Lhx2 Regulates the Development of the Forebrain Hem System.

18.9 MB·English

by  Roy, Achira

CerebralCortexMay2014;24:1361–1372
doi:10.1093/cercor/bhs421
AdvanceAccesspublicationJanuary10,2013
Lhx2 Regulates the Development of the Forebrain Hem System
AchiraRoy1,MiriamGonzalez-Gomez2,AlessandraPierani3,GundelaMeyer2andShubhaTole1
1DepartmentofBiologicalSciences,TataInstituteofFundamentalResearch,Mumbai,India,2DepartmentofAnatomy,Facultyof
Medicine,UniversityofLaLaguna,Tenerife,Spainand3InstitutJacquesMonod,CNRS,UniversityofParisDiderot,Paris,France
AddresscorrespondencetoShubhaTole,B-304,DepartmentofBiologicalSciences,TataInstituteofFundamentalResearch,HomiBhabhaRoad,
Colaba,Mumbai400005,India.Email:[email protected];GundelaMeyer,DepartmentofAnatomy,FacultyofMedicine,UniversityofLa
Laguna,38071LaLaguna,Tenerife,Spain.E-mail:[email protected]
Early brain development is regulated by the coordinated actions of Thetelencephalicchoroidplexusisflankedrostrallybythe
multiple signaling centers at key boundaries between compart- septum, on itstelencephalic side by the cortical hem, and on
ments. Three telencephalic midline structures are in a position to itsdiencephalicsidebythethalamiceminence.
play such roles in forebrain patterning: The cortical hem, the Though the septum, hem, and thalamic eminence have
septum, and the thalamic eminence at the diencephalic–telence- beenstudiedinliteratureasindependentstructures,theyhave
phalic boundary. These structures express unique complements of certain fundamental similarities with respect to anatomy and
signaling molecules, and they also produce distinct populations of function. Anatomically, these 3 structures surround the tele-
Cajal–Retzius cells, which are thought to act as “mobilepatterning ncephalic choroid plexus, being physically connected with it
units,”migratingtangentiallytocoverthetelencephalicsurface.We (Figs 1 and 2). They are each positioned at strategic bound-
show that these 3 structures require the transcription factor Lhx2 aries in the forebrain and have known or suggested roles as
todelimittheirextent.IntheabsenceofLhx2function,all3struc- secondary organizers. Finally, each of these structures
turesaregreatlyexpanded,andtheCajal–Retziuscellpopulationis produce Cajal–Retzius cells, which are the sources of Reelin
dramaticallyincreased.Weproposethatthehem,septum,andthal- that is necessary for cortical development (D’Arcangelo et al.
amiceminencetogetherforma“forebrainhemsystem”thatdefines 1995;Ogawaetal.1995).
and regulates the formation of the telencephalic midline. Disrup- ThecorticalhemisaWntandbonemorphogeneticprotein
tionsintheforebrainhemsystemmaybeimplicatedinseverebrain (Bmp) rich signaling centre that is connected to the choroid
malformations such as holoprosencephaly. Lhx2 functions as a plexus on one side and the cortical neuroepithelium on the
central regulator of this system’s development. Since all com- other (Grove et al. 1998). The hem was recently found to act
ponents of the forebrain hem system have been identified across as a secondary organizer that induces the formation of the
severalvertebratespecies,themechanismsthatregulatethemmay hippocampus in the adjacent cortical neuroepithelium
have played a fundamental role in driving key aspects of forebrain (Mangaleetal.2008),drawing aremarkableparallel withthe
evolution. roofplateofthespinalcord.Attherostralendofthetelence-
phalon, the cortical hem is contiguous with another midline
Keywords:Cajal–Retziuscells,human,Lhx2,midline,mouse,patterning, structure, the septum, a site that is enriched in Fgf family
thalamiceminence signaling molecules (Hoshikawa et al. 1998) and known to
function as an organizer for cortical areal patterning
(Fukuchi-ShimogoriandGrove2001).Thethalamiceminence
isatransientmedialstructureatthediencephalic–telencepha-
lic boundary (DTB) of the forebrain. Based on its position
andexpressionofsignalingmolecules,thethalamiceminence
Introduction
hasbeenhypothesizedtoalsoserveasanorganizeroraputa-
Themidlineofthecentralnervoussystemarisesasaresultof
tive signaling centre (Abbott and Jacobowitz 1999; Kim et al.
complex morphogenetic and inductive interactions. In the
2001; Fotaki et al. 2008). Though the septum continues into
spinal cord,theroofplateisformeddorsallyby thefusionof
maturity, the hem and thalamic eminence are not discernible
the 2 lateral margins of the neural plate, whereas the floor bylateembryoniclife:Thehemgiveswaytothefimbria,while
plate is induced ventrally by signals from the underlying no-
the thalamic eminence gets subsumed in diencephalic deriva-
tochord. The roof plate and the floor plate, in turn, function
tives(Groveetal.1998;AbbottandJacobowitz1999).
