Subpallial Boundary Patterning by Pax6 and Gsh2

15 Pages·2009·3.15 MB·English

CerebralCortexApril2009;19:745--759
doi:10.1093/cercor/bhn123
AdvanceAccesspublicationAugust12,2008
Differential Regulation of Telencephalic Rosalind S.E. Carney1,Laura A.Cocas1,2, Tsutomu Hirata1,
Kevin Mansfield1and Joshua G. Corbin1
Pallial--Subpallial Boundary Patterning by
Pax6 and Gsh2 1Center for Neuroscience Research, Suite5340, Children’s
Research Institute, Children’s NationalMedical Center,
Washington, DC 20010, USAand 2Interdisciplinary Program in
Neuroscience, Georgetown University MedicalCenter, 3970
Reservoir Rd.,NW, Washington, DC 20057, USA
In the embryonic telencephalon, the pallial--subpallial boundary regionally expressed transcription factors endow telence-
D
(PSB)separatesthedorsalPax61palliumfromtheventralGsh21 phalic progenitor populations with the intrinsic potential to o
w
subpallium. Previous studies have revealed that this region is generate distinct neuronal cell types (reviewed in Wonders nlo
asourceofcellsthatwillpopulateboththeolfactorybulbandbasal and Anderson 2006; Molyneaux et al. 2007). ad
e
telencephalic limbic system. However, the level of progenitor cell Fromearlystagesoftelencephalicdevelopment(byapprox- d
heterogeneity and developmental genetic regulation of this pro- imately embryonic day 10.0 [E10.0]), the pallium and sub- fro
m
genitor region remains to be fully elucidated. In this study pallium are identified by the expression of the homeodomain h
we carried out a comprehensive analysis of gene expression containing genes Pax6 and Gsh2, respectively. The pallial-- ttp
s
patterns at the PSB, in addition to an examination of the subpallial boundary (PSB, also known as the cortico-striatal ://a
combinatorial function of Pax6 and Gsh2 in the specification of border)istheregionwherehighPax6expression(Pax6isalso ca
d
the PSB. First, we reveal that the PSB is comprised of a complex expressedatalowlevelinthedorsal-mostaspectofthelateral e
m
mixofmolecularlydistinctprogenitorpools.Inaddition,byanalysis ganglion eminence (LGE) [dLGE] ventricular zone [VZ]) abuts ic
of single Sey, Gsh2, and Sey/Gsh2 double mutant mice, we Gsh2 expression near the cortico-striatal angle. The PSB is .ou
p
demonstrate that both Pax6 and Gsh2 are directly required for comprised of neural progenitor cells from both the ventral- .c
o
major aspects of PSB progenitor specification. Our analysis also mostaspectofthepallium(VP)anddLGE(Puellesetal.1999; m
reveals that the establishment of the epidermal growth factor Puelles et al. 2000; Yun et al. 2001; Stenman, Toresson, et al. /ce
receptor positive lateral cortical stream migratory route to the 2003). In addition, previous studies have also revealed that rco
bwaeslal-lcthealreanccteeprihzaeldoncirsosPsa-xre6pdreespseinvdeenrot.leTshuins,dinorasdadl/ivtieonntrtaoltphaetir- pDrboxg1en(iVtoPr)ancedllSsp8o,fmTthshe1(PdSLBGEt)ratnhsaitencotllylecetixvperlyesmsarSkfrtph2is, r/article
terningouranalysesrevealimportantnovelfunctionsofGsh2and region as molecularly distinct from the rest of the telenceph- -a
b
Pax6 in the regulation of PSB progenitor pool specification and s
patterning. aWloancla(wPueelltesale.t2a0l.0260)0.0E;mKeimrgientgale.v2id0e0n1c;eCafurboimt etouarl. 2w0o0r5k; tract/1
(Carney et al. 2006) and that of others (Fernandez et al. 9
Keywords:cortico-striatal border, development, gene expression, /4
lateralcorticalstream, mutant,small eye(Sey) 1998;Puellesetal.1999;Puellesetal.2000;Hirataetal.2002; /74
Stenman, Yu, et al. 2003; Bai et al. 2008) has revealed that 5
/2
a major target of lateral cortical stream (LCS) migrating PSB 7
9
neurons (VP and dLGE) is the basal telencephalic limbic 52
2
Introduction system, most prominently the amygdala and piriform cortex. b
y
Theembryonictelencephaloncanbeparcellatedinto2broad Moreover,progenitorsfromthePSBgenerateoneoftwomain g
u
subgroupsofCajal--Retziusneuronsinthepallium(Bielleetal. e
progenitor zones, the pallium and subpallium, which are s
responsible for generating, in a highly precise and complex 2005), as well as OB interneurons derived from the rostral t on
manner, telencephalic excitatory and inhibitory neurons, migratory stream (RMS) (Waclaw et al. 2006; Kohwi et al. 1
1
respectively (reviewed in Corbin et al. 2001; Marı´n and 2007).Thus,thisregionisanimportantsourceoftelencephalic A
p
Renutbiaelnastnedinte2m0p0o1;raWllyonredgeurslataenddeAxnpdreersssoionn2p0a0t6te)r.nTshoefdpiaflfleiar-l celPlrpeovpiouulastisotnusd.ies have revealed that Pax6 and Gsh2 are ril 20
1
(e.g., Pax6, Emx1, Ngn2) and subpallial (e.g., Gsh2, Mash1, required for proper positioning of the PSB by functioning to 9
Dlx family members) genes define discrete progenitor geneticallycrossrepresseachother(Toressonetal.2000;Yun
domains that give rise to neuronal diversity in several etal.2001;Corbinetal.2003;Stenman,Yu,etal.2003;Yunetal.
telencephalic regions such as the cerebral cortex (Nery 2003). As such, the lack of Pax6 in Sey (Small eye; Hill et al.
etal.2002;Lo´pez-Benditoetal.2004;Xuetal.2004;Buttetal. 1991)mutantmiceresultsinanexpansionofsubpallialmarkers
2005; Flames et al. 2007; Fogarty et al. 2007; Miyoshi et al. such as Dlx1, Dlx2, Gsh2, Mash1, Vax1 into the pallium
2007), olfactory bulb (OB) (Wichterle et al. 1999; Stenman, (Stoykova et al. 1996; Corbin et al. 2000; Stoykova et al. 2000;
Toresson, et al. 2003; Kohwi et al. 2005; Vergan˜o-Vera et al. Toressonetal.2000;Yunetal.2001;KrollandO’Leary2005).
