA). For immunoblotting, we followed estab-lished protocols (17, 31).

RESULTS

DLX1 and DLX2 Homeobox Proteins Bind to a Neuropilin-2Promoter Region in Embryonic Forebrain in Vivo—Marin et al.(15) established that theClass III semaphorins, semaphorins 3Aand 3F, and their receptors, Neuropilin-1 and Neuropilin-2,play important roles in the sorting of interneurons that aremigrating to the neocortex and the striatum. The promoters ofboth of these genes, Neuropilin-1 (Nrp-1) and Neuropilin-2(Nrp-2), contain putative TAAT/ATTA homeodomain DNAbinding sites. Hence, we consideredNrp-1 andNrp-2 as candi-date transcriptional targets of several homeobox genes region-ally expressed in the subcortical telencephalon, includingmembers of the Dlx gene family. To test this hypothesis, weassayed for the direct binding to both Nrp promoters by DLX1and/or DLX2 in vivo. E13.5 ganglionic eminences were treatedwith formaldehyde to cross-link protein-DNA complexes.Using a modified ChIP procedure, soluble nucleoprotein com-plexes of �100–300 base pair fragments were immunoprecipi-tated using anti-DLX1 and/or DLX2 antibodies. We then usedPCR to amplify candidate homeodomain binding regions in theNrp-1 and Nrp-2 loci. These regions were chosen based on thepresence of consensus homeodomain binding motifs and weredesignatedNrp-1i (nucleotides 104–283), Nrp-1ii (nucleotides711–900), Nrp-2i (nucleotides 138–255), and Nrp-2ii (nucleo-tides 444–562) (Fig. 1A). Notably, ChIP assays revealed that

both DLX1 and DLX2 bound only to Nrp-2 promoter region 2(Nrp-2ii) in embryonic striatum but there was no evidence forbinding with the other promoter regions in vivo (Fig. 1B). Theresulting amplicons were subcloned and sequenced to verifytheir identity and for subsequent biochemical analyses. Asexpected, control ChIP assays performed without antibody orwith anti-DLX antibodies and chromatin derived from embry-onic hindbrain tissues where Dlx genes are not expressed werenegative.DLX1 and DLX2 Bind to the Neuropilin-2 Promoter in Vitro—

To determine whether DLX1 and DLX2 specifically bind to theNrp-2 promoter region 2 in vitro, we used recombinant DLX1and DLX2 proteins and radiolabeled Nrp-2ii promoter regionsisolated from the ChIP assay. An electrophoretic gel mobilityshift assay (EMSA) showed binding of both DLX1 and DLX2 toNrp-2ii (Fig. 2A, lanes 2, 6) that was competitively inhibited byunlabeled Nrp-2ii probe (lanes 3, 7). Moreover, the addition ofspecific anti-DLX1 or anti-DLX2 antibodies to the protein-DNAcomplex resulted in significant bandmobility shifts (lanes4, 8), whereas a nonspecific polyclonal antibody failed to pro-duce such a “supershift” (lanes 5, 9). Neither DLX1 nor DLX2bind to the first TAAT motif of the Nrp-2ii in vitro. However,both recombinant Dlx proteins bind to the second motif (Fig.1A, box, supplemental Fig. S2, and data not shown). Theseexperiments demonstrate that DLX1 and DLX2 specificallybind to theNrp-2 promoter region 2 in vitro. As well, binding ofDLX1 and DLX2 to this region may be mutually exclusive.EMSAwith nuclear extracts derived from embryonic gangli-

onic eminences showed that endogenousDLX1 andDLX2 pro-teins can bind to the Nrp-2 promoter (Fig. 2B, lane 2). Unla-beled probe can competewith radiolabeled oligonucleotides forboth proteins (lane 3). Experiments using specific DLX1 orDLX2 antibodies and a control antibody confirmed the identityof theDlx complexes (lanes 4–6). The recombinant and endog-enous DLX proteins do not have identical molecular weights,perhaps reflecting different binding partners or post-transla-tional modifications such as phosphorylation in vivo (32).4These experiments demonstrate that DLX1 and DLX2 specifi-cally bind to the Nrp-2 promoter region 2 in vitro.DLX1 and DLX2 Repress Neuropilin-2 Promoter Expression

in Vitro—The functional significance of DLX1 andDLX2 bind-ing to the Nrp-2 promoter was assessed using luciferasereporter gene experiments. We co-transfected human embry-onic kidney 293 cells or P19 embryonal carcinoma cells (datanot shown) with an expression vector encoding Dlx1 or Dlx2and a vector in which region 2 of theNrp-2 promoter (444–562nt) drives luciferase expression. Co-transfection with eitherwild-typeDlx1 orDlx2 expression constructs resulted in signif-icant reductions of luciferase activity compared with controls(�1.7-fold for Dlx1 and �6-fold for Dlx2, p � 0.001; Fig. 3C),indicating that both DLX1 and DLX2 proteins can act as tran-scriptional repressors of Nrp-2 promoter expression.We generated several different mutant constructs of DLX1

andDLX2 homeodomain proteins to explore the consequencesof modifying specific DLX protein domains on activity of the