as secondary organizers, to regulate dorso-ventral identity
Adistinctivefeatureoftheseptum,hem,andthalamicemi-
along the entire length of the spinal cord (Yamada et al. nenceisthattheyeachproduceauniquepopulationofCajal–
1991;BriscoeandEricson1999;Liemetal.1997).Inthefore-
Retzius cells, which are among the earliest born neurons of
brain, the dorsal midline is more complicated as a result of
the developing forebrain (Meyer et al. 2002; Takiguchi-
the prosencephalic vesicle giving rise to paired telencephalic
Hayashi et al. 2004; Bielle et al. 2005; Yoshida et al. 2006;
evaginations and a central diencephalic vesicle. The choroid
Cabrera-Socorro et al. 2007; Abellan and Medina 2009;
plexus has a central position in the forebrain: It forms at the
Abellan et al. 2010; Tissir et al. 2009; Meyer 2010). Though
medial edges of the telencephalic neuroepithelium (the tele-
ncephalic choroid plexus) and also at the roof of the dience- the Cajal–Retzius cells arising from these different origins are
phalon. An additional site of choroid plexus formation is the molecularly distinct, all secrete Reelin, a glycoprotein known
roof of the hindbrain. In this study, we examine 3 key to be critical for cortical lamination (D’Arcangelo et al. 1995;
forebrain structuresconnectedwiththe telencephalicchoroid Ogawa etal.1995).The expression ofReelin hasbeenexam-
plexus. inedinextantvertebratesandappearstohavebeenamplified
©TheAuthor2013.PublishedbyOxfordUniversityPress.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by-nc/3.0/),whichpermitsnon-
commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Forcommercialre-use,[email protected]
in mammals, consistent with a role in the evolution of staged after legal abortions, following national guidelines in Spain
migratory mechanisms that led to the formation of laminated and Belgium and in accordancewith the institutional medical ethics
neocortex (Baret al. 2000; Tissiret al. 2002). In the develop- committeeguidelines(Meyeretal.2000).Fetalbrainswereobtained
after spontaneous abortions, under the same ethical guidelines. The
ing human brain, which has a protracted migratory period,
wholeheadswerefixedimmediatelyuponcollection,inBouin’sfixa-
Cajal–Retzius cells display complex morphologies and extend tive,embeddedinparaffin,andcutinseriesof10μmsections.They
a distinctive Reelin-positive axonal plexus at the interface of were immunostained with antibodies to calbindin and Tbr1, as well
boththemarginalzoneandthecorticalplate(MeyerandGof- as other antibodies, as described in Meyer et al. (2000) and
finet 1998). Furthermore, human Cajal–Retzius cells specifi- re-examinedforthepresentstudy.
cally express the Human accelerated region gene, HARF1,
indicating human-specific traits of this cell type, further sup-
porting an important role for these cells in neocortical evol- HistochemicalProcedures
ution (Pollard et al. 2006). Finally, experiments using the In situ hybridization was performed as described previously in
Bulchand et al. (2003). Briefly, the hybridization was performed
combinations of transgenic and knockout mice have
suggested that Cajal–Retzius cells mayact as “mobile pattern- overnight at 70°C in 4× sodium chloride-sodium citrate-citric acid
buffer(SSC),50%formamide,and10%sodiumdodecylsulphate.Post
ing units” providing regionalization cues to the developing
hybridization washes were at 70°C in 2× SSC and 50% formamide.
telencephalon (Griveau et al. 2010). The septum, hem, and These were followed by washes in 2× SSC, 0.2× SSC, and then
thalamic eminence, being the sites of the origin of Cajal– Tris-buffered salinewith 1% Tween-20 (TBST). Anti-digoxigenin Fab
Retziuscells,arethereforeimportantnotonlyfromadevelop- fragments(Roche)wereusedat1:5000inTBST,andthecolorreaction
mental,butalsoanevolutionaryperspective. using 4-nitro blue tetrazolium chloride + 5-bromo-4-chloro-3-indolyl
phosphate (Roche) was performed according to the manufacturer’s
In earlier work, we showed that the cortical hem expands
instructions. Immunohistochemistry were performed as described
intheabsenceofLIM-homeodomaintranscriptionfactorLhx2
in Subramanian et al. (2011). Primary antibodies used are mouse
(Bulchand et al. 2001; Mangale et al. 2008). Here, we show anti-5-bromo-2′deoxyuridine (anti-BrdU; Roche; 1:100) and rabbit
that loss of Lhx2 also results in the expansion of septum and polyclonalanti-Ki67(Abcam;1:50).Immunohistochemistryonhuman
thalamic eminence. Concomitantly, there is a dramatic over- embryonic and fetal sections was performed as described in
production of Cajal–Retzius cells in the Lhx2 mutant. Thus Meyer et al. (2000) using a rabbit polyclonal calbindin antibody
(Swant, Bellinzona, Switzerland) and Tbr1 antibody (kind gift from
Lhx2,acriticalregulatorofearlyforebraindevelopment,con-
R.Hevner).
trols the extent of the septum, hem, and thalamic eminence,
and as aconsequence, also the numberof Cajal–Retzius cells
produced. Since these 3 midline structures are developmen-
QuantitativeAnalysis
tally and functionally linked, we propose that they together
Forareameasurementstudies,datawerecollectedfrom3contiguous
form a “forebrain hem system” that serves as a multicompo-
sectionstakenfrom3mousebrainsatE12.5.Theareawascalculated
nent patterning and organizing center for the medial using ImageJ software (NIH). For labeling index and quit fraction
forebrain. studies,confocalmicroscopywasused.Individualcellsinthesections
Defective formation of the forebrain midline can give rise were imaged at a magnification of 40× using Zeiss LSM 510 Meta.
to severe malformations such as holoprosencephaly, one of Image stacks were generated by scanning at intervals of 0.44μm
usingfiltersoftheappropriatewavelengths.Thecellswerequantified
the most common defects in brain development. Here, we
usingImageJsoftware.Theproliferatingpoolofcellsinthetelence-
comparethesestructuresintheembryonicmouseandhuman
phalon is known to include not only neuroepithelial stem cells,
forebrain. Our interpretation of the septum, hem, and thal- butalsomicrogliaandendothelialcells.However,themicrogliahave
amic eminence as a unified forebrain hem system offers a only just begun to invade the cortex at E10.5–E11.5 (Swinnen et al.