2006; Waclaw et al. 2006; Kohwi et al. 2007), amygdala Conversely,inGsh2mutantmice (Szucsik et al. 1997) pallial
(Wichterle et al. 2001; Gorski et al. 2002; Nery et al. 2002; markers such as Pax6, Ngn2, Math2, Tbr1 extend ventrally
Remedios et al. 2007), and adult subventricular zone (SVZ) into the LGE (Corbin et al. 2000; Toresson et al. 2000;
(DeMarchisetal.2007;Kohwietal.2007;Youngetal.2007). Toresson and Campbell 2001; Yun et al. 2001). Thus, in Sey
Emerging evidence also interestingly indicates that in mutant mice VP and lateral pallial (LP) progenitors acquire
a manner similar to the spinal cord, combinatorial codes of subpallial properties, whereas in Gsh2 mutant mice the dLGE
(cid:1)TheAuthor2008.PublishedbyOxfordUniversityPress.Allrightsreserved.
Forpermissions,pleasee-mail:[email protected]
is respecified to a pallial identity. Loss of VP markers, Sfrp2, Genotyping
Dbx1, and Tgfa in Sey mutant mice has suggested a direct GenomicDNAwasisolatedbyphenol:chloroformextraction.Genotyp-
requirement of Pax6 for their expression (Kim et al. 2001; ing of Gsh2 adult mice and embryos was carried out by PCR using
previously described methods (Stenman,Wang,et al.2003). PCR was
Yun et al. 2001; Assimacopoulos et al. 2003; Stenman, Yu,
performed using 2 primers (for adult genotyping) or 3 primers (for
et al. 2003). Similarly, dLGE development and gene expres-
embryogenotyping)usingaGCrichkit(Roche,Indianapolis,IN).Sey
sion is severely affected in Gsh2 mutants (Corbin et al. 2001; mutantembryoswereidentifiedbylackofeyesandabnormalcranio-
Yun et al. 2001; Waclaw et al. 2006). However, the facial features(Hillet al. 1991). Additional confirmationof genotypes
interpretation of the genetic dependence of Pax6 for PSB was carried out on sections by analysis of known, for example, Dlx2
gene expression in Sey mutants in confounded by ectopic (RNA or X-gal stainingof Dlx2+/tauLacZ sections) (Corbin et al. 2000)
expressionchangesinGsh2andSeymutantembryos.Sey/Gsh2double
Gsh2 expression in the VP and LP. Conversely, in Gsh2
mutantsectionswereconfirmedbythelackofPax6andGsh2protein
mutants, a direct function of Gsh2 cannot be dissociated
inadditiontothelackofeyesandPCRforGsh2mutantandwild-type
from ectopic Pax6 expression in the dLGE. alleles.MaleheterozygousDlx5/6–cre-iresEGFP(Stenman,Toresson,etal.
The purpose of this study was 2-fold: First, we sought to 2003,hereinreferredtoasDlx5/6-GFP)micewerecrossedwithSwiss
D
systematicallyanalyzetheexpressionpatternsofPSB-specific Webster females to generate Dlx5/6-GFP+ embryos. The adults were o
w
genesduringkeydevelopmentaltimepointsofneurogenesis. genotypedby PCRforCre (CRE-F:GCGGTCTGGCAGTAAAAACTATC; n
Second,wewantedtocomprehensivelydissecttheindividual CRE-R: GTGAAACAGCATTGCTGTCACTT) and the embryos were loa
and combinatorial roles of Pax6 and Gsh2 in PSB patterning, identifiedbyGFPfluorescenceusingadissectingmicroscope. ded
thereby providing an in-depth understanding of the genetic fro
requirement for the establishment of neural diversity in PSB- NonradioactiveDioxygenin-LabeledRNAInSituHybridization m
derivedstructures.OurexpressionstudiesrevealthatthePSB Wholeheads(E12.5,E13.5)orisolatedbrains(E15.5)werefixedat4(cid:2)C http
in 4% paraformaldehyde (PFA) (in 0.1 M phosphate buffer, pH 7.4) s
is a highly complex and dynamic telencephalic progenitor (4% PFA) overnight then cryoprotected in graded sucrose concen- ://a
zone, comprised of multiple molecularly distinct progenitor trations (10%, 20% then 30% overnight) before embedding in Tissue- ca
pools.Furthermore,geneexpressionanalysisinsingleSeyand TekOCTcompound(SakuraFinetekUSA,Inc.,Torrance,CA).Coronal de
m
Gsh2mutantmiceandSey/Gsh2doublemutantmice,reveals sections at a thickness of 20 (E12.5, E13.5) or 30 lm (E15.5) were ic
that in the absence of both Pax6 and Gsh2 all examined preparedusingacryostat(MicromHM505E,GMI,Inc.,Ramsey,MN). .o
u
ainsdpieccattsesotfhaptatttheersneinggenoefs,tihnecoPmSBbinreatmioanin, aarebnroerqmuairl.edThfoisr moSdeicfitcioantionRsNAof ipnrevsiiotuuslhyybdreisdcirziabteiodnprwoatsococlasrr(ieSdchaoeurtenb-Wyiesmligehrst p.com
correct positioning and expression of PSB-specific genes. andGerfin-Moser1993;WilkinsonandNieto1993).RNAprobeswere /c
preparedusingdioxygenin(DIG)RNAlabelingkits(Roche).Air-dried e
Moreover, analysis of markers that individually label the VP cryostatsectionswerepostfixedin4%PFAfor10minfollowedby235 rco
and dLGE further reveals a complex and novel differential min rinses in phosphate-buffered saline (PBS). Proteinase K (Roche) r/a
regulation of PSB progenitor pool specification by Gsh2 and digestion(20lg/mLinPBS)wascarriedoutfor6minfollowedby135 rtic
Pax6,whichchangesovertimeduringdevelopment.Further- minrinseinPBS,refixingfor5minin4%PFAandanotherPBSrinse. le-a
more, we show that epidermal growth factor receptor The sections were acetylated for 10 min (2.2 g triethanolamine bs