4 X. Qiu and D. Eisenstat, unpublished observations.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

reporter gene (Fig. 3A). The first set of constructs included amutation that converts glutamine (Q) to glutamate (E) at aminoacid position 50 (Q50E) of the DLX homeodomain. This Q50Emutant homeodomain is predicted to abrogate DNA binding,and the mutant protein may act in a dominant negative fashion(33). EMSA assays confirmed that the Q50E mutants do notbind to theNrp-2 promoter (Fig. 3B). In co-transfection assays,mutantDlx1 andDlx2 (Q50E) were unable to repress luciferasegene expressionwhen comparedwith thewild-typeDlx expres-sion constructs (Fig. 3C). The DNA binding mutant affects asimilar level of transcriptional activity for both Dlx1 and Dlx2;however, because the repression ofDlx1on theNrp-2promoteris less than by Dlx2 in the first place, the difference in Dlx1rescue (from repression) is not as significant as we found inDlx2 rescue by the Q50E DNA binding mutants. The second

group of constructs included anN-terminal domain exchange ofDLX1 and DLX2 with either a VP16activator domain or an Engrailedrepressor domain (30). N-terminalDLX1 and DLX2-Engrailed chi-meric fusion proteins furtherrepressed transcription of theNrp-2promoter region 2 compared withthe wild-type Dlx constructs. Con-versely, N-terminal DLX1 andDLX2-VP16 chimeric fusion pro-teins activated transcription of theNrp-2 promoter region, overcom-ing all or some of the transcriptionalrepression resulting from the wild-type Dlx constructs, by �3.7- and1.7-fold, respectively (p � 0.001,Fig. 3D). In this assay, Dlx2 is astronger repressor; hence thisfunction is not completely allevi-ated by replacement by VP16 at itsN terminus. Hence, from thesedata we can conclude that thedirect interaction of DLX1 orDLX2 proteins with the Nrp-2promoter results in the repressionof Neuropilin-2 transcription.DLX1 or DLX2 and Neuropilin-2

Expression Patterns in the Develop-ing Forebrain—Dlx homeoboxgenes are expressed in interneuronsthat express �-aminobutyric acid(GABA) in the embryonic rostralforebrain (5, 6, 34, 35). During theearly stages of embryonic telen-cephalon development, the subpal-lium expresses Dlx1 and Dlx2 pri-marily in the ventricular andsubventricular zones (1, 2, 36, 37)with a clear limitation of expressionat the LGE/neocortex (subpallial-pallial) boundary. Dlx1 and Dlx2

expression are both well established by E10.5 (1). Expression ofNRP-2 is reported to be primarily restricted to the mantle zoneof the MGE as well as the olfactory cortex (15). We found thatendogenous expression of Nrp-2 becomes well establishedfrom E13.5 in the mantle zone of the subcortical telencephalonusing in situ hybridization (supplemental Fig. S5). Further-more, we also detected NRP-2 protein expression as early asE13.5 using immunofluorescence (Figs. 4 and 5A). NRP-2expression becomes more restricted to the mantle zone of theMGE and paleocortex at age E16.5 and E18.5 or postnatal day 0(Figs. 5, B and C, supplemental Figs. S3 and S4). Co-expressionstudies of DLX1 or DLX2 and NRP-2 at E13.5 (for E16.5 andE18.5, see supplemental Figs. S3 and S4) show only a minimaloverlapping pattern with majority populations of DLX1/DLX2and NRP-2 single positive cells in the basal telencephalon (Fig.

FIGURE 1. Neuropilin-2, but not Neuropilin-1, is a DLX homeoprotein target in vivo. A, the sequences of candi-date regulatory elements within the mouse Neuropilin-1 (GenBankTM AF482432) and Neuropilin-2 (GenBankTM

AF022854) promoters, designated Nrp-1i, Nrp-1ii, Nrp-2i, and Nrp-2ii, respectively, contain putative homeodo-main DNA binding sites (TAAT/ATTA) in italics. Oligonucleotide primers used for PCR are underlined, and theTAAT motif located in Nrp-2ii required for binding to DLX1 and DLX2 is boxed. B, ChIP assays were performed onE13.5 mouse forebrain tissues using affinity-purified polyclonal DLX1 and DLX2 antibodies following cross-linking of protein-DNA complexes with 1% paraformaldehyde. Specific bands were evident for Nrp-2ii but notNrp-1i, Nrp-1ii, or Nrp-2i (left panel). Negative controls included performing ChIP without the addition of eitherprimary antibody or the use of E13.5 hindbrain, tissue that does not express Dlx genes (right panel). Positivecontrols were mouse genomic DNA (gDNA) and primer pairs for the Dlx5/Dlx6 intergenic enhancer (data notshown) (17). PCR bands were subcloned and confirmed by DNA sequencing.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

4). Combined in situ RNA hybridization (Nrp-2) and immuno-histochemistry (DLX1 or DLX2) experiments at E13.5 andE16.5 confirmed that only subsets of cells co-expressed Nrp-2

and DLX1/DLX2 within this neuroanatomic region (supple-mental Fig. S5). Because most cells do not co-express DLX andNrp-2, these results are consistent with the potential role ofDLX proteins as repressors of Neuropilin-2 expression in thedeveloping forebrain.Neuropilin-2 Is Ectopically Expressed in theAbsence ofDlx1and

