Identification of a Pax6-dependent epidermal growth factor family signaling source at the lateral edge of the embryonic cerebral cortex.

Abstract

In an emerging model, area patterning of the mammalian cerebral cortex is regulated in part by embryonic signaling centers. Two have been identified: an anterior telencephalic source of fibroblast growth factors and the cortical hem, a medial structure expressing winglessint (WNT) and bone morphogenetic proteins. We describe a third signaling source, positioned as a mirror image of the cortical hem, along the lateral margin of the cortical primordium. The cortical antihem is identified by gene expression for three epidermal growth factor (EGF) family members, Tgf(alpha), Neuregulin 1, and Neuregulin 3, as well as two other signaling molecules, Fgf7 and the secreted WNT antagonist Sfrp2. We find that the antihem is lost in mice homozygous for the Small eye (Pax6) mutation and suggest the loss of EGF signaling at least partially explains defects in cortical patterning and cell migration in Small eye mice.

Full text

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BriefCommunication
IdentificationofaPax6-DependentEpidermalGrowth
FactorFamilySignalingSourceattheLateralEdgeofthe
EmbryonicCerebralCortex
StavroulaAssimacopoulos,ElizabethA.Grove,andCliftonW.Ragsdale
DepartmentofNeurobiology,Pharmacology,andPhysiology,TheUniversityofChicago,Chicago,Illinois60637
Inanemergingmodel,areapatterningofthemammaliancerebralcortexisregulatedinpartbyembryonicsignalingcenters.Twohave
beenidentified:ananteriortelencephalicsourceoffibroblastgrowthfactorsandthecorticalhem,amedialstructureexpressingwingless-
int(WNT)andbonemorphogeneticproteins.Wedescribeathirdsignalingsource,positionedasamirrorimageofthecorticalhem,along
the lateral margin of the cortical primordium. The cortical antihem is identified by gene expression for three epidermal growth factor
(EGF) family members, Tgf?, Neuregulin 1, and Neuregulin 3, as well as two other signaling molecules, Fgf7 and the secreted WNT
antagonist Sfrp2. We find that the antihem is lost in mice homozygous for the Small eye (Pax6) mutation and suggest the loss of EGF
signalingatleastpartiallyexplainsdefectsincorticalpatterningandcellmigrationinSmalleyemice.
Keywords:corticalpatterning;Tgf?;Neuregulin;Sfrp2;Fgf7;Smalleye;Pax6;Emx2
Introduction
Recent studies indicate that regional specification and growth
control of the cerebral cortex is initiated by signaling centers
operating on an originally homogeneous embryonic field
(Fukuchi-ShimogoriandGrove,2001;RagsdaleandGrove,2001;
Hebertetal.,2002;Gareletal.,2003).Anteroposteriorpositionis
conferred by an anterior fibroblast growth factor (FGF) source
(Fukuchi-ShimogoriandGrove,2001),andmediolateralpattern
is regulated by the cortical hem, a medial signaling center en-
richedinbonemorphogeneticproteins(BMPs)andwingless-int
(WNT)proteins(Groveetal.,1998;Leeetal.,2000;Ragsdaleand
Grove, 2001; Hebert et al., 2002). Given the size and complexity
ofthefinalcorticalareamap,however,additionalcorticalsignal-
ing sources are almost certain to exist.
We hypothesized that the lateral edge of the cortical primor-
dium,wherethedorsalandventraltelencephalonmeet,mightbe
thesiteofanadditionalsignalingcenter.Thelateralcorticalmar-
ginisreadilyidentifiedbygeneexpressionforaWNTantagonist,
thesecretedfrizzled-relatedproteinSfrp2(seeFig.1)(Ragsdaleet
al., 2000; Kim et al., 2001). Sfrp2 expression is detected in this
territoryasearlyasembryonicday10.5(E10.5)(datanotshown)
and, by E12.5, describes a curve that laterally mirrors the medial
corticalhem,itselfidentifiablebytheexpressionofmultipleWnt
genes (see Fig. 1A). Together, these territories mark the lateral
and medial limits of the cortical ventricular zone (VZ) (see Fig.
1C,D). Two-color in situ hybridization experiments suggest a
pincerarrangementbetweenthehemandtheSfrp2-richantihem;
the two territories are well separated in rostral telencephalon but
approach one another to meet in the caudal telencephalon (see
Fig. 1C).