new framework in which such defects may be interpreted, 2012). Consistent with the description of microglial morphology
leadingtoabetterunderstandingofmidlinepathologies. (Kettenmannetal.2011),cellswithcompact4′,6-diamidino-2-pheny-
lindole (DAPI)-positive nuclei surrounded by DAPI-negative cyto-
plasmwerefoundinoursections,mostlynearthepialsurface.Neural
progenitors in contrast had large DAPI-positive nuclei that occupied
MaterialsandMethods mostofthevolumeofthecell.Endothelialcellsenterthecortexfrom
capillaries,andattheearlystagesweexaminedwouldbeexpectedto
AnimalsandSamplePreparation beincloseassociationwiththem.Wedidnotuseregionswithcapil-
lariesinourcounts.Eventhoughbothmicrogliaandendothelialcells
Mice peakinnumberonlylater,itispossiblethatourcellcountsincluded
AllproceduresfollowedInstituteAnimalEthicsCommitteeguidelines some cells other than neuroepithelial cells. Sections from 3 brains
and National Institutes of Health guidelines for the care and use of werescored.Foreachbrain,600–1000cellswerecountedforthela-
animals (mice). Timed pregnant Lhx2+/− mice (gift of F.D. Porter, belingindexand500–700cellsforthequitfraction.Statisticalanalysis
NIH)wereused.PregnantdamswereobtainedfromtheTataInstitute wasdoneusinga2-tailedStudent’st-testinMicrosoftExcel.
animal breeding facility. A tamoxifen-inducible CreER line (strain
name:B6;129-Gt(ROSA)26Sortm1(cre/ERT)Nat/J;stocknumber:004847)
was obtained from the Jackson Laboratory. The floxed Lhx2 line
(Lhx2lox/lox)wasobtainedfromE.S.Monuki’slab.Forinsituhybrid- 3-DModelingandVideo
ization, the mouse embryos were harvested in phosphate buffer The 3-dimensional (3-D) model of the forebrain hem system was
salineatE11,E11.5,andE12.5,fixedin4%paraformaldehyde(PFA), developed by using the software BioViz3D version 3.0. 42 serial
equilibratedin30%sucrosemadein4%PFA,andsectionedat30μm sections(25 µm each) of an E12.5wild-typebrain were imagedand
onafreezingmicrotome. arranged in the rostrocaudal order. Contours were drawn in each
section,basedon morphologyforthe choroid plexus,andbasedon
theexpressionofspecificmarkersforthecorticalhem,septum,and
Human thalamic eminence. The 3-D reconstruction was created using these
Sevenhumanembryonicandfetalbrains(from6to10/11gestational contours for the entire stack. The moviewas made using the movie
weeks, GW) were examined. Embryonic brains were obtained and makersoftwareHypercam2.
1362 Lhx2RestrictsForebrainHemSystem (cid:129) Royetal.
Results eminence collectively encircle it. These 3 structures at
key forebrain boundaries mark the transition from the
TheMorphologicalRelationshipsofMedial telencephalic choroid plexus to the rest of the forebrain:
ForebrainStructures The septum and hem connecting the choroid plexus to the
The septum, hem, and thalamic eminence share the telencephalon, and the thalamic eminence connecting it to
expression of several transcription factors and signaling mol- thediencephalon.
ecules. In E12.5 coronal sections, the septum appears at To examine the forebrain systemin intact preparations, we
rostrallevels,whereasthehemandthalamiceminenceappear prepared whole-mount “hemi forebrains” at E11.5–E12.5, by
atmidandcaudallevels.Inrostralsections,Wnt8bexpression making a cut along the diencephalic midline, such that each
is seen in the medial telencephalon extending into the pallial telencephalic hemisphere remains attached to a hemi-
septum, but excludes the subpallial septum (Fig. 1F). Wnt8b diencephalon. In such preparations, in situ hybridization
isalsoexpressedinthethalamiceminenceandhem(Fig.1G, reveals Wnt2b expression in the cortical hem, and AP2α in
H). At caudal levels, the thalamic eminence is contiguous the thalamic eminence; Lhx1 expression labels both these
withthecaudalganglioniceminence(CGE)inthetelencepha- structuresandtheseptum(Fig.2B,C).
lon (Fig. 1C,H,L). Horizontal sections at E12.5 are useful to The relative position of these structures is revealed in the
examine all 3 structures simultaneously. In such sections, sections of human embryonic and early fetal brains, with an
Wnt8b expression on either side of the choroid plexus E12.5 mouse section alongside for comparison, in which the
suggeststhatthethalamiceminencemaybeconsideredtobe overallstageofdevelopmentappearssimilartothatseenina
a“diencephalichem,”acounterparttothetelencephaliccorti- 7-GW human embryo at the preplate stage (Fig. 2E,F). A
cal hem (Fig. 1I). The expression of transcription factor Zic2 definitive marker of the choroid plexus is transthyretin (Ttr;
is seen in a broad region of the medial forebrain, including Herbert et al. 1986; Grove et al. 1998), which identifies this
the septum, hem, and thalamic eminence (Fig. 1J–M; Okada structure in the lateral ventricles and in the third ventricle of
et al. 2008). Zic2 is a majorcandidate gene implicated in ho- an E12.5 mouse section (Fig. 2E). In the human brain, the
loprosencephaly, and alterations in Zic2 function may affect entire hem system and choroid plexus express calbindin
theentireforebrainhemsystem. (Meyer 2010). From 7 to 11 GW, the human choroid plexus
A rostrocaudal series of E12.5 coronal sections were col- extensively expands, maintaining its connection with the
lated into a 3-D model, and animated into a movie (M1; still hem, septum, and thalamic eminence (Fig. 2F–H). This con-
images in Figs 1E and 2D) that begins with the choroid nection is schematized in Figure 2I. The choroid plexuses of
plexus and illustrates how the septum, hem, and thalamic lateral and third ventricles also appear fused to each other in
Figure1. Medialforebrainstructures.(A–C)Schematicsof3coronallevelsofsectioningintheE12.5mousebrain,correspondingto“i,”“ii,”and“iii”inN,respectively.“i”isat
thelevelofseptum(yellow);“ii”and“iii”showthecorticalhem(red)andthethalamiceminence(TE;blue).TEiscontiguouswiththeCGEasseeninlevel“iii.”(D)Schematic
depictingthepositionsofseptum,corticalhem,andTEinahorizontalsection,correspondingto“iv”inN.The3rostro-caudalcoronallevels(i,ii,andiii)and1horizontallevel
(iv)ofsectioningintheE12.5wholeforebrainareschematizedinN.(E)Dorsalviewofa3-DmodelofE12.5brainshowingthelocationsofseptum(yellow),hem(red),andTE
(blue),encirclingthechoroidplexus(green).Wnt8bisexpressedinthemedialneuroepitheliumofthedorsaltelencephalon,includingthepallialseptum(F),corticalhem(open
arrowhead), and also in the ventricular zone of TE (asterisk; G–I). (J–M) Zic2 is expressed in the entire medial forebrain, including the forebrain hem system. CGE, caudal
ganglioniceminence;h,hem;s,septum;TE,thalamiceminence.Scalebar:300µm.