Dlx2 Gene Function—The above assays show that DLX proteinsbind Nrp-2 in vivo and in vitro, that they repress the Nrp-2 pro-moter in vitro, and that DLX1/DLX2 and Nrp-2 are primarilyexpressed in separate cell populations in vivo. However, theseexperiments do not reveal whether DLX proteins are required torepressNrp-2 in vivo. To address this key issue,wedetermined theexpression patterns of Neuropilin-1 and Neuropilin-2 in vivo byanalyzingNrp-1RNA,NRP-2 protein, andNrp-2RNAexpressionin the forebrainsofDlx1/Dlx2doublemutantmice.Nodifferencesin Neuropilin-2 expression were observed between wild-type andDlx1/Dlx2mutant telencephalon at E13.5 (Fig. 5A). Similarly, theexpression pattern of Nrp-1 is unaffected throughout develop-ment of the embryonic forebrain toP0 (supplemental Fig. S7B anddata not shown). Significantly, immunofluorescence and in situRNA hybridization experiments demonstrate that there is pro-gressive accumulationofNeuropilin-2-expressing cells in the SVZof the LGE and MGE after E13.5 through to birth (15) when themutant mice die (Figs. 5, B and C, and 6). These results are con-sistentwith the hypothesis thatwith the absence ofDlx1 andDlx2function there is loss of transcriptional repression by DLX1 orDLX2 leading to theaberrant expressionofNRP-2, resulting in theformation of SVZ ectopias in subcortical cells of the mutant gan-glionic eminences (Fig. 6, arrows, panels b, d).

DISCUSSION

This study is the first to report that DLX homeoproteinsmayfunction as transcriptional repressors in vivo. DLX proteinshave been previously characterized as transcriptional activators(17, 38, 39), although it was shown that DLX proteins couldrepress transcription of several reporter plasmids in vitro (40).In addition, BP-1, an isoform of DLX7, represses the �-globingene in vitro (41). We have demonstrated that both DLX1 andDLX2 bind to specific homeodomain binding motifs in a cis-regulatory domain of the Neuropilin-2 (Nrp-2), but not theNrp-1 promoter in vivo, and repress transcription of a reportervector containing these sequences in vitro. We have also con-firmed that the homeodomain of DLX1 and DLX2 is necessaryfor DNA binding and that this binding is essential for Dlxrepression of Nrp-2 expression. A single base pair mutation ofglutamine (Q) to glutamate (E) at amino acid position 50(Q50E) of the 60-amino acid homeodomain is sufficient toeliminate the DNA binding ability of the DLX1/DLX2 home-odomain and subsequent transcriptional repression of areporter gene in vitro (Fig. 3,B andC). Residual DNAbinding inthe Q50E mutant proteins due to other possible DNA bindingsites localized within the homeodomain (not detected byEMSA) or by binding of DLX1/2 to other proteins bound to theNrp-2 promoter could account for the ability ofDlx1/2Q50E tostill marginally repress (although this repression was not statis-tically significant). Consistent with the evidence that Dlx1 andDlx2 function as repressors when bound to theNrp-2 promoteris the observation that Nrp-2 expression is significantly

FIGURE 2. DLX1 and DLX2 proteins specifically bind to a regulatory ele-ment within the Neuropilin-2 promoter in vitro and in vivo. A, EMSAs showbinding of recombinant DLX1 or DLX2 to Nrp-2 promoter region ii oligonu-cleotides containing homeodomain binding sites in vitro. Radiolabeled Nrp-2ii oligonucleotide probes were incubated alone (lane 1), with recombinantDLX1 protein (lanes 2–5), with recombinant DLX2 protein (lanes 6 –9), withunlabeled Nrp-2ii probes (lanes 3, 7), with affinity-purifed DLX antibodies(lanes 4, 8), and with nonspecific antibodies (lanes 5, 9). B, EMSA using embry-onic forebrain demonstrates that endogenous DLX1 and DLX2 proteins bindto Nrp-2ii in vivo. Radiolabeled Nrp-2ii oligonucleotides were incubated alone(lane 1), with nucleoprotein extracts from E13.5 ganglionic eminences (lanes2– 6), with unlabeled Nrp-2ii probe (lane 3), with affinity-purified DLX antibod-ies (lanes 4, 5), and with nonspecific antibodies (lane 6). Gel shifts, denotingspecific binding of DLX proteins to DNA, are indicated with solid arrows.Supershifts with specific DLX antibodies are indicated by broken arrows.Nrp-2, Neuropilin-2; r, recombinant; 1, DLX1/anti-DLX1; 2, DLX2/anti-DLX2; I,nonspecific polyclonal antibody.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

increased in the basal ganglia ofDlx1/Dlx2 doublemutantmice(15); Figs. 5, B and C, and 6).In contrast to our finding thatDLX1 andDLX2 are transcrip-

tional repressors of theNrp-2 promoter, DLX1 andDLX2 act astranscriptional activators of a specific target, the Dlx5/Dlx6intergenic enhancer in vitro and in vivo (17, 39) and gonado-tropin-releasing hormone regulatory elements in vitro (42).Furthermore, theDlx familymember DLX3 also acts as a trans-activator of a model target gene construct, Dlx3-CAT (43) andthe osteocalcin gene promoter (44). In support of a repressorfunction of the Dlx genes, DLX1, DLX2, and DLX5 interactwith a homeodomain binding sitewithin theWnt-1 enhancer invitro (2, 45). Mutation of this site results in extension of the