Iftheantihemisanimportantembryonicsignalingcenter,itis
expected from studies of other cortical signaling centers to ex-
press multiple members of at least one secreted signaling mole-
culefamily.Twolinesofresearchpromptedasurveyofepidermal
growth factor (EGF) family members. First, a classic series of in
vitro experiments implicates EGF family members in the devel-
opment of cerebral cortical areas linked to the limbic system
(Ferri and Levitt, 1993; Levitt et al., 1997). The limbic system-
associated membrane protein LAMP is expressed in limbic cor-
tical areas (Levitt et al., 1997) and is upregulated in cells from
non-limbic cortical domains in response to EGF family ligands
(Ferri and Levitt, 1995). Second, EGF receptor-mediated signal-
ing controls dorsoventral neuronal specification in the develop-
ment of the Drosophila ventral nerve cord (Skeath, 1998; von
Ohlen and Doe, 2000). Two EGF ligands are involved in this
dorsoventral signaling: Spitz, a Tgf?-like molecule, and Vein,
which is similar in structure to the Neuregulin proteins, a sub-
family of vertebrate EGF ligands (Golembo et al., 1999).
MaterialsandMethods
Gene expression patterns were studied in CD-1 mice, Small eye (Sey, or
Pax6Sey-Neu) mice maintained on a C3H/He background, and Emx2-
targeted mice maintained on a C57BL/6 background. The day of plug
discovery was designated E0.5. In situ hybridization of E9.5–E18.5 fore-
brains and genotyping of the Sey and Emx2 mice were performed as
described previously (Pellegrini et al., 1996; Xu et al., 1997; Grove et al.,
1998).AntihemmarkergeneswerestudiedwithmousecDNAsforAreg/
ReceivedDec.12,2002;revisedMay7,2003;acceptedMay16,2003.
This work was supported by a research grant from the National Alliance for Research on Schizophrenia and
Depression(E.A.G.)andbygrantsfromtheNationalInstitutesofHealth(E.A.G.,C.W.R.).WethankD.Anderson,E.
Boncinelli, S. Dey, P. Godowski (Genentech, South San Francisco, CA), D. Lee, J. Nathans, R. Nusse, J. Pascall, J.
Rubenstein,andD.Taylor(Bristol-MyersSquibb,Princeton,NJ)forgiftsofplasmidDNA,P.GrussandD.O’Learyfor
theEmx2mutantmice,R.MaasfortheSeymice,andAnnaMaeGreenleeandEunPaikfortechnicalassistance.We
thankDanGeschwindandAngelikiLouviforvaluablediscussions.
CorrespondenceshouldbeaddressedtoCliftonW.Ragsdale,DepartmentofNeurobiology,Pharmacology,and
Physiology,TheUniversityofChicago,947East58thStreet,Chicago,IL60637.E-mail:c-ragsdale@uchicago.edu.
Copyright©2003SocietyforNeuroscience 0270-6474/03/236399-05$15.00/0
TheJournalofNeuroscience,July23,2003 • 23(16):6399–6403 • 6399

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Amphiregulin (Das et al., 1995), Egf (Pascall and Brown, 1988), Hegfl/
Heparin-binding EGF-like growth factor/Diphtheria toxin receptor (IM-
AGE Consortium, GenBank accession number W80035), Fgf7/
Keratinocyte growth factor (IMAGE Consortium, GenBank accession
number BF159111), Nrg1 (IMAGE Consortium, GenBank accession
number AI197081), Nrg2 (IMAGE Consortium, GenBank accession
number AW476657), Nrg3 (Zhang et al., 1997), Nrg4 (IMAGE Consor-
tium, GenBank accession number AA238077), Sfrp2 (Rattner et al.,
1997), Tgfa/Tgf? (Vaughan et al., 1992; Kornblum et al., 1997), Tmeff1/
Transmembrane protein with EGF-like and two follistatin-like domains 1
(IMAGE Consortium, GenBank accession number BF147745) and
Tmeff2/Tomoregulin (IMAGE Consortium, GenBank accession number
AI098476), and a rat cDNA for Ereg/Epiregulin (Taylor et al., 1999).