positive (EGFR+) cells, that mark the LCS migratory popula- hydrochloride[AcrosOrganics,Geel,Belgium],540lLof10NNaOH tra
[FisherScientific,Pittsburgh,PA],300lLofaceticanhydride[Sigma,St c
tionfromthePSB,arealsodifferentiallyregulatedbyPax6and Louis,MO]in60mLofmoleculargradewater[Cellgro,Herndon,VA], t/1
9
Gsh2. Thus, our study provides important insights into the priorto335minrinsesinPBS).RNAprobes,preparedatadilutionof2 /4
genetic requirement for the generation of the PSB, a major lL/mLofhybridizationsolution(50%formamide[Invitrogen,Carlsbad, /74
progenitorregionforthegenerationofOBandlimbicsystem CA], 10%dextransulfate,1%1003 Denhart’s,250lg/mLyeasttRNA, 5/2
cell diversity. 0.3MNaCl,20mMTris--HCl,pH8,5mMethylenediaminetetraacetic 79
acid[EDTA],10mMNaPO4,1%sarcosyl[allfromSigmaexceptwhere 52
indicated] in diethylpyrocarbonate-treated H2O [Invitrogen]), were 2 b
incubatedat80(cid:2)Cfor2min.Thereafter,250lLoftheprobemixwas y
Materials and Methods appliedtoeachslide,coverslippedwithHybri-slips(Sigma)andplaced gu
e
inasealedboxhumidifiedwith50%formamideandH Oandincubated s
AnimalUse at 55 (cid:2)C overnight. The next day, the Hybri-slips we2re floated off by t o
n
Allanimalsusedinthestudyweremaintainedaccordingtoprotocols placingtheslidesin53saline-sodiumcitratebuffer(Cellgro),priorto 1
1
approved by the Animal Welfare and use committee at Children’s a30-minhighstringencywashinprewarmed50%formamide,23SSCat A
NwaetlifoarneallaMwesd.iFcoarlsCteagnitnegr,oWftahsheienmgtborny,oDs,Cm,aidnddaaydohfetrhinegdtaoyaolflvaangiimnaall 6b5uff(cid:2)eCr.(N0.e5xMt, tNhaeCsle,c1t0iomnMs wTerrise--HriCnsl,epdHin7.35,35m10MmEiDnTrAin)s,efosllionwReNdabsey pril 2
0
plug detection was considered as E0.5. Previously published Gsh2 RNaseA(Roche)treatment(20lg/mLinRNasebuffer)for30minand 1
9
(Szucsiketal.1997)andPax6(Sey)mutants(Hilletal.1991)were one 15 min rinse in RNase buffer, all at 37 (cid:2)C. The high stringency
used in this study. Adult Gsh2+/– and Sey/+ heterozygotes were washeswererepeatedtwicefor20mineachat65(cid:2)C,followedbya15
maintainedbycrossestoSwissWebstermice(Taconic,Albany,NY). minrinsein23SSC,then0.13SSC,bothat37(cid:2)CandaPBT(PBS+0.1%
In all cases, single Gsh2–/–, Sey/Sey (referred to as Sey mutant), or Tween20;Sigma)rinsefor15minatRT.Thesectionswereblocked
Sey/Sey;Gsh2–/–doublehomozygousmutant(referredtoasSey/Gsh2 with10%goatseruminPBTfor1hatRT,priortoa3-hincubationwith
doublemutant)embryosweregeneratedbyheterozygouscrossesas analkalinephosphatase-coupledanti-DIGantibody(1:5000in1%goat
previously described (Corbin et al. 2000; Toresson et al. 2000; Yun seruminPBT;Roche)inahumidifiedchamberatRT.Then,thesections
et al. 2001; Waclaw et al. 2004, 2006). In some crosses, mice also were rinsed extensively in PBT at RT (4 3 15 min rinses) and then
carried the Dlx2+/tauLacZ allele, and in these cases embryos and underwent2310minrinsesinfreshlypreparedNTMTbuffer(100mM
brainswerestainedforX-galtovisualizeDlx2(Corbinetal.2000). NaCl,100mMTris--HCl,pH9.5,50mMMgCl , 0.1%Tween20). The
2
Fortheinsituhybridizationanalyses,theGsh2–/–,Sey/Sey,andSey/ sections were then placed in a light-protected humidified chamber
Gsh2doublemutantgenotypeswereeachprocessedindependently with approximately 400 lL of BM-purple AP substrate (Roche)
withlittermateheterozygoteandwild-typemiceusedascontrolsto containing ~0.25 mg/mL levamisol (Sigma) until satisfactory staining
ensure correct RNA probe labeling. A minimum of 3 mutants and was achieved, typically overnight. Finally, the sections were rinsed
controls were used for all genotypes, n numbers are given in the twice in PBS, coverslipped using Crystal mount aqueous mounting
figurelegends. media(Sigma)andphotographedimmediately.
746 Pax6andGsh2inPallial--SubpallialBoundaryPatterning d Carneyetal.
Thefollowingprobeswereusedinthisstudy:Dbx1(Luetal.1992; tolow-caudalgradient(WaltherandGruss1991;Stoykovaand
Yunetal.2001),Dlx2(Porteusetal.1991),Dlx5(Eisenstatetal.1999), Gruss 1994), we carried out this analysis at 2 rostro-caudal
Er81(Stenman,Toresson,etal.2003),Gsh1(ToressonandCampbell, levels for eachmarker.
2001), Sfrp2 (Kim et al. 2001), Sp8 (Bell et al. 2003; Waclaw et al.
2006), Tgfa (Vaughan et al. 1992; Assimacopoulos et al. 2003), and
mTsh1(Caubitetal.2005). Pax6andGsh2areRequiredfortheCorrectExpressionof
Er81atthe PSB
Er81isamemberoftheETStranscription factorfamilyandis
Immunohistochemistry
Sectionspreparedasdescribedaboveforinsituhybridizationanalysis highlyexpressedinthedLGESVZ,VPVZ,andotherpallialand
wereair-driedandunderwentmicrowaveheatantigenretrieval(except subpallial regions (Fig. 1A,E,I,M) (Stenman, Wang, et al. 2003;
forDlx5/6-GFP+brainswhichwerefixedfor4handdidnotrequire Flames et al. 2007). In Gsh2 mutants at both E13.5 and E15.5,
antigen retrieval) in 10 mM sodium citrate (Fisher Scientific) pH 6.0 Er81 expression in the dLGE SVZ is missing and there is an
prior to several rinses in PBS. Subsequently, blocking of nonspecific
expansionofthedomainofVZexpressionatE13.5(Fig.1B,F,J,N).