rostral boundary of Wnt-1/lacZ expression in transgenic ani-mals. The authors suggest that this site maymediate repressionof Wnt-1 expression in the forebrain, although neither DLX1nor DLX2 has been implicated in transcriptional repression ofWnt-1 in vivo (45). The amino termini of both DLX1 andDLX2may mediate, in part, the transcriptional repression activity ofthese homeodomain proteins, as demonstrated by the results ofsubstituting their N termini with the Engrailed repressor orVP16 activation domains in the reporter gene assays. We havefound DLX2 to be a better repressor than DLX1; therefore, thereplacement of the DLX1 N-terminal domain with the VP16activation domain is able to overcome the “weak” repressivefunction of DLX1. In contrast, DLX2 is a stronger repressor;

FIGURE 3. Transcriptional repression of Neuropilin-2 expression by DLX1 or DLX2 in vitro. A, schematic diagram of DLX fusion protein constructs. TheN-terminal domains of wild-type (wt) DLX1 or DLX2, leaving the nuclear localization sequence intact, were replaced with the transactivation domain of herpessimplex virus VP16 (VP16-Dlx construct) or the transcriptional repression domain of D. melanogaster Engrailed (Eng-Dlx construct, (30)). In addition, theglutamine residue at amino acid position 50 of the homeodomain, critical for binding of the homeodomain to DNA (33), was mutated to glutamate (Q50E) inthe full-length wild-type and fusion protein constructs. DB, DNA binding mutant. B, both DLX1 and DLX2 Q50E recombinant proteins (left panel) fail to bindNrp-2ii oligonucleotides (right panel), confirming these proteins as DNA binding mutants. Arrows indicate specific DLX1 or DLX2/Nrp-2ii complexes. C, reportergene assays following transient co-transfections with Nrp-2ii DNA sequences fused to a pGL3-Luciferase reporter, in the absence or presence of DLX1 or DLX2expression in human embryonic kidney 293 cells. Q50E mutations of the DLX homeodomain resulted in an almost complete reversal of transcriptionalrepression of the Nrp-2 promoter evident with wild-type Dlx constructs. D, DLX homeodomains fused with N-terminal Engrailed repressor or VP16 activatordomains modify transcriptional activity of Neuropilin-2 promoter. Compared with wild-type DLX proteins, transcriptional activity of Nrp-2ii was furtherrepressed by Eng-DLX fusion proteins but was reversed by using VP16-DLX fusion proteins (more so for DLX1 than DLX2; refer to “Discussion”). For panels C andD, data shown are the mean � S.D. of at least three trials. �-galactosidase activity was used as an internal control. Because each construct has a different basallevel of reporter gene expression, luciferase activities were normalized. * denotes a p value � 0.001.

FIGURE 4. Patterns of DLX1 or DLX2 and Neuropilin-2 expression in the basal telencephalon. Cryosections of E13.5 forebrain were labeled with specificantibodies against DLX1 (a), DLX2 (d), and NRP-2 (b, e). The left panels (a, d) show DLX1- or DLX2-positive cells in lateral ganglionic eminence (LGE), medialganglionic eminence (MGE), and the anterior preoptic area (POa). The center panels (b, e) show NRP-2-positive cells within the mantle zone of the pallidalanlagen. In the right panels, image overlays demonstrate minimal overlap of DLX1 or DLX2 with NRP-2 expression. However, the majority of DLX1/DLX2-positive cells are not expressed with NRP-2-positive cells. Scale bar, 200 �m. H, hippocampus; NCx, neocortex; PCx, paleocortex; Str, striatum.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

hence, this function is not completely alleviated by replacementby VP16 at its N terminus. Certainly, the VP16-DLX2 mutantrepresses less than the wild-type protein. Perhaps the fusionprotein did not sufficiently eliminate all of the repressordomains ofDLX2. For example, theremay be another repressordomain at the C terminus of DLX2, given that the chimericVP16-DLX2 construct was unable to transactivate reportergene expression to levels seen with the VP16-DLX1 construct.Subsequently, fusion of the DLX2 DNA binding domain alonewith VP16may have been able to activate better than the VP16-DLX2mutant used in this study. BothN andC termini of DLX3are required for mediating transcriptional activation in vivousing Xenopus embryo expression assays (43). Further analysiswill better delineate functional domains of DLX proteins otherthan the homeodomain that are important for the modulationof transcription.DLX2 is more robust than DLX1 as a transcriptional activa-

tor (17) or transcriptional repressor (Fig. 3, C and D). Further-more, co-transfection of Dlx1 and Dlx2 wild-type or chimericconstructs is neither additive nor synergistic, yielding resultsthat are similar to that of transfection of Dlx2 constructs alone(17); data not shown). This lack of potentiation suggests thattheremay be greater affinity of DLX2 than DLX1 for binding totheir specified homeodomain DNA binding sites and/or slowerrates of dissociation. In the embryonic telencephalon, it is alsopossible that DLX2 interacts with one ormore co-repressors orco-activators with which DLX1 does not interact. The Evf-2noncoding RNA is a recently identified DLX2 transcriptionalcoactivator transcribed from the Dlx5/Dlx6 intergenic region(46). Although DLX2 and DLX5 form homodimers and alsoheterodimerize withMsx repressor proteins in vitro and in vivo(38), heterodimerization of Dlx family members has not beenestablished.The DNA binding specificity of MSX1 is determined by its