Results
We examined mRNA expression of 11 EGF family members: Egf
itself, Tgf?, Neuregulins 1–4, Amphiregulin, Epiregulin,
Heparin-binding EGF-like growth factor, Tmeff1, and Tmeff2.
Our screen disclosed that the lateral margin of the cortical pri-
mordium is enriched in expression of three Spitz and Vein ho-
mologs, Tgf?, Neuregulin1 (Nrg1), and Nrg3. At E12.5, Tgf? ex-
pression marks the antihem, which appears as a curve of strong
Tgf? expression when viewed from the lateral face of the cortical
primordium (Figs. 1,2A). Coronal sections show that Tgf? ex-
pressionisintheVZofproliferatingcells(Fig.3).Nrg1andNrg3
showoverlappinghighexpressioninthesamecurvingband(Fig.
2B,C). All three genes display some graded expression in the
corticalVZ,whichismostpronouncedinNrg3preparations.For
both Nrg genes, expression in the cortical primordium increased
as development proceeded (last age examined, E18.5). For all
three genes, however, the cortical antihem is the peak of expres-
sion in the cortical primordium.
Other EGF ligands, including Egf itself, were at least weakly
detectedintheembryonictelencephalon(Figs.2E,3K,L)(Korn-
blum et al., 1997) but not concentrated in the antihem. At E10.5,
E12.5, and E14.5, Amphiregulin, Egf, Epiregulin, Hegf1, Nrg2,
Nrg4, Tmeff1, and Tmeff2 are expressed in the ventral telenceph-
alon, usually increasing in intensity with age. Most EGF family
members are expressed in the dorsal telencephalon from E12.5
onward, with the exception of Nrg2, which is detectable at E10.5.
No striking patterns of expression were detected except for Epi-
regulin and Tmeff1, which display expression gradients in far lat-
eral and medial embryonic cortex.
In a screen of Fgf gene expression in and near the cortical
primordium, we found that Fgf7 gene expression marks the an-
tihembyE13.5(Fig.2D).TransientembryonicexpressionofFgf7
was noted previously in a lateral embryonic cortical region (Ma-
sonetal.,1994).Thus,togetherwithSfrp2,fivesecretedsignaling
molecules are expressed along the lateral edge of the cortical
primordium.
The cortical hem is part of the true cortical primordium as
characterized by progenitor cell behavior and gene expression
(Grove et al., 1998). Tgf? expression at the antihem also lies
within cortical primordium, defined by expression of Neuroge-
nin2 (Ngn2) (Fig. 3A,B; two-color in situ data not shown). Sfrp2
expression is within the Tgf? domain and marks the extreme
margin of the cortical primordium (Fig. 3G; Ngn2/Sfrp2 two-
color in situ data not shown). Fgf7 expression, which is strongest
in the posterior antihem, overlaps that of Sfrp2 (Fig. 3J). In con-
trast, expression of Nrg1 and Nrg3 is not restricted to the cortical
VZbutstretchesintotheVZofthebasalforebrain(Fig.3C,H,I).
Figure1.
cessedwithone-ortwo-colorinsituhybridizationandviewedfromthemedial(A)orlateral(B,
C)faces.Anteriortotheleft.D,CoronalsectionthroughE12.5cerebralhemisphereprocessed
with two-color in situ hybridization. A, B, The “swoosh” of the medial cortical hem (A, blue
arrow)ismirroredbythelateralantihem(B,redarrow).Together,theyformapincerarrange-
mentaroundthecorticalprimordium(C).ThecorticalhemismarkedbystrongWnt3aexpres-
sion(purplein A,C,D),andtheantihemisidentifiedbyexpressionofSfrp2(purpleinB;reddish-
browninC,D).Arrowhead(C)indicatesmeetingofthehemandantiheminthecaudalcerebral
hemisphere.