CerebralCortexMay2014,V24N5 1363
Figure2. Connectionofthechoroidplexuswithcomponentsoftheforebrainhemsystem.(A)SchematicofthemedialviewofanE11.5hemi-forebrainshowingthepositionof
the forebrain hem system (yellow), encircling the choroid plexus (cp; green). The dashed line (A and C) correspondsto the diencephalic midline that was bisected for the
hemi-forebrain preparation (B). Whole mount in situ hybridization of an E12.5 hemi-forebrain reveals Wnt2b expression, specific to the cortical hem (purple), and AP2α
expression,specifictothethalamiceminence(asterisk;brown).(C)Lhx1isexpressedintheE11.5septumandthalamiceminence(asterisk)andalsoweaklyinthecortical
hem.(D)Lateralviewofasinglehemispherein3-DmodelofE12.5brainshowingthelocationoftheseptum(yellow),hem(red),andthalamiceminence(blue),encirclingthe
choroidplexus(green).(E)E12.5mousebrainsectionsprocessedformarkerTtrshowthepresenceofthechoroidplexusadjacenttoboththelateralventriclesandthethird
ventricle.(F–H) Human sectionsimmunostainedby calbindinantibodyshow the development of choroid plexus from 7 to 10/11GWs. (H) Anoblique sectionof 10/11-GW
human brain that showsthe connection of choroid plexus with both the septum and cortical hem (open arrowheads), as also depicted in I. cp, choroid plexus; h, hem; s,
septum;TE,thalamiceminence;IIIV,thirdventricle.Scalebar:200µm(BandC).
the human, seen in a horizontal section of a 6-GW embryo, acquired an identity corresponding to the subpallial septum.
publishedinCabrera-Socorroetal.(2007). Wequantitatedtheseptalareaandfoundittobesignificantly
Inpriorwork,wereportedthatLhx2actstosuppresscorti- increasedintheLhx2mutantatE12.5(P<0.01;Fig.3I).
cal hem fate and is required to restrict the cortical hem to its In our previous studies, we reported of the expansion of
medial location (Bulchand et al. 2001; Mangale et al. 2008). thehemintoterritorythatisnormallyoccupiedbythecortical
Furthermore, the telencephalic choroid plexus, which arises neuroepithelium (Bulchand et al. 2001; Mangale et al. 2008).
from the cortical hem (Louvi et al. 2007), is greatly increased This occurs throughout the telencephalon except at extreme
intheLhx2mutantmice(Monukietal.2001).Thismotivated rostrallevels,wheretheneuroepitheliumtakestheidentityof
anexaminationoftheotherderivativeofthecorticalhem,the theseptummediallyandhemdorsally,asshowninFigure3H.
Cajal–Retzius cells. Furthermore, since the septum and thal- The hem expresses both Zic2 and Ngn2, which accounts for
amic eminence also produce Cajal–Retzius cells (Meyer et al. theiroverlapinFigure3F.
2002; Bielle et al. 2005; Tissir et al. 2009), we examined the
roleofLhx2inthedevelopmentofthesemedialstructuresas
Cajal–RetziusCellsareIncreasedintheLhx2Mutant
well. Lhx2 expression is seen in the septum, thalamic emi-
nence, and hem at E11.0, but expression declinestoweakor Cajal–Retziuscellsareearly-bornneuronsthatarisefrommul-
undetectablelevelsbyE12.5(SupplementaryFig.S1). tiple origins and are thought to control radial migration of
cortical neurons via the reelin-Dab1 signaling pathway
(Howell et al. 1999; Tissir and Goffinet 2003). These cells
TheSeptumExpandsintheAbsenceofLhx2 arise from the cortical hem, septum, and thalamic eminence
The septumfunctions as a rostral signaling center in the tele- (Meyeretal.2002; Takiguchi-Hayashietal.2004;Bielle etal.
ncephalon, expressing multiple members of the Fgf familyof 2005; Cabrera-Socorro et al. 2007; Abellan and Medina 2009;
signaling molecules and their targets (Hoshikawa et al. 1998; Abellanetal.2010;Tissiretal.2009;Meyer2010).