association with cofactors, such as PIAS1, that direct thishomeoprotein to subnuclear compartments where its targetgenes are located (47). It is likely that the specificity of tran-scriptional regulation byDlx genesmay involve other factors inunique protein-protein complexes with different transcrip-tional activities. Whether DLX proteins function as activatorsor repressors of target gene expression may depend on cooper-ationwith other transcription factors as demonstrated for otherhomeoboxproteinswith paired-type homeoproteins (48) or theTATA-binding protein (49). DLX transcriptional activity mayalso depend on post-translational modifications such as phos-phorylation as shown for DLX3 (32) or interaction with pro-teins such as the PDZ protein GRIP1 (40) and the MAGE pro-tein Dlxin (4, 50). It has been shown that DLX1 interactsthrough its homeodomain with the co-Smad Smad4 duringhematopoietic differentiation (51). It will also be interesting todeterminewhetherDLX1 andDLX2have non-overlapping setsof downstream gene targets at various developmental timepoints or in specified tissues where Dlx genes are expressed.

FIGURE 5. Neuropilin-2-expressing interneurons accumulate in the stria-tal anlagen in the Dlx1/Dlx2 double mutant. A, at E13.5, NRP-2 expressionremains unchanged in the mantle zone of the embryonic striatum when com-paring wild-type and Dlx1/Dlx2 double mutant littermates. B and C, at E16.5(B) and P0 (C) in the absence of Dlx1 and Dlx2 gene function, there is increasedNRP-2 expression with extension in the mutant ganglionic eminences andaccumulation within the striatal anlagen. Coronal sections, scale bars, 200

�m. H, hippocampus; LGE, lateral ganglionic eminence; MGE, medial gangli-onic eminence; NCx, neocortex; PCx, paleocortex; POa, anterior preoptic area;Str, striatum.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Although a consensus DNA binding sequence has beenestablished for paired-type homeoproteins such as PAX6,5�-TAATN3ATTA-3� (48), and HOX proteins, 5�-(C/G)TAATTG-3� (52), one specific forDlx familymembers remainsto be established (nucleotides conserved among confirmedDLX targets are underlined). Feledy et al. identified an 8-baseconsensus 5�-(A/C/G)TAATT(G/A)(C/G)-3� for DLX3 DNAbinding sites (43). This sequence was conserved at specific sitesof theDlx5/Dlx6 intergenic enhancer bound by DLX1 or DLX2in vitro or in situ (17, 39), although there was discordance at themost 3�-nucleotide. As well, only seven nucleotides match forthe region of the Neuropilin-2 promoter, ATAATTAT, boundby DLX1 or DLX2 in vitro (Fig. 1A). These results suggest thatalthough there are sequence similarities between DLX1, DLX2,andDLX3 binding sites, there are currently insufficient verifiedDLX targets to establish a consensus DNA binding site for theDlx gene family.In the developing forebrain, although cell migration along

radial glia is predominant, tangential migration is also animportant means for other sets of differentiating cells to reachtheir destinations. For instance, immature GABAergic inter-neurons produced in the ganglionic eminences (subpallium)migrate tangentially to the neocortex and hippocampus (pal-

lium) (5, 14, 15, 53–55). Interneu-rons migrating to the cortex ariseprimarily from the MGE and theentopeduncular area and follow twomajor pathways as they traverse theLGE/striatum: a superficial subpialroute and a deep route adjacentto the SVZ (25, 34, 37).The molecular mechanisms thatguide and sort these migrating cellsare becoming elucidated (25).Recently, ErbB/neuregulin signal-ing has been implicated in support-ing migration through the LGEtoward the cerebral cortex (22, 23,56). Glial cell line-derived neurotro-phic factor signaling via GFR�1 hasa role in the differentiation andmigration of cortical GABAergiccells from the MGE (57). Likewise,chemorepellant tissues and factorshave been identified that participatein directing these migrations (24,26). There is evidence that striatalexpression of semaphorin 3A and3F repels tangentially migratinginterneurons (15). Semaphorin 3Acan also act as a chemoattractant invitro (58, 59), whereas anothersecreted class III semaphorin, sema-phorin 3B, mediates both attractionand repulsion in vivo (60). Sema-phorins interact with receptor com-plexes consisting of neuropilins(ligand binding subunits) and class