bindingsiteswasachievedusing10%normalserumdilutedinPBSwith
0.3%Triton-X(PBST;Sigma)toaidpermeabilization.Primaryantibodies, InSeymutantsatbothages,Er81expressionisabsentintheVZ Do
goatanti-b-galactosidase(b-gal)(1:200,Biogen,Cambridge,MA),sheep oftheVPthatiscombinedwithanexpandedexpressioninthe w
n
anti-EGFR (1:20; Millipore, Bedford, MA), rabbit anti-Gsh2 (1:2000; SVZinboththemutantdLGEandVPdomains (Fig. 1C,G,K,O). lo
a
Toressonetal.2000;kindgiftofK.Campbell),mouseanti-Pax6(1:2000; In Sey/Gsh2 double mutants, Er81 expression remains d
e
DIoewvaeloCpitmy,enIAta),lrSatbubdiitesanHtiy-Pbarixd6om(1a:1B0a0n0k; C[DovSaHnBc]e, URnesiveearrscihtyPorofdIuocwtas,, athbantorombsaelravnedd,ianlthsionugglehSseliyghmtluytlaenstsssaetvebroet,hclEo1s3e.l5y arensdemE1b5le.5s d from
Berkeley,CA),mouseanti-RC2(1:20;DSHB),rabbitanti-Tbr1(1:1000; (Fig. 1D,H,L,P). Interestingly, Er81 expression reveals nascent h
rHsienersvuenmseriinnetPPaBlBS.TS2,0at0nh1de;iknsicenucdbtigaotinfetsdoowfvRee.rrenHiegivnhncteuarbt)a4twe(cid:2)edCre.wFdoitillhluotwethdineigna31p%3pro1n0porrmimatianel ppaarllaiuvemnt(rKicruollalraencdtoOp’iLaeaarsyp2r0ev0i5o)u,swlyhdicehscarribeeadlsionoSbesyermveudtanint ttps://a
c
secondaryCy3-(1:200)orfluoresceinisothiocyanate-(1:50)conjugated the double mutant. a
d
antibodies (serum and secondary antibodies were from Jackson e
m
Immunoresearch,WestGrove,PA),dilutedasfortheprimaryantibodies, ic
inthedarkfor2hatRT.AfterfurtherrinsesinPBS,nuclearcounterstaining Differential Regulation ofdLGE Sp8andmTsh1 .o
u
was performed by incubation in To-Pro-3 iodide (To-Pro-3; 1:100, Expression byGsh2 and Pax6 p
.c
Invitrogen)for10minatRT.FinalrinsesinPBSwerecarriedoutpriorto Next, we examined 2 zincfinger transcription factors, Sp8 and om
coverslippingusingGelMountaqueousmountingmedia(Sigma). mTsh1,whichareexpressedintheSVZofthedLGEaspectofthe /c
e
PSB. Recent studies have revealed that Sp8 is required for the rc
o
DataAnalysis specification,migrationandsurvivalofOBinterneurons(Waclaw r/a
Analysis of in situ hybridization experiments was performed using etal.2006)andplaysaroleincorticalarealization(Saharaetal. rtic
brightfield microscopy (Olympus BX51, Olympus, Center Valley, PA) 2007;Zembrzyckietal.2007).Themouseteashirtfamilyofgenes le-a
andhigh-resolutiondigitalimageswerecapturedundera34objective (mTsh1-3)encodezincfingerDNAbindingproteinsthatarealso bs
usinganOlympusD570camera.Forfluorescence,digitalphotographs expressedinthedevelopingbrain(Caubitetal.2005). tra
wereobtainedfromepifluorescencemicroscopy(OlympusBX61)and c
selectedforfurther analysis usinga Zeiss (Thornwood, NY) LSM 510 At E13.5, Sp8 is expressed at very high levels in the SVZ of t/1
9
META confocal microscope. For confocal analysis, each fluorophore the dLGE and at lower levels in the progenitor zones of the /4
was scanned sequentially and Z-stacks of the images obtained were dorsolateral and dorsomedial cortex (Fig. 2A,E) (Waclaw et al. /74
collapsed into a single projection image, or presented as individual 2006).IthaspreviouslybeenshownthatinGsh2mutantmice 5/2
opticalsectionsusingtheLSM510software(Zeiss).Toassessimmuno- Sp8 protein is missing at E12.5, with a partial recovery of this 79
colocalization, 3 sections from at least n = 2 brains (values given in expressionbyE16.5(Waclawetal.2006).Consistentwiththis 52
figure legends) were examined. Figures were prepared using Adobe observation, we find a striking reduction in Sp8 expression at 2 b
PhotoshopCSsoftware(AdobeSystems,SanJose,CA).Adjustmentsto y
contrast were applied across each image as a whole and equally to E13.5 in Gsh2 mutants (Fig. 2B,F). In contrast, in Sey mutant gu
e
controlandmutantbrains. mice,thereisanexpansionofSp8mRNAintotheSeymutant s
pallium at E13.5 (Fig. 2C,G). In Sey/Gsh2 double mutants, t on
although there appears to be some improvement of ectopic 11
Results Sp8 expression, in general Sp8 expression remains abnormal A
p
The PSB is defined as the location of the border between (Fig. 2D,H). ril 2
progenitor cells of pallial (Pax6+) and subpallial (Gsh2+) Similar to Sp8, mTsh1 expression marks the dLGE SVZ. 0
1
identity(Yunetal.2001;Corbinetal.2003;Stenman,Yu,etal. However, in contrast to Sp8, mTsh1 expression persists in 9
2003; Yun et al. 2003). To examine the regulation of PSB presumptive migrating cells that emanate from the dLGE
patterning by Gsh2 and Pax6, we used a series of well- and form the LCS migratory route to the basal telencephalon
characterized in situ hybridization probes that mark the VZ (Fig.2I,M).TheexpressionofmTsh1ismaintainedinthedLGE
and/or SVZ progenitor cell compartments of either both the and cells of the presumptive LCS of Gsh2 mutants at E13.5
pallial and subpallial aspects of the PSB (Er81), dLGE (Sp8, (Fig. 2J,N), which is in surprising contrast to the loss of Sp8
mTsh1), or the VP (Sfrp2, Dbx1, Tgfa). These analyses were expression (Fig. 2B,F) thereby indicating a differential regula-
carried out at 2 developmental time points, E13.5 and E15.5, tion of Sp8 and mTsh1 by Gsh2. In the Sey mutant
which represent the peak of severity of patterning defects in telencephalon at E13.5, mTsh1 expression expands into the
Gsh2andSeymutants(Corbinetal.2000;Toressonetal.2000; pallium(Fig.2K,O).Inaddition,therealsoappearstobeectopic
Yun et al. 2001; and data shown here) and partial recovery of expressionalongtheouterventraltelencephalonintheareaof
the striatal phenotype in Gsh2 mutants (Corbin et al. 2000; the presumptive piriform cortex. Similar to Sp8 expression in
ToressonandCampbell2001;Yunetal.2001;Yunetal.2003), Sey/Gsh2 double mutant mice, although partially recovered,
respectively.AstheexpressionofPax6occursinahigh-rostral mTsh1 expressionremains abnormal (Fig. 2L,P).