Fukuchi-Shimogori and Grove 2001; Fukuchi-Shimogori and InE12.5Lhx2mutantforebrain,thereisanoverallincrease
Grove 2003; Okada et al. 2008). Transcription factors, Six3 in Cajal–Retzius cells as seen by expression of Reelin, Lhx1,
and Zic2, are also expressed in the septum, with Six3 being Lhx5, and ΔNp73, a definitive marker of Cajal–Retzius cells
restricted to its subpallial component (Oliver et al. 1995; arising from the forebrain hem system (Fig. 4A–D,I,J). Exam-
Inoue et al. 2007; Fig. 3B,E). Fgf17 expression is specific to inationoflabeledcellsaftera2-hpulseofBrdUadministered
the entire septum (Fig. 3G). Emx1 and Ngn2, definitive at E11.5 revealsthat the labeling index in control and mutant
markers of the pallial neuroepithelium, are expressed in the septum (45% and 47%, respectively) and hem (46% and 45%,
pallial component of the septum and the adjacent medial respectively) is similar. This indicates that cell division rates
pallium.IntheLhx2mutant,expressionofthesegenesisnot are unaffected in the absence of Lhx2. However, when BrdU
seen in the medial pallium at rostral levels, suggesting a loss injection at E11.5 is followed by BrdU/Ki67 double labeling
of pallial identity. Instead, Zic2, Six3, and Fgf17 expression in brains harvested 1day later, at E12.5, the quit fraction of
extend into the territory from which Emx1 and Ngn2 have the mutant septum (57%) and hem (57%) is significantly in-
receded (Fig. 3A–F). The expansion of Six3, in particular, creased when compared with the control septum (31%) and
indicates that, at rostral levels, the medial pallium has hem (35%; Fig. 4E–H,K–N). These data indicate that, in the
1364 Lhx2RestrictsForebrainHemSystem (cid:129) Royetal.
Figure3. TheseptumexpandsintheLhx2mutant.(AandD)In controlbrainsatE12.5,Emx1(A)andNgn2(D)areexpressedinthepallium(dorsaltelencephalon),their
expressionextendingmediallytoincludethepallialseptum.(B)Six3isexpressedinthesubpallialcomponentoftheseptumandalsointhesubpallium(E).Zic2isexpressedin
bothcomponentsoftheseptumandalsoadjacentmedialpallialregions.InLhx2mutantbrains,Emx1andNgn2expressionrecedesfromthemedialtelencephalon,whereas
Zic2andSix3expressionexpandtofillthisterritory(A,B,D,E),asseeninfalse-coloroverlaysofadjacentsectionsshowingEmx1/Six3(C)andNgn2/Zic2(F)expression.Ngn2
andZic2expressionsoverlapinthecorticalhem(FandH).ArrowheadsinA,B,andCmarkthedorsallimitoftheseptumandthebeginningoftheEmx1-expressingdomain(A)
in control and Lhx2 mutant sections.(G) Fgf17 is expressed in both pallial and subpallial components of control septum shown at E11.5 and E12.5. In Lhx2 mutant, Fgf17
expressionexpandsdorsally(arrowhead),whileitsventrallimitremainsnormallypositioned(openarrowhead).(H)SchematicofLhx2mutantsectionatlevel“i”ofFigure1F,
showingtheextentoftheexpandedseptum(s)andhem(h).(I)AreaoccupiedbytheseptumissignificantlyincreasedinLhx2mutantbrainscomparedwiththatofcontrol
brains(*P<0.01).Scalebar:300µm.
Lhx2mutantseptumandhem,cellsexitthecellcycleprema- analyzed diencephalic and telencephalic domains adjacent to
turely,leadingtoanoverproductionofpostmitoticcells. the thalamic eminence in the Lhx2 mutant. The loss of Lhx2
does not appear to affect the position, or the extent of the
prethalamus, or the thin Lhx1/5-expressing domain adjacent
TheThalamicEminenceExpandsintheLhx2Mutant tothezonalimitansintrathalamica(Fig.5C,D;Supplementary
Thethalamiceminenceisseenincoronalsectionsastheneu- Fig.S2).However,intheLhx2mutant,theCGE,whichisnor-
roepithelium connecting the telencephalic choroid plexus to mally located adjacent to the thalamic eminence, is undetect-
the diencephalon. Postmitotic cells of the thalamic eminence able in terms of the expression of Dlx2 and its downstream
express a battery of transcription factors including Tbr1, targetArx.Whileboththesegenesdisplaynormalexpression
Lhx1, Lhx5, and Lhx9 (Bulfone et al. 1995; Sheng et al 1997; in the mutant lateral and medial ganglionic eminences (LGE
Retaux et al. 1999; Yamazaki et al. 2004; Abellan et al. 2010; and MGE; Fig. 5E,F) as well as in the prethalamus, what
Fig. 5A; Supplementary Fig. S2). Whereas these markers are remainsinthelocationoftheCGEisahighlyshrunkenDlx2-
also expressed elsewhere in the forebrain, transcription and Arx-negative structure (black asterisk, Fig. 5A–D). Thus,
factor,Mab21l2,expressionuniquelymarksthethalamicemi- the expansion of the thalamic eminence is accompanied by
nence (Wong etal. 1999;Yamadaetal.2004;Fig.5B).In the shrinkage of the adjacent CGE, suggesting a parallel with the
absence of Lhx2, each of these markers reveals the thalamic expansionofthecorticalhemandseptum.