A plexins (signal transduction subunits) (61–63). In Neuropi-lin-2 null mice increased numbers of tangentially migratingcells enter the striatum. Similarly, interneurons that express adominant negative form of Neuropilin-1 also aberrantly enterthe striatum (15).Previous studies demonstrated that Dlx1 and Dlx2 function

is necessary for migration of more than 75% of tangentiallymigrating interneurons to the murine cortex (5), olfactory bulb(13), and hippocampus (16). Upon loss of Dlx1 and Dlx2 func-tion, there is ectopic accumulation of cells with molecularproperties of cortical interneurons within the SVZ of the gan-glionic eminences. These cells, which express high levels ofNrp-2 (15) (Figs. 5, B and C, and 6) are presumed to be collec-tions of interneurons that have failed to migrate to the cortex.Normally, Neuropilin-2 expression patterns minimally overlapwith Dlx1 and Dlx2 expression (Fig. 4) and with the pathwaysfollowed by tangentially migrating interneurons (64). Thesedata support our hypothesis in which DLX1- and/or DLX2-expressing cells could down-regulate Nrp-2 expression afterE13.5, enabling later born interneurons, most derived from theMGE and the anterior entopeduncular area, to take the deeproute to the striatum toward the neocortex. Repression ofNRP-2 expression may therefore allow these interneurons to

FIGURE 6. Interneurons in the Dlx1/Dlx2 double mutant express ectopic Neuropilin-2 in the SVZ of thebasal telencephalon. Digoxigenin in situ RNA hybridization confirms ectopic expression of Nrp-2 in the Dlx1/Dlx2 null ganglionic eminences at E16.5 (b) and E18.5 (d) compared with matched wild-type tissue sections(a, c), respectively. The asterisk in panel b denotes the anterior entopeduncular area located in this more caudalcryosection. Arrows demarcate small “collections” of interneurons expressing ectopic Nrp-2 that resembleperiventricular heterotopias or ectopias most evident at E18.5 (b, d). Scale bars in panels a and b, 400 �m and inpanels c and d, 500 �m. H, hippocampus; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence;NCx, neocortex; PCx, paleocortex; POa, anterior preoptic area; Str, striatum.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

migrate through semaphorin-expressing cells in the striatumand at the pallial-subpallial interface (15, 25, 65). Late borninterneurons that aberrantly express NRP-2 in the absence ofDlx1 and Dlx2 function could fail to migrate to neocortex dueto the repulsive guidance cues from semaphorin-3F-expressingcells mediated via NRP-2 and accumulate as subventricularectopias (15, 25, 65). In theDlx1/Dlx2mutant, loss of DLX-de-pendent repression ofNrp-2 transcriptionmay explain, in part,the ectopic accumulation of GABAergic interneurons in theganglionic eminences. These studies do not exclude the likelypossibility that other molecules, some directly regulated byDlxgenes and others independent ofDlx function, contribute to thetangential migrations of interneurons especially after they havepassed the pallial/subpallial junction (15). Other transcrip-tional factors, in addition to Dlx-dependent repression, maycontribute to repress Nrp-2 expression in interneurons enter-ing the striatum, as evidenced by the presence of semaphorin-expressing domains and the lack of Neuropilin-2 expression insorted striatal interneurons (15). However, our results suggestthat Dlx1 and Dlx2 repression of Nrp-2 may contribute to thetangential migration of late born inhibitory interneurons fromthe subcortical telencephalon.

Acknowledgments—We thank Drs. D. Ginty, D. Kessler, A. Kolodkin,J. Rubenstein, and M. Tessier-Lavigne for antibodies and probes, J.Rubenstein for the Dlx1/Dlx2 heterozygous mice, D. Chateau for sta-tistical advice, and S. Anderson, R. Bremner, J. deMelo, and J. Ruben-stein for helpful discussions and review of our manuscript.

REFERENCES1. Eisenstat, D. D., Liu, J. K., Mione, M., Zhong, W., Yu, G., Anderson, S. A.,

Ghattas, I., Puelles, L., and Rubenstein, J. L. (1999) J. Comp. Neurol. 414,217–237

2. Liu, J. K., Ghattas, I., Liu, S., Chen, S., andRubenstein, J. L. (1997)Dev. Dyn.210, 498–512

3. de Melo, J., Qiu, X., Du, G., Cristante, L., and Eisenstat, D. D. (2003)J. Comp. Neurol. 461, 187–204

4. Panganiban,G., andRubenstein, J. L. (2002)Development129, 4371–43865. Anderson, S. A., Eisenstat, D. D., Shi, L., and Rubenstein, J. L. (1997)

Science 278, 474–4766. Anderson, S. A., Qiu, M., Bulfone, A., Eisenstat, D. D., Meneses, J., Peder-

sen, R., and Rubenstein, J. L. (1997) Neuron 19, 27–377. Qiu, M., Bulfone, A., Ghattas, I., Meneses, J. J., Christensen, L., Sharpe,

P. T., Presley, R., Pedersen, R. A., and Rubenstein, J. L. (1997) Dev. Biol.185, 165–184

8. de Melo, J., Du, G., Fonseca, M., Gillespie, L. A., Turk, W. J., Rubenstein,J. L., and Eisenstat, D. D. (2005) Development 132, 311–322

9. Long, J. E., Garel, S., Depew, M. J., Tobet, S., and Rubenstein, J. L. (2003)J. Neurosci. 23, 568–578

10. Robledo, R. F., Rajan, L., Li, X., and Lufkin, T. (2002) Genes Dev. 16,1089–1101

11. Cobos, I., Calcagnotto,M. E., Vilaythong, A. J., Thwin,M.T., Noebels, J. L.,Baraban, S. C., and Rubenstein, J. L. (2005) Nat. Neurosci. 8, 1059–1068

12. Wonders, C., and Anderson, S. (2005) Nat. Neurosci. 8, 979–98113. Bulfone, A., Wang, F., Hevner, R., Anderson, S., Cutforth, T., Chen, S.,

Meneses, J., Pedersen, R., Axel, R., and Rubenstein, J. L. (1998)Neuron 21,1273–1282

14. Letinic, K., Zoncu, R., and Rakic, P. (2002) Nature 417, 645–64915. Marin, O., Yaron, A., Bagri, A., Tessier-Lavigne, M., and Rubenstein, J. L.