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Figure1. ExpressionofEtstranscriptionfactorEr81atE13.5andE15.5.Er81isexpressedintheSVZofthedLGEandVZoftheVPasshownatE13.5incontrols(A,E).VZ 5/2
expressionexpandsventrallyinGsh2mutantmice(B,F,arrowheads).InSeymutants,SVZexpressionextendsectopicallyintothecerebralcortex(CTX,alsotermedthepallium) 7
9
(C,G,arrows).InSey/Gsh2doublemutants,ectopicpallialexpressionofEr81resemblestheSeymutantphenotype,butisweaker(D,H,arrows).AtE15.5,thedLGESVZ 5
2
expressionofEr81incontrols(I,M)ismissingintheGsh2mutant(J,N,asterisks).Conversely,inSeysinglemutants(K,O)andSey/Gsh2doublemutants(L,P),Er81is 2
b
ectopicallyexpressedinthepallium(arrows)andformsnascentparaventricularectopia(K,L,O,P,emptyarrowhead).IntheSeymutant,abasalstreamofEr81expressionis y
alsoobserved(K,O,arrowheads),whichisnotpresentinthedoublemutant(L,P,arrowhead).nnumbersareasfollows:controls,n55[E13.5],n55[E15.5];Gsh2(cid:1)/(cid:1),n56 gu
[E13.5],n55[E15.5];Sey/Sey,n55[E13.5],n55[E15.5];Sey/Sey;Gsh2(cid:1)/(cid:1),n55[E13.5],n55[E15.5].Scalebar:200lm. es
t o
n
1
1
At E15.5, Sp8 is still strongly expressed in the dLGE SVZ reduced (Fig. 3J,N), revealing that mTsh1 expression is A
p
(Fig.3A,E),whichshowspartialrecoveryofSp8expressionin temporally regulatedbyGsh2 (Fig.3J,N). ril 2
thedLGEofGsh2mutants(Fig.3B,F)ascomparedwithE13.5 Ectopic pallial and basal expression of mTsh1 persists at 0
1
(Fig. 2B,F), similar to that observed at E16.5 (Waclaw et al. E15.5intheSeymutant(Fig.3K,O).Moreover,doublemutant 9
2006). In contrast, the Sey mutant telencephalon continues analysis shows that the loss of ectopic Gsh2 in Sey mutants
to exhibit robust ectopic Sp8 expression in the pallium does not significantly rescue the abnormal phenotype
(Fig.3C,G),andatmedialtelencephaliclevelsanectopicbasal (Fig.3L,P).TheformationofmTsh1+paraventricularectopia
streamofSp8expressionisobserved(Fig.3G).Thisdomainof shows that dorsal telencephalic progenitors in the Sey
ectopicpallialSp8expression,althoughlesssevere,persistsin mutant pallium are respecified to not only an Sp8
Sey/Gsh2mutantsatE15.5(Fig.3D,H).Sp8expressionreveals lineage (Kroll and O’Leary 2005; this study), but to mTsh1
paraventricular ectopia in Sey/Gsh2 double mutants and Er81 lineages. Furthermore, removal of Gsh2 from the
(Fig. 3D,H) as in the case of the Sey single mutant (Kroll and Sey mutant pallium is not sufficient to rescue the respeci-
O’Leary,2005; this study). fication of dorsal telencephalic precursors to these dLGE
At E15.5, similar to E13.5, mTsh1 is expressed in the dLGE identities.
SVZ and in the LCS migratory route (Fig. 3I,M). Interestingly, In summary, this analysis reveals that 1) Sp8 and mTsh1
although mTsh1 expression in Gsh2 mutants was normal at appear to be differentially regulated by Gsh2 in a temporally
E13.5, at E15.5 expression in the dLGE and LCS is markedly dependent manner and 2) altered expression of Sp8 and
748 Pax6andGsh2inPallial--SubpallialBoundaryPatterning d Carneyetal.
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Figure2. ExpressionofdLGEmarkersSp8andmTsh1atE13.5.Sp8isstronglyexpressedinthedLGESVZincontrolsatE13.5(A,E).Thelevelofexpressionismarkedly 5/2
diminishedinGsh2mutants(B,F,arrows).ThelossofPax6inSeymutantsresultsinectopicpallialexpressionofSp8(C,G,arrowheads).InSey/Gsh2doublemutantmice,this 7
9
ectopicexpression,althoughimprovedovertheSeysinglemutantphenotype,persists(D,H,arrowheads).AtE13.5,mTsh1isexpressedinthedLGESVZandalsotheputative 5
2
LCSmigratoryroute(I,M,arrows).ThisexpressionisgenerallyunaffectedinGsh2mutants(J,N,arrows).Conversely,intheSeymutant,ectopicmTsh1expressionisobserved 2
inboththepallium(K,O,arrowheads)andbasally(K,O,arrows).ThismispatterningisgenerallysimilarintheSey/Gsh2doublemutants(L,P,arrows,arrowheads).Abbreviation: by
nnumbersforeachprobeareasfollows:controls,n54[Sp8],n56[mTsh1];Gsh2(cid:1)/(cid:1),n54[Sp8],n54[mTsh1];Sey/Sey,n54[Sp8],n55[mTsh1];Sey/Sey;Gsh2(cid:1)/(cid:1), g
u
n55[Sp8],n55[mTsh1].Scalebar:200lm. e
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mTsh1 in Sey/Gsh2 double mutants broadly resembles that of 5D,H). Thus, we reveal that the Pax6-regulated expression of A
p
singleSey mutants. Sfrp2isindependent of Gsh2. ril 2
Dbx1 is a homeodomain gene that, similar to Sfrp2, is 0
1
expressed in the VZ of the VP aspect of the PSB (Yun et al. 9
Differential Regulation ofVentral Pallial Markers Sfrp2 2001;Medinaet al. 2004;Bielleet al. 2005).The VPgenerates
andDbx1byPax6andGsh2 cells destined for the pallium (Bielle et al. 2005) and the
Sfrp2,aninhibitoroftheWNTsecretedgrowthfactorprotein basolateral and lateral amygdala (Stenman, Yu, et al. 2003;
isexpressedintheVZoftheVP,asshownatE13.5andE15.5 Carney et al. 2006). We analyzed the expression of Dbx1 at
(Figs 4 and 5A,E). In Gsh2 mutants at E13.5, the expression E12.5,afterwhichVPexpressionisdownregulated(Fig.4I).In
domain of Sfrp2 expands ventrally (Fig. 4B,F). This expansion Gsh2 mutants, Dbx1, similar to Sfrp2 at E13.5, is ectopically
remains present at E15.5 (Fig. 5B,F). In agreement with expressed in the dLGE (Fig. 4J). We further observe that Sey