eminence to be dramatically expanded. We compared the Anexaminationoflabelingaftera2-hBrdUpulseadminis-
area of the thalamic eminence in control and Lhx2 mutant teredatE11.5revealsasimilarlabelingindexincontrol(39%)
brains and found it to be significantly greater in the absence and mutant brains (42%; Fig. 5J), indicating that cell division
ofLhx2(P<0.005;Fig.5I). rates are unaffected in the absence of Lhx2. However, when
We examined whether the increase in the area of the thal- BrdU injection at E11.5 is followed by BrdU/Ki67 double la-
amic eminence was at the expense of the surrounding tissue beling in brains harvested 1 day later, at E12.5, the mutant
asisthecasewiththecorticalhemandseptum.These2struc- thalamic eminence is seen to have a significantly increased
tures expand at the expense of the adjacent cortical neuroe- quitfraction (62%)when compared withthe control thalamic
pithelium (Fig. 3; Bulchand et al. 2001). Therefore, we eminence (48%, Fig. 5K). This indicates that similar to the
CerebralCortexMay2014,V24N5 1365
Figure4. LossofLhx2resultsinanincreaseinCajal–Retziuscells.(A–D)ExpressionofReelin(A),Lhx1(B),Lhx5(C),andΔNp73(D)atE12.5revealsaconsiderableincrease
intheCajal–RetziuscellsaccumulatedattheseptumofLhx2mutants(asterisks)comparedwithcontrolbrains(arrowheads).(IandJ)Similarly,themantleofthemutanthem
(asterisks)alsoshowsincreasedCajal–Retziuscellmarkerexpressioncomparedwithcontrols(arrowheads).(E–H)correspondstosectionsandcellcountsfortheseptumand
(K–N),forthehem.(EandK)SectionsofE11.5controlandLhx2mutantbrains,aftera2-hBrdUpulse,processedforBrdUandDAPIstaining.Labelingindices(BrdU+cells/
totalDAPI+cells)atE11.5aresimilarforcontrolandLhx2mutantseptum(45%and47%,respectively;EandG)andhem(46%and45%,respectively;KandM).(FandL)
Sections of E12.5 Lhx2 control and mutant brains harvested 1day after BrdU administration at E11.5 and processed for BrdU–Ki67 double immunohistochemistry. The quit
fraction (BrdU+; Ki67− cells/total BrdU+ cells) is significantly greater in the Lhx2 mutant septum (57%; **P<0.005) and hem (57%; ***P<0.0005) than in the control
septum(31%)andhem(35%).Scalebars:300µm(A–D);50µm(E,F,K,L);100µm(I,J).
septum and hem, cells exit the cell cycle prematurely in the the stage at which the first neurons of the cortex are born,
Lhx2 mutant thalamic eminence as well. Interestingly, the and the septum, hem, and thalamic eminence can be ident-
expression of Reelin and ΔNp73 (Supplementary Fig. S3) is ified by marker gene expression in the mouse (Grove et al.
only modestly increased in the Lhx2 mutant thalamic emi- 1998; Zembrzycki et al. 2007; Fotaki et al. 2008; Supplemen-
nence compared with that of Tbr1 or Mab21l2 (Fig. 5A,B). taryFig.S4).
Therefore,itappearsthat,intheabsenceofLhx2,otherpost-
mitoticderivativesofthethalamiceminenceareproducedto- ForebrainHemSysteminExtantVertebrates
getherwithReelinandp73-expressingCajal–Retziuscells. The thalamic eminence and septum are thought to be evolu-
tionarily ancient structures, having been described in fish
ACriticalPeriodforLhx2FunctioninRestricting (Wullimann and Rink 2002; Wullimann and Mueller 2004),
theForebrainHemSystem amphibians(Broxetal.2002,2003),reptiles(Cabrera-Socorro
Lhx2 expression in the septum, hem, and thalamic eminence etal.2007),birds(Puellesetal.2000;Cobosetal.2001),and
is diminished by E11.0 and undetectable by E12.5 (Sup- mammals (Abbott and Jacobowitz 1999; Puelles et al. 2000).
plementaryFig.S1).Inanearlierstudy,weusedaconditional Iftheseindeed playafundamental roleindevelopment,then
knockoutlinetodemonstratethatdeletionofLhx2afterE10.5 they would be expected to be conserved in humans as well.
did not result in an expansion of the cortical hem (Mangale We examined human brains at 6 and 7 GWs and made mor-
et al. 2008). We used the same conditional knockout line phological comparisons of the components of the forebrain
crossed to CreER and administered tamoxifen at E10.5. We hem system between human and mouse (Fig. 7). The cortical
examined these conditional knockout embryos at E12.5 and hem and thalamic eminence flank the telencephalic choroid
findthatneithertheseptumnorthethalamiceminenceisex- plexus in the human embryo just as they do in the mouse
panded (Fig. 6). Therefore, the role of Lhx2 in restricting the (Fig. 7E–H). In addition to calbindin, we present a compari-
extent of the forebrain hem system ends by E10.5, which is son of 7-GW human and E12 mouse embryos using Tbr1
1366 Lhx2RestrictsForebrainHemSystem (cid:129) Royetal.
Figure 5. The thalamic eminence expands in the Lhx2 mutant. (A and B) Tbr1 and Mab21l2 expression in the mantle of the thalamic eminence in E12.5 control brains
(arrowheads)revealaprofoundexpansionofthisterritoryintheLhx2mutant(whiteasterisk).(A–D)TheLhx2mutantCGEatE12.5appearsmorphologicallyreducedandlacks
Arxexpressioninitsmantle(blackasterisk,C)andDlx2expressioninitsventricularzoneandmantle(blackasterisk,D).(EandF)ArxandDlx2expressionappearnormalinthe
LGEandMGE(EandF)andprethalamus(PTh;CandD).ArrowheadsincontrolsectionsandwhiteasterisksinLhx2mutantsectionsinA–Dindicatethethalamiceminence.(G
andJ)SectionsofE11.5controlandLhx2mutantbrains,aftera2-hBrdUpulse,processedforBrdUandDAPIstaining.Labelingindices(BrdU+cells/totalDAPI+cells)atE11.5
aresimilarinthethalamiceminenceofcontrol(39%)andLhx2mutant (42%)brains.(HandK)SectionsofE12.5Lhx2controlandmutantbrainsharvested1dayafterBrdU
administrationatE11.5;andprocessedforBrdU–Ki67doubleimmunohistochemistry.Thequitfraction(BrdU+;Ki67−cells/totalBrdU+cells)issignificantlygreaterintheLhx2
mutantthalamiceminence(62%;P<0.05)whencomparedwithcontrolbrains(48%).(I)AreaoccupiedbythethalamiceminenceissignificantlyincreasedinLhx2mutant
brainscomparedwiththatofcontrols(**P<0.005).Scalebars:300µm(A–F)and50µm(GandH).
immunostaining, Tbr1 being expressed in the postmitotic signaling molecules (Fgfs, Wnts, and Bmps) (Furuta et al.
neurons of the entire forebrain hem system in both species 1997; Grove et al. 1998; Hoshikawa et al. 1998; Kimura et al.