(2001) Science 293, 872–87516. Pleasure, S. J., Anderson, S., Hevner, R., Bagri, A., Marin, O., Lowenstein,

D. H., and Rubenstein, J. L. (2000) Neuron 28, 727–740

17. Zhou, Q. P., Le, T. N., Qiu, X., Spencer, V., de Melo, J., Du, G., Plews, M.,Fonseca, M., Sun, J. M., Davie, J. R., and Eisenstat, D. D. (2004) NucleicAcids Res. 32, 884–892

18. Stoykova, A., Treichel, D., Hallonet, M., and Gruss, P. (2000) J. Neurosci.20, 8042–8050

19. Yun, K., Potter, S., and Rubenstein, J. L. (2001)Development 128, 193–20520. Shinozaki, K., Miyagi, T., Yoshida, M., Miyata, T., Ogawa, M., Aizawa, S.,

and Suda, Y. (2002) Development 129, 3479–349221. Stenman, J., Yu, R. T., Evans, R. M., and Campbell, K. (2003)Development

130, 1113–112222. Flames, N., Long, J. E., Garratt, A. N., Fischer, T. M., Gassmann, M.,

Birchmeier, C., Lai, C., Rubenstein, J. L., andMarin, O. (2004)Neuron 44,251–261

23. Lopez-Bendito, G., Cautinat, A., Sanchez, J. A., Bielle, F., Flames, N., Gar-ratt, A. N., Talmage, D. A., Role, L. W., Charnay, P., Marin, O., and Garel,S. (2006) Cell 125, 127–142

24. Marin, O., Plump, A. S., Flames, N., Sanchez-Camacho, C., Tessier-Lav-igne, M., and Rubenstein, J. L. (2003) Development 130, 1889–1901

25. Marin, O., and Rubenstein, J. L. (2003) Annu. Rev. Neurosci. 26,441–483

26. Wichterle, H., Alvarez-Dolado, M., Erskine, L., and Alvarez-Buylla, A.(2003) Proc. Natl. Acad. Sci. U. S. A. 100, 727–732

27. Chen, H., Bagri, A., Zupicich, J. A., Zou, Y., Stoeckli, E., Pleasure, S. J.,Lowenstein, D. H., Skarnes, W. C., Chedotal, A., and Tessier-Lavigne, M.(2000) Neuron 25, 43–56

28. Cloutier, J. F., Giger, R. J., Koentges, G., Dulac, C., Kolodkin, A. L., andGinty, D. D. (2002) Neuron 33, 877–892

29. Giger, R. J., Cloutier, J. F., Sahay, A., Prinjha, R. K., Levengood, D. V.,Moore, S. E., Pickering, S., Simmons, D., Rastan, S.,Walsh, F. S., Kolodkin,A. L., Ginty, D. D., and Geppert, M. (2000) Neuron 25, 29–41

30. Kessler, D. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13017–1302231. Porteus,M. H., Bulfone, A., Liu, J. K., Puelles, L., Lo, L. C., and Rubenstein,

J. L. (1994) J. Neurosci. 14, Pt. 1, 6370–638332. Park, G. T., Denning, M. F., and Morasso, M. I. (2001) FEBS Lett. 496,

60–6533. Gehring, W. J., Qian, Y. Q., Billeter, M., Furukubo-Tokunaga, K., Schier,

A. F., Resendez-Perez, D., Affolter,M.,Otting,G., andWuthrich, K. (1994)Cell 78, 211–223

34. Anderson, S. A., Kaznowski, C. E., Horn, C., Rubenstein, J. L., and McCo-nnell, S. K. (2002) Cereb. Cortex 12, 702–709

35. Marin, O., Anderson, S. A., and Rubenstein, J. L. (2000) J. Neurosci. 20,6063–6076

36. Anderson, S., Mione, M., Yun, K., and Rubenstein, J. L. (1999) Cereb.Cortex 9, 646–654

37. Anderson, S. A., Marin, O., Horn, C., Jennings, K., and Rubenstein, J. L.(2001) Development 128, 353–363

38. Zhang, H., Hu, G., Wang, H., Sciavolino, P., Iler, N., Shen, M. M., andAbate-Shen, C. (1997)Mol. Cell. Biol. 17, 2920–2932

39. Zerucha, T., Stuhmer, T., Hatch, G., Park, B. K., Long, Q., Yu, G., Gam-barotta, A., Schultz, J. R., Rubenstein, J. L., and Ekker, M. (2000) J. Neuro-sci. 20, 709–721