previous findings at E13.5 (Kim et al. 2001; Muzio et al. mutants lack Dbx1 expression at the PSB (Fig. 4K). Interest-
2002),weobservethatthetelencephalicexpressionofSfrp2at ingly,incontrasttoSfrp2,removalofectopicGsh2intheSey
thePSBismissinginSeymutantmiceatE13.5andE15.5(Figs4 mutant pallium re-initiated expression of Dbx1 in the LGE of
and5C,G). At E13.5and E15.5, lack of bothPax6 and Gsh2 in Sey/Gsh2 double mutants (Fig. 4L). However, this expression
Sey/Gsh2 double mutant mice does not restore Sfrp2 appearstobeectopicallylocalizedtotheSVZ.Thisrevealsthat
expression at the PSB at rostral or medial levels (Figs 4 and Pax6 is required for Dbx1 expression at the VP and Gsh2
CerebralCortexApril2009,V19N4 749
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Figure3. ExpressionofdLGEmarkersSp8andmTsh1atE15.5.AtE15.5,Sp8stronglymarksthedLGESVZ(A,E).InGsh2mutants,thisexpressionpatternislargelynormal(B, 5/2
F,arrowheads).SeymutantsdisplaysignificantectopicexpressionofSp8inthepallium(C,G,arrows),withanenlargedbasalstreamatmoremediallevels(G,arrowhead)thatis 7
9
notobservedmorerostrally(C,arrowhead).Sey/Gsh2doublemutantsdisplayectopicpallialexpression,thoughatdiminishedlevelsfromtheSeymutants(D,H,arrows),with 5
2
nascent paraventricular ectopia (D, H, empty arrowheads). Sey/Gsh2 double mutants also do not display a basal stream of Sp8 expression (D, H, arrowheads). mTsh1 is 2
expressedinthedLGESVZandputativeLCS(arrows)(I,M).InGsh2mutants,thereisdiminishedmTsh1inthedLGESVZ(arrows)withalackofexpressionintheputativeLCS by
(J,N,asterisk).IntheSeymutant,ectopicmTsh1expressionisfoundinthepallium(arrows)andformsparaventricularectopia(emptyarrowheads)(K,O).Basal-directed g
u
putativemigrationisobservedatrostral(K,arrowheads)andmedial(O,arrowheads)levels.Sey/Gsh2doublemutantsalsoexhibitpallialectopicmTsh1expression(arrows)with e
s
paraventricularectopia(emptyarrowheads)andputativebasal-directedmigration(arrowheads)(L,P).nnumbersforeachprobeareasfollows:controls,n56[Sp8],n56 t o
[mTsh1];Gsh2(cid:1)/(cid:1),n55[Sp8],n55[mTsh1];Sey/Sey,n55[Sp8],n56[mTsh1];Sey/Sey;Gsh2(cid:1)/(cid:1),n54[Sp8],n55[mTsh1].Scalebar:200lm. n
1
1
A
p
functionstorepressectopicDbx1expressionintheLGE.Thus, theputativeLCS(Fig.6N).Inagreementwithpreviousfindings ril 2
similartotheabovedifferentialregulationofmTsh1andSp8by (Assimacopoulos et al. 2003), Tgfa expression is absent in the 0
1
Gsh2 and Pax6, Sfrp2, and Dbx1 are also regulated in VPinSeymutantsatbothages(Fig.6C,G,K,O).SimilartoSfrp2, 9
a complexmanner byGsh2 and Pax6. but in contrast to the VP marker Dbx1, expression of Tgfa is
not restored at the PSB in the Sey/Gsh2 double mutants at
Regulation ofTgfaExpression byGsh2 andPax6 either age (Fig. 6D,H,L,P).
atE13.5 andE15.5
Next, we examined expression of the secreted factor Tgfa, Interneuron markers Dlx2andDlx5
which is a ligand for EGFR in the developing forebrain TheDlxfamilyofhomeoboxtranscriptionfactorscomprises6
(Kornblum et al. 1997). At E13.5 and E15.5, Tgfa is strongly genes, 4 of which are expressed in the developing forebrain
expressed in the VP aspect of the PSB (Fig. 6A,E,I,M) (Bulfone, Puelles, et al. 1993; Simeone et al. 1994; Anderson
(Assimacopoulos et al. 2003). In Gsh2 mutants at E13.5, Tgfa etal.1997;Liuetal.1997).Dlx1andDlx2areexpressedinthe
expressionectopicallyextendsintotheLGE(Fig.6B,F),similar subpallial VZ and SVZ, whereas Dlx5 and Dlx6 are strongly
to otherVP markers, Sfrp2 and Dbx1 (Fig. 4B,F,J). At E15.5in expressed in the subpallial SVZ and postmitotic regions
Gsh2 mutants, Tgfa expression remains slightly expanded (Eisenstat et al. 1999). We have previously shown that in the
(Fig. 6J,N) along with ectopic expression along the route of absence of Gsh2, there is a lack of Dlx2+ cells along the LCS
750 Pax6andGsh2inPallial--SubpallialBoundaryPatterning d Carneyetal.
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Figure4. ExpressionofVPmarkersSfrp2andDbx1atE13.5andE12.5.Sfrp2isexpressedintheVPasshownatE13.5(A,E).Thisexpressiondomainisexpandedventrallyin rco
Gsh2mutants(B,F,arrowheads).Sfrp2expressionisabsentatthePSB(asterisk)intheSeymutant(C,G,asterisks).InSey/Gsh2doublemutantsexpressionisnotrestored(D, r/a
H,asterisks)Dbx1isexpressedintheVPatE12.5(I).LossofGsh2resultsinectopicDbx1expressionintheLGE(J,arrows).InSeymutants,Dbx1ismissingintheVP(K, rtic
asterisk).InSey/Gsh2doublemutantsDbx1expressionisre-expressed,butmislocalizedinthedLGESVZ(L,arrows).nnumbersforeachprobeareasfollows:controls,n56 le
[Sfrp2],n53[Dbx1];Gsh2(cid:1)/(cid:1),n54[Sfrp2],n53[Dbx1];Sey/Sey,n54[Sfrp2],n52[Dbx1];Sey/Sey;Gsh2(cid:1)/(cid:1),n54[Sfrp2],n52[Dbx1].Scalebar:200lm. -a
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Figure5. ExpressionofSfrp2atE15.5.Sfrp2isexpressedintheVPandlayerVneuronsofthecortex(A,E,arrows).InGsh2mutants,theVPexpressiondomainisexpanded(B,
F,arrowheads)butreducedinthelateralcortex(B,F,arrows).InSeymutants,VPexpressionofSfrp2ismissing(asterisks)andlayerVexpressionisdrasticallyreduced(arrows)
(C,G).InSey/Gsh2doublemutants,Sfrp2expressionisnotrestoredintheVP(asterisks),andisseverelyreducedinthelateralcortex(arrows)(D,H).nnumbersareasfollows:
controls,n56;Gsh2(cid:1)/(cid:1),n54;Sey/Sey,n54;Sey/Sey;Gsh2(cid:1)/(cid:1),n54.Scalebar:200lm.