(Fig.7E,F).AhorizontalsectionofanE11.5mousebrainwith 2005). These structures were also previously known or pro-
a morphology similar to that of the human section is shown posed to play major roles in forebrain patterning events, and
alongside for the comparison of the location of the thalamic to produce Cajal–Retzius cells, which are implicated in
eminence and hem in the 2 species (Fig. 7G,H). The com- aspects of patterning and cell migration in the forebrain
ponents of the forebrain hem system reported in extant ver- (Meyeretal.2002;Takiguchi-Hayashi etal.2004; Bielleetal.
tebrates are summarized in Figure 7J. The cortical hem 2005; Yoshida et al. 2006; Cabrera-Socorro et al. 2007;
appearstobeanevolutionarilynewerstructure,reportedonly Abellan and Medina 2009; Abellan et al. 2010; Tissir et al.
in reptiles and mammals and being particularly prominent in 2009; Meyer 2010). We discovered a key feature that unifies
the human brain (Grove et al. 1998; Meyer et al. 2002; these structures, namely that transcription factor Lhx2 acts to
Cabrera-Socorroetal.2007;Meyer2010). limittheirextent.Weobserveall3structurestoexpandinthe
Lhx2 mutant. The major patterning disruptions occur in the
Lhx2 mutant involving the expansion of the septum, hem,
Discussion
and thalamic eminence into adjacent territories (Bulchand
In this study, we examine 3 structures located at important et al. 2001; Mangale et al. 2008; this study). This expansion
morphological boundaries in the medial forebrain, septum, appears to be due to a fate change of the adjacent neuroe-
hem, and thalamic eminence and find that they are unified pithelium: The rostral cortex in the case of the septum;
by significant common features. It was already known that the mid level cortex in the case of the hem; and the CGE
these boundary structures express specific complements of in the case of the thalamic eminence. These expanded
CerebralCortexMay2014,V24N5 1367
Figure 6. Critical period for Lhx2 to regulate the forebrain hem system ends by E10.5. Conditional deletion of Lhx2 using a tamoxifen-inducible CreER driver. Tamoxifen
administrationatE10.5(T10.5)doesnotinterferewithforebraindevelopmentincontrol(CreER;Lhx2lox/+)embryos(A–I).AprobeagainstLhx2exon2,3indicatesthatLhx2
deletionhasoccurredthroughoutthebrainofthefloxed(CreER;Lhx2lox/−)embryos(C,F,I).(A–C)TheseptuminthefloxedembryosappearsnormalasevidentfromSix3and
Fgf17 expression (bars; A and B). (D and E) Thalamic eminence expressing Mab21l2 and Tbr1 appear similar in the floxed embryos asthat in controls (asterisk). Similarly,
expressionofWnt8binthecorticalhem,medialpallium,andthalamiceminenceaswellasofWnt3ainthecorticalhemiscomparablebetweenfloxedandcontrolembryos(G
andH).Scalebar:300µm.
neuroepithelial domains also display an enhanced quit frac- expense of the CGE, which is an important source of inter-
tion, resulting in a great excess of postmitotic cells produced neuronsforthecaudalcerebralcortexandhippocampus(Nery
from the septum, hem, and thalamic eminence. We propose et al. 2002; Yozu et al. 2005). We suggest that this structure
that these structures function as a multicomponent medial mayfunctionasadiencephalichem,toregulatekeyaspectsof
organizingsystem,theforebrainhemsystem,whichregulates forebrainpatterninganddevelopment.
thedevelopmentoftheforebrain. Our new perspective unifies 3 key forebrain midline struc-
Our forebrain hem system model builds upon and extends turesandmay provideawayofunderstandingholoprosence-
theideaofthecorticalhemandtheseptumasmedialorgani- phaly, the most common congenital malformation of the
zers in the telencephalon. It is well established that the human forebrain with a prevalence of 1 in 250 conceptions
Wnt-rich cortical hem acts as an organizer for the adjacent and 1 in 16000 live births (Oliver et al. 1995; Brown et al.
cortical neuroepithelium to become hippocampus. In mosaic 1998; Wallis and Muenke 1999; Arauz et al. 2010; Paulussen
embryos consisting of Lhx2 null and wild-type cells, multiple et al. 2010; Solomon et al. 2010). Key genes that are impli-
hems are formed within the cortical neuroepithelium, and cated in holoprosencephaly are transcription factors Zic2,
ectopic hippocampi are induced adjacent to each hem Six3, and signaling molecules Fgf8 and Bmps, which are
(Mangaleetal.2008).Fgf8,expressedintheseptum,iscritical expressedinpartoralloftheforebrainhemsystem,andShh,
inregulatingcorticalarealization. An ectopicsourceof Fgf8in which is expressed in adjacent ventral regions. Six3,
the caudal telencephalon is sufficient to produce a mirror- expressed in the subpallial septum and other ventral struc-
imageduplicationofthecorticalareamap(Fukuchi-Shimogori tures, is a major locus affected in human holoprosencephaly
andGrove2001).Thethalamiceminenceisknowntosecretea (Oliveret al. 1995; Wallis and Muenke 1999). Shh is acritical
composite set of signaling molecules (Kimura et al. 2005) and ventral signaling molecule and its absence results in holopro-
is suggested to be a putative signaling centre at the DTB sencephaly with malformation/reduction of only ventral
(Abbott and Jacobowitz 1999; Kim et al. 2001). The thalamic midline structures; in contrast, the Bmpr1a/b double mutant
eminence is also the site through which the thalamocortical displays dorsal holoprosencephaly (midline interhemispheric
axons cross the diencephalon to enter the telencephalon. holoprosencephaly),includingaseverereductionofthehem,
In the Lhx2 mutant, the thalamic eminence expands at the absence of the choroid plexus, and partial fusion of the