Locationofthecorticalhemandantihem.A–C,E12.5cerebralhemispheres,pro-
Figure2.
bralhemispheresviewedfromthelateralface;anteriortotheleft(A–C,E,E12.5;D,E13.5).F,
E18.5hemisphereviewedfromtheinsidelookinglaterally.A–C,PeaksofexpressionofTgf?,
Nrg1,andNrg3markthecurvinglongitudinallateralbandoftheantihem(arrowsinA–C).Tgf?
expressionismaintainedforseveraldaysinthisposition(arrow,F).D,Fgf7isalsoexpressedin
theantihematE13.5.E,ThefoundingmemberoftheEGFfamily,Egfitself,isexpressedinthe
ventraltelencephalonandinthecorticalprimordiumwithoutapeakofexpressionattheanti-
hem.Asterisksmarkexpressioninventraltelencephalon(C,E,F).
EGFfamilymembersareexpressedinthecorticalantihem.A–E,Embryoniccere-
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The transcription factor genes Emx1 and Dlx2 are differen-
tiallyexpressedindorsalandventraltelencephalon.AtE12.5,the
boundariesofexpressionofEmx1andDlx2leaveanintermediate
wedge clear of expression of either gene in the VZ of the lateral
margin of the cortical primordium (Fig. 3E) (Fernandez et al.,
1998). Two-color in situ hybridization shows that this wedge-
shapedzoneisantihemterritory:itisfilledbydenseexpressionof
Tgf?, and Sfrp2 expression marks its ventral limit (Fig. 3D–G).
Although the cells that compose the antihem do not express
Emx1atE12.5,theyappeartodosoatalaterstageofdevelopment
(Fernandez et al., 1998). Genetic fate mapping of Emx1-
expressing cells suggests that cells in this region give rise to por-
tions of the amygdala and lateral cortex (Gorski et al., 2002).
The antihem shows both similarities and differences with
other cortical signaling sources. For example, expression of EGF
family genes are not detectable in the antihem at E10.5, an age at
which Fgf, Wnt, and Bmp expression is prominent at other corti-
cal signaling sources. Nrg1 and Tgf? are only barely detectable in
the lateral cortical primordium at E11.5. Moreover, EGF gene
expression does not overlap as neatly as the expression of multi-
ple Wnt genes in the cortical hem or even of Fgf genes at the
anterior pole. Particularly for the Nrg genes, mRNA expression
extendsasagradientfromtheantihemintothemoredorsolateral
corticalVZ.Nonetheless,theantihemissimilartoothersignaling
sources in representing a peak of expression of several members
of a single signaling molecule family.
TheSmalleye(Sey)homozygotemutantmouselacksfunction
of the transcription factor Pax6 and displays impaired cortical
neurogenesis, cell migration, and patterning (Chapouton et al.,
1999;Bishopetal.,2000;Stoykovaetal.,2000),aswellaslocalized
defects in the antihem region, losing Sfrp2 expression (Wawersik
etal.,1999;Ragsdaleetal.,2000;Kimetal.,2001).Tgf?andNrg1
expressionisalsolostintheantihem(Fig.4A,B,E–H),suggesting
a complete absence of this signaling center in the homozygote
mutant.Becauseofapossiblegeneraldevelopmentaldelayinthe
mutant, these mice and littermate controls were analyzed for
gene expression from E12.5 to E16.5 for Sfrp2 (n ? 9 homozy-
gotes; n ? 18 controls), Nrg1 (n ? 6 homozygotes; n ? 10 con-
trols),andTgf?(n?12homozygotes;n?21controls).AtE14.5,
Tgf? expression marks the antihem in controls but is eliminated
in homozygote mutants (Fig. 4A,B,E,F). Expression of Nrg1 in
the lateral cortical VZ is almost undetectable in the homozygote
Sey mouse, with the result that the gradient of expression is re-
versed, medial to lateral, with highest Nrg1 expression in the
hippocampal primordium (Fig. 4H, asterisk). Dense expression
ofNrg1appearsattheantihemregioninlittermatemiceatE14.5
(Fig. 4E), with a stronger gradient in the rest of the cortical pri-
mordium than in CD-1 mice (compare with Fig. 2B).
Mice lacking the transcription factor Emx2 also show wide-
spread defects in neurogenesis and patterning (Bishop et al.,
2000; Mallamaci et al., 2000) with concomitant defects in signal-
ing sources (Muzio et al., 2002). WNT signaling in the cortical
hem region is affected, as is anteroposterior gene expression of
the FGF receptor Fgfr3 (Muzio et al., 2002), suggesting that sig-
naling along both mediolateral and anteroposterior axes is ab-
normal.Wefoundthattheantihem,markedbyTgf?expression,
is retained in the Emx2 homozygote mutant but appears dorsally
displaced (Fig. 4C,D; data not shown).