migratoryroutetothebasaltelencephalon(Carneyetal.2006). (Fig. 2J,N). To this end, we sought to examine the expression
Therefore, the presence of mTsh1-positive cells along the patterns of Dlx2 and Dlx5, which in addition to marking
putative LCS in the Gsh2 mutant in this study is surprising subpallial cells, demonstrate migrating cells of the LCS. At
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Figure6. ExpressionofsecretedfactorTgfaatE13.5andE15.5.TgfaisexpressedintheVPandbasaltelencephalonatE13.5(A,E,arrow).InGsh2mutants,VPexpressionis 5/2
expandedventrally(arrowheads)andbasalexpressionismaintained(arrow)(B,F).InSeymutants,VPexpressionismissing(asterisks)whereasbasalexpressionisunaffected 7
9
(arrows,C,G).VPexpressionremainsabsentinSey/Gsh2doublemutants(asterisks),thoughbasalexpressionispresent(arrows)(D,H).AtE15.5,expressionisobservedinthe 5
2
VPanddifferentiatingstriatum(STR,arrows)(I,M).InGsh2mutants,VPexpressionisexpanded(arrowheads),andaputativeectopicbasalmigrationisobserved(empty 2
arrowheads).Asmallerdomainofexpressionisobservedinthestriatum(J,N,arrows).InSeymutants,VPexpressionismissing(asterisks),althoughstriatalexpressionremains by
(arrows)(K,O).InSey/Gsh2doublemutants,expressionattheVPisnotrestored(asterisks),andremainspresentinthestriatum(arrows)(L,P).nnumbersareasfollows: g
u
controls,n54[E13.5],n54[E15.5];Gsh2(cid:1)/(cid:1),n53[E13.5],n53[E15.5];Sey/Sey,n53[E13.5],n54[E15.5];Sey/Sey;Gsh2(cid:1)/(cid:1),n53[E13.5],n53[E15.5].Scalebar: e
s
200lm. t o
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E13.5, Dlx mRNA and protein expression is confined to the In addition to putative migratory defects along the LCS as ril 2
subpallial progenitor zones and postmitotic cells undergoing definedbyDlx2andDlx5expressioninbothSeyandSey/Gsh2 0
1
tangential migration (Supplemental Figs 1 and 2A,E) (Porteus double mutants, we also observe alterations in cortical 9
etal.1991;Bulfone,Kim,etal.1993;Porteusetal.1994;Puelles patterning that is generally consistent with previous observa-
et al. 2000; Nery et al. 2003; Carney et al. 2007; Flames et al. tions. At both E13.5 and E15.5, in the Sey mutants, there is
2007). At E15.5, Dlx2 and Dlx5 streams are clearly observed a large ectopic expression of Dlx2 and Dlx5 in the cerebral
alongtheLCS(SupplementalFigs1and2I,M).InGsh2mutant cortex indicative of a broad ventralization of the dorsal
mice, Dlx2 and Dlx5 expression is truncated along the telencephalon (Supplemental Figs 1 and 2C,G,K,O). In Sey/
emanating LCS (Supplemental Figs 1 and 2J,N) (Szucsik et al. Gsh2 double mutants this phenotype is slightly improved
1997;Corbinetal.2000;Neryetal.2002;Carneyetal.2006).In (Supplemental Figs 1and 2D,H) (Toresson et al.2000).
the Sey mutant, there appears to be a large ectopic stream of
LCS cells destined for the basal telencephalon that is also Alterations in LCScellmigration inGsh2, Sey,andSey/
consistentwithotherfindings(SupplementalFigs1and2K,O) Gsh2 double mutants atE15.5
(Caubit et al. 2005). This abnormal putative migratory stream To examine defects in cell migration along the LCS in Gsh2,
appears to persist in Sey/Gsh2 double mutants (Supplemental Sey, and Sey/Gsh2 double mutant mice, we carried out
Fig.1Land Supplemental Fig.2L,P). immunohistochemical analysis for EGFR, which is expressed
752 Pax6andGsh2inPallial--SubpallialBoundaryPatterning d Carneyetal.
atthePSBandinmigratingcellsoftheLCS(Caricetal.2001). both rostral and medial levels at E13.5, and at rostral levels at
First, we further characterized the cell types of the PSB that E15.5(Fig.8C,G,K).Incontrast,atmediallevelsatE15.5,alow
expressEGFR.Tothisend,weperformeddualimmunolabeling level of Gsh1 expression expands into the pallium in Sey
experiments to label VZ and SVZ/mantle cells that are pallial- mutants (Fig. 8O). In Sey/Gsh2 double mutants at E13.5 and
derived (Pax6+ and Tbr1+) and subpallial-derived (Dlx2+ and E15.5, the dorsal limit of Gsh1 expression resembles that of
Dlx5/6+) at E15.5, which represents the time point of robust Gsh2 mutants (Fig. 8D,H,L,P). Thus, Pax6 and Gsh2 act
LCScellmigrationofbothpopulations(Carneyetal.2006).We combinatorially to repress Gsh1 expression in the ventral and
observe that many EGFR+ cells at the PSB also express Pax6 lateral pallium.
(Fig. 7A,B) and a minority colocalize with the pallial mantle
marker Tbr1 (Fig. 7C,D). To analyze whether the subpallial
Discussion
progenitors also coexpress EGFR, EGFR immunolabeling was
combined with b-gal immunohistochemistry in sections from Gene expression studies have revealed that the embryonic
Dlx2+/tauLacZ mice or colocalization with endogenous GFP telencephalon can be parcellated into refined molecular maps
from Dlx5/6-GFP mice. WefoundthatmanyEGFR+cellsatthe (Puelles et al. 2000; Medina et al. 2004; Flames et al. 2007). Do
w
PSBarealsob-gal+,indicatingthatthesecellsareDlx2+(Fig.7E,F). Combined with in utero transplantation and genetic fate n
Incontrast,Dlx5/6-GFP+cellsdonotexpressEGFR(Fig.7G,H). mapping studies this work has unraveled the correlation loa
d
Although this was somewhat surprising, it has been previously between specific subpallial progenitor pools and the diversity ed
shownthatnotallDlx2+cellsalsoexpressDlx5(Eisenstatetal. of cortical interneurons in the adult brain (Nery et al. 2002; fro
1999),thustheDlx2+/EGFR+populationmaybeasubsetofthis Buttetal.2005;Flamesetal.2007;Fogartyetal.2007;Wonders m
h
subpallial Dlx2+/Dlx5/6- population. Alternatively, this EGFR+ et al. 2008, Xu et al. 2008). This restricted potential of neural ttp
PpSoBputhlaattioarnemalsaoyPaalsxo6+be(Cpaarrnteoyfetthael.2su0b0s6e)t.ToafkDenlxt2o+gectehlelsr,atthtehsee p20ro0g7e)n.itMoorsreiosvearls,otomaainltaarigneedexintenatdumlthajooorda(sMpeecrtksleofetthael. s://ac
a
datasuggestthatEGFR+cellsoftheLCSarisefromprogenitorsof functionofbothintrinsicandextrinsickeyplayersthatpattern de
boththepallialandsubpallialaspectofthePSB. the telencephalon have been elucidated. However, our un- mic
We next examined the status of the EGFR+ LCS population derstanding of the genetic mechanisms that pattern specific .o
u
and the radial glial scaffold in Gsh2, Pax6, and Sey/Gsh2 telencephalicprogenitorzones,suchasPSB,remainunknown. p.c