1368 Lhx2RestrictsForebrainHemSystem (cid:129) Royetal.
Figure7. Presenceoftheforebrainhemsystemcomponentsinextantvertebrates.(A–F)Morphologicalcomparisonsbetweenstage-matchedmouseandhumanembryonic
sectionsatrostral(AandB),middle(CandD),andcaudal(EandF)levels.Tbr1immunostainingmarksthethalamiceminenceinbothE12mouseand7-GWhumanfetus.
(GandH)AhorizontalsectionofanE11.5mousebrainappearsmorphologicallycomparablewitha6-GWhumanfetalbrainsection.Bothspeciesrevealtheheminthecaudal
telencephalon(openarrowhead)andthethalamiceminence(arrowhead)atthetransitionofthecaudaltelencephalontothediencephalon.(I)Schematicofthemousebrain
sectioninA,depictingthepositionsoftheseptum(yellow),corticalhem(red),andthalamiceminence(blue).(J)Tabulationofthepresenceofthe3medialsourcesofCajal–
Retziuscellsinthedevelopingforebrainofdifferentvertebrates(Groveetal.1998;AbbottandJacobowitz1999;Puellesetal.2000;Cobosetal.2001;Broxetal.2002,2003;
Meyeretal.2002;WullimannandRink2002;WullimannandMueller2004;Cabrera-Socorroetal.2007).
hemispheres (Fernandes et al. 2007). The absence of Zic2 An interesting feature of the hem and septum is that they
results in both dorsal and ventral midline defects (Brown appear to cross-regulate each other, after they are formed.
et al. 1998). Zic2 levels appear to regulate fundamental mor- This regulation is mutually repressive in which BMPs from
phogenetic events such asthe timing of neurulation and roof the hem curtail the extent of the septum, and Fgf8 from the
plate formation (Nagai et al. 2000). Mechanistically, Zic2 is septumcurtailstheextentoftheWntexpressiondomain,that
thought to act in part via the regulation of β-catenin activity, is,thehem(Shimogorietal.2004).ButsincetheroleofLhx2
which is a major downstream mediator of Wnt signaling is complete by E10.5, itis unlikely that thesecross-repressive
(Pourebrahim et al. 2011). Our results show that Zic2 is ex- interactions might mediate the function of Lhx2. Rather, they
pressed in the entire forebrain hem system and provides a appeartoacttomaintaininternalboundarieswithinthefore-
new framework in which phenotypes associated with its brainhemsystem.HowLhx2suppressesarangeofalternative
deficiency can be interpreted. Broadly, the defects associated fates to preserve the extent of the cortex, or the forebrain at
withholoprosencephalyareduetodeficienciesinthespecifi- largeisacompellingquestionforfuturestudies.
cationof,orsignalingfromkeymidlinestructures.Incontrast, An important property of the forebrain hem system is that
the absence of Lhx2 results in an *expansion* of the same all its components are sources of the Cajal–Retzius cells
midline structures. We propose that our interpretation of the which,inturn,arethemainsourceofReelinsignalinthede-
forebrain hem system as an entity may lead to different veloping telencephalon. Each component of the forebrain
approachestoanalyzingholoprosencephalies. hem system is also connected to the choroid plexus, and at
OurfindingspositionLhx2inafundamentalroleinregulat- least one component, the hem, is thought to produce the
ing forebrain development by controlling the formation of choroid plexus as well (Louvi etal. 2007). Amechanistic link
thisorganizersystem,actingnotonlytodelimititsextent,but between Cajal–Retzius cells and the choroid plexus has been
alsotomaintainitsneuroepithelialstemcellsinaproliferative discoveredinthemouse,inwhichNgn2andHesgenesantag-
mode.InMangaleetal.(2008),weexaminedmosaicembryos onistically regulate the specification of these 2 fates, respect-
that contained Lhx2 mutant and wild-typepatches of neuroe- ively(Imayoshietal.2008).Bothhavecriticalrolesincortical
pithelium and showed that the expansion of the hem is due development: Cajal–Retzius cells being sources of Reelin
to a cell autonomous role of Lhx2. We expect a similar (D’Arcangelo et al. 1995; Ogawa et al. 1995) and choroid
cell-autonomous function for Lhx2 in both the septum and plexusbeingthesourceofthecerebrospinalfluid,whichcon-
thalamic eminence as well, with a critical period that ends at tains a complex cocktail of proteins that nourishes the brain
E10.5 by which time these structures are identifiable (Fig. 6). (Thouvenot et al. 2006; Marques et al. 2011). The forebrain
Supporting this interpretation, Lhx2 expression in the fore- hem system is therefore positioned to regulate the develop-
brain hem system drops significantly by this stage, consistent ment of the telencephalon, and potentiallyalso its expansion
with a scenario in which Lhx2 must be downregulated in inevolution.AssummarizedinFigure7J,componentsofthis
orderforthestructurestogaintheiridentity. system have been identified in several vertebrate species.
CerebralCortexMay2014,V24N5 1369
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Funding Cajal-Retzius cells and the cortical hem in lizard, mouse and
human.BrainRes.1132:59–70.
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theTataInstituteofFundamentalResearchtoS.T. Funding to 239:46–67.
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