40. Yu, G., Zerucha, T., Ekker, M., and Rubenstein, J. L. (2001) Brain Res. Dev.Brain Res. 130, 217–230

41. Chase, M. B., Fu, S., Haga, S. B., Davenport, G., Stevenson, H., Do, K.,Morgan, D., Mah, A. L., and Berg, P. E. (2002) Mol. Cell. Biol. 22,2505–2514

42. Givens, M. L., Rave-Harel, N., Goonewardena, V. D., Kurotani, R., Berdy,S. E., Swan, C. H., Rubenstein, J. L. R., Robert, B., and Mellon, P. L. (2005)J. Biol. Chem. 280, 19156–19165

43. Feledy, J. A., Morasso, M. I., Jang, S. I., and Sargent, T. D. (1999) NucleicAcids Res. 27, 764–770

44. Hassan, M. Q., Javed, A., Morasso, M. I., Karlin, J., Montecino, M., vanWijnen, A. J., Stein, G. S., Stein, J. L., and Lian, J. B. (2004)Mol. Cell. Biol.24, 9248–9261

45. Iler, N., Rowitch, D. H., Echelard, Y., McMahon, A. P., and Abate-Shen, C.(1995)Mech. Dev. 53, 87–96

46. Feng, J., Bi, C., Clark, B. S.,Mady, R., Shah, P., andKohtz, J. D. (2006)GenesDev. 20, 1470–1484




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

47. Lee, H., Quinn, J. C., Prasanth, K. V., Swiss, V. A., Economides, K. D.,Camacho,M.M., Spector, D. L., andAbate-Shen, C. (2006)Genes Dev. 20,784–794

48. Mikkola, I., Bruun, J. A., Holm, T., and Johansen, T. (2001) J. Biol. Chem.276, 4109–4118

49. Zhang, H., Catron, K. M., and Abate-Shen, C. (1996) Proc. Natl. Acad. Sci.U. S. A. 93, 1764–1769

50. Masuda, Y., Sasaki, A., Shibuya, H., Ueno, N., Ikeda, K., andWatanabe, K.(2001) J. Biol. Chem. 276, 5331–5338

51. Chiba, S., Takeshita, K., Imai, Y., Kumano, K., Kurokawa, M., Masuda, S.,Shimizu, K., Nakamura, S., Ruddle, F. H., and Hirai, H. (2003) Proc. Natl.Acad. Sci. U. S. A. 100, 15577–15582

52. Pellerin, I., Schnabel, C., Catron, K. M., and Abate, C. (1994) Mol. Cell.Biol. 14, 4532–4545

53. Stuhmer, T., Puelles, L., Ekker, M., and Rubenstein, J. L. (2002) Cereb.Cortex 12, 75–85

54. Wichterle, H., Garcia-Verdugo, J. M., Herrera, D. G., and Alvarez-Buylla,

A. (1999) Nat. Neurosci. 2, 461–46655. Wichterle, H., Turnbull, D. H., Nery, S., Fishell, G., and Alvarez-Buylla, A.

(2001) Development 128, 3759–377156. Hanashima, C., Molnar, Z., and Fishell, G. (2006) Cell 125, 24–2757. Pozas, E., and Ibanez, C. F. (2005) Neuron 45, 701–71358. Carmeliet, P., and Tessier-Lavigne, M. (2005) Nature 436, 193–20059. Song, H., and Poo, M. (2001) Nat. Cell Biol. 3, E81–E8860. Julien, F., Bechara, A., Fiore, R., Nawabi, H., Zhou, H., Hoyo-Becerra, C.,

Bozon, M., Rougon, G., Grumet, M., Puschel, A. W., Sanes, J. R., andCastellani, V. (2005) Neuron 48, 63–75

61. Guan, K. L., and Rao, Y. (2003) Nat. Rev. Neurosci. 4, 941–95662. Pasterkamp, R. J., and Kolodkin, A. L. (2003) Curr. Opin. Neurobiol. 13,

79–8963. Garrity, P. A. (2005) Nat. Neurosci. 8, 1635–163664. Tamamaki, N., Fujimori, K., Nojyo, Y., Kaneko, T., and Takauji, R. (2003)

J. Comp. Neurol. 455, 238–24865. Marin, O., and Rubenstein, J. L. (2001) Nat. Rev. Neurosci. 2, 780–790




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

D. EisenstatTrung N. Le, Guoyan Du, Mario Fonseca, Qing-Ping Zhou, Jeffrey T. Wigle and David

Forebrain by Direct Repression of the Semaphorin Receptor Neuropilin-2 Homeobox Genes Promote Cortical Interneuron Migration from the BasalDlx

doi: 10.1074/jbc.M607486200 originally published online January 26, 20072007, 282:19071-19081.J. Biol. Chem.

10.1074/jbc.M607486200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

http://www.jbc.org/content/suppl/2007/01/30/M607486200.DC1

http://www.jbc.org/content/282/26/19071.full.html#ref-list-1

This article cites 65 references, 28 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M607486200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;282/26/19071&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/282/26/19071

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=282/26/19071&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/282/26/19071

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/suppl/2007/01/30/M607486200.DC1

http://www.jbc.org/content/282/26/19071.full.html#ref-list-1

http://www.jbc.org/

dlx homeobox genes promote cortical interneuron migration from

Documents