mutantsbydualimmunolabelingforEGFRandRC2atE15.5.In In this study, we examined the development of the PSB, an om
controls,strongRC2expressiondefinestheradialglialscaffold important progenitor domain for the OB, amygdala and early /c
e
of the LCS migratory route to the basal telencephalon, and cerebral cortical Cajal--Retzius populations. Our studies reveal rc
o
EGFR+ cells are observed at the PSB and along the LCS that the PSB is a highly complex and dynamic telencephalic r/a
(Fig. 7I,M). In Gsh2 mutant mice, the RC2+ radial glia scaffold progenitor zone, comprised of multiple molecularly distinct rtic
appears to occupy a larger domain at the PSB, concomitant progenitor pools. We also provide novel insight into the le-a
with a wider domain of EGFR+ cells in the LGE and the LCS geneticregulation of their specification byPax6and Gsh2. bs
(Fig. 7J,N). Conversely, in Sey mutants, the fasciculated RC2+ tra
c
processes of the LCS radial glia scaffold are missing, and RC2 Combinatorial Codes ofGene Expression DefineUnique t/1
9
labelingappearsdiffuse(Fig.7K,O)(Stoykovaetal.1997).Also, Progenitor Domains atthe PSB /4
thereisastrikingabsenceofEGFRexpressionbothatthePSB Theimportanceofintrinsicspecificationforneuronaloutputin /74
5
and in the LCS in Sey mutants (Fig. 7K). In Sey/Gsh2 double the cerebral cortex was first suggested by the ‘‘protomap’’ /2
mutants,theRC2+immunolabelingalsoappearsdiffuse,similar theory (Rakic 1988). Furthermore, the parcellation of pro- 79
5
totheSeymutant.Interestingly,EGFRexpressionisrestoredin genitor domains and the generation of restricted neuronal 2
2
the double mutant, although EGFR+ cells appear scattered in diversitywerefirstappreciatedinthespinalcord(reviewedin b
y
the LGE and LCS (Fig. 7L,P). This observation is similar to the Jessell 2000). Collectively, these studies defined a paradigm in gu
e
restoration of Dbx1 expression in the LGE in Sey/Gsh2 whichregionalexpressionofkeygenes(typicallytranscription s
mutants (Fig. 4), and reveals that the loss of EGFR expression factors) instruct cell fate decisions in a cell-autonomous t on
at the PSB and LCS in Sey mutant mice is a consequence of manner, before or at the last cell division (reviewed in Dehay 11
ectopicGsh2 expression. andKennedy2007).Amajorpredictionofthesestudiesisthat A
p
progenitorpoolsexpresscombinationsoftranscriptionfactors, ril 2
Gsh1expressioninsingleSey,Gsh2,andSey/Gsh2double whichendowthesecellswithuniquefatepotentials.Basedon 0
1
mutants this idea, recent studies have comprehensively explored gene 9
Previously it has been shown that Gsh1 can rescue many, but expression patterns of putative instructive factors in the
not all, of the subpallial patterning defects in found in Gsh2 subpallial proliferative zones. One study defined at least 18
mutants starting at around E14 (Toresson and Campbell 2001; molecular distinct progenitor domains in the embryonic
Yun et al. 2001, 2003). As described previously (Toresson and subpallium(Flamesetal.2007).Moreover,atembryonicstages,
Campbell 2001), and shown here at E13.5 and E15.5, Gsh1 is postmitotic subpallial-derived cells express genes which
normally expressed at high levels in the medial ganglionic encode proteins of a characteristically mature neuronal
eminence (MGE) and ventral LGE, with intense expression in phenotype as early as E13.5 (Batista-Brito, Machold, et al.
theVZandinscatteredcellsintheSVZ(Fig.8A,E,I,M).InGsh2 2008). Collectively, these analyses have provided both an
mutant mice at E13.5, high levels of expression extend to the important reference and context to consider the spatio-
PSB, most prominent at rostral levels (Fig. 8B,F). At E15.5 in temporal and genetic origins of neural diversity. However, to
Gsh2mutantmice,highlevelsofGsh1expressionextendinto a large extent such studies have focused on the MGE, which
the VP (Fig. 8J,N). In contrast, in the Sey mutant, this dorsal generates the majority of cortical interneurons (Sussel et al.
limit of expression of Gsh1 is located much more ventrally at 1999).
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Figure7. AlterationsinLCScellmigrationinGsh2,Sey,andSey/Gsh2doublemutantsatE15.5.LowmagnificationofdualimmunolabelingforPax6(green,A),Tbr1(green,C), ril 2
Dlx2(green,E)andEGFR(red)showscoexpressionofasubsetofcellsatthePSB(A,C,E).Highermagnificationofboxedregionsin(A,C,E)showindividualdoubleEGFRþcells 0
1
(arrows)colocalizedwithPax6(B,arrows),Tbr1(D,arrows),orDlx2(F,arrows)intermingledwithEGFRþcellsnotexpressinganyofthesemarkers(arrowheads).Incontrast, 9
thereisnocolocalizationofendogenousDlx5/6-GFPlabeling(green,arrows)andEGFRimmunolabeling(red,arrowheads)(G,H).RC2(green)andEGFR(red)dualimmunolabeling
revealstheLCSradialglialscaffold(arrows)andEGFRþmigratorycells(arrowheads)emanatingfromthePSB(emptyarrowheads)(I,M).InGsh2mutants,theVZEGFRþ
domainappearsexpanded(emptyarrowheads),withamorediffuseRC2immunolabeling(arrows)(J,N).EGFRþcells(arrowheads),althoughdisorganized,remainpresentalong
theLCS.InSeymutants,EGFRþcellsarecompletelyabsent,andtheRC2þradialgliaalongtheLCSmigratoryrouteismissing(K,O,arrows).InSey/Gsh2doublemutants,
RC2þradialglia(arrows)resembleSeymutants,butEGFRþcellsarenowpresentalongtheLCS(arrowheads)andoccupyanexpandeddomainatthePSB(emptyarrowheads)
(L,P).To-Pro-3counterstaining(blue)isshowninthemajorityofpanels.nnumbersareasfollows:EGFRandPax6orDlx2orTbr1,n52;EGFR/Dlx5/6-GFP,n52;EGFR/RC2:
controls,n53;Gsh2(cid:1)/(cid:1),n53;Sey/Sey,n54;Sey/Sey;Gsh2(cid:1)/(cid:1),n53.Scalebar:A,C,E,G,M--P:100lm,I--L:40lm,B,D,F,H:30lm.
In this study, we sought to extensively examine the gene expression. These combinatorial expression patterns
molecular code of gene expression of the PSB. Our analysis highlight the importance of the PSB as a unique and highly
using a battery of markers that delineate the dLGE (mTsh1, heterogeneous telencephalic progenitor domain. Moreover
Sp8), VP (Sfrp2, Tgfa, and Dbx1), or both (Er81) subdomains considering that the PSB is a major source of cells that
reveals that the PSB also contains highly regulated patterns of generates neuronal cell diversity in the limbic system and OB,
754 Pax6andGsh2inPallial--SubpallialBoundaryPatterning d Carneyetal.