Discussion
Several roles can be hypothesized for the cortical antihem. A
likelypossibilityisthattheantihemservesasabarrierbetweenthe
dorsal and ventral telencephalon, in both pattern formation and
cell migration. Localized Sfrp2 may limit the spread of WNT
signaling between dorsal and ventral telencephalon (Ragsdale et
al.,2000;Kimetal.,2001).Morebroadly,bothattheantihemand
within the cortical primordium, EGFs may antagonize BMP and
WNTsignaling.GeneticstudiesofDrosophiladevelopmentshow
that EGF receptor-mediated signaling attenuates both Decapen-
taplegic (BMP) and Wingless (WNT) signals (O’Keefe et al.,
1997;Szutsetal.,1997;Kubotaetal.,2000).Thus,thelateralEGF
sourcemayassistinmaintainingadistinctionbetweenthedorsal
and ventral telencephalon and in regional patterning of the cere-
bral cortex. The latter role is supported by the ability of Tgf? to
convert nonlimbic to limbic cortex in vitro (Ferri and Levitt,
1995).
EGFs may also regulate cortical cell migration. In rodents, a
large proportion of cortical interneurons derived from the ven-
traltelencephalonmigratepasttheregionoftheantihemtopop-
ulatethecorticalprimordium(Andersonetal.,2001).Duringfly
Figure 3.
telencephalon.A–L,CoronalsectionsthroughE12.5andE13.5(J)cerebralhemispherespro-
cessedforone-ortwo-colorinsituhybridization.A,Ngn2expressionmarkstheboundariesof
corticalneuroepithelium.B,TheTgf?-expressingdomainlieswithinthecorticalprimordium
(compareA,B).C,Nrg1expressionpeaksintheTgf?-richdomainbutextendsasadecreasing
gradientintodorsolateralcorticalneuroepithelium,withanadditionalzoneofexpressioninthe
medialcorticalprimordium.D–F,AtE12.5,awedge-shapedterritorylacksexpressionofEmx1
or Dlx2 (between arrows). This territory is filled by Tgf? expression (D), with Sfrp2 and Fgf7
expressionnestedintheTgf?domain(F,G,J).Tgf?,Fgf7,andSfrp2expressionremainwithin
corticalneuroepitheliumatthissite(D,F,J),butNrg1andNrg3extendintotheVZofthebasal
telencephalon(H,I).Asterisks(G–J)marktheSfrp2-expressingzone,themostlateralmargin
of the cortical neuroepithelium. K, L, At E12.5, Egf (K) and Tmeff1 (L), like other EGF family
members,donotshowgeneexpressionpeaksinthecorticalantihem.Arrow(L)notesincreased
Tmeff1expressioninmedialembryoniccortex,asseeninNrg1-labeledsections(C).
Location of the antihem relative to the transition between dorsal and ventral
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oogenesis, the Tgf?-like ligand Gurken guides dorsal migration
ofbordercells(DuchekandRorth,2001).Bothoverexpressionof
EGF family ligands and misexpression of a constitutively acti-
vated form of the EGF receptor inhibit border cell migration
(Duchek and Rorth, 2001). Therefore, by analogy with fly devel-
opmentalmechanisms,theantihemregioninmouse,richinEGF
family members, could promote migration of the correct ventral
telencephalic cells or inhibit the migration of incorrect cells. In
retroviral studies of rodent telencephalic development, Caric et
al. (2001) have found that increasing the EGF receptor levels in
VZcellspromotestheirradialmigrationawayfromtheVZ.Thus,
neurons born in the antihem region, which are thought to mi-
gratetowardventrolateralcorticalareas(Bayeretal.,1991),could
be guided in part by the EGF ligands of the antihem.
Eachoftheabovehypothesesreceivessupportfromanalysisof
the Sey/Sey mutant mouse, which lacks functional Pax6 and also
appears to lack an antihem. First, patterning defects in the pre-
sumptive area map are seen in Sey/Sey cerebral cortex just before
birth,withshrinkageoflateralandrostralcorticaldomains(Bish-
op et al., 2000). Second, gene expression patterns normally con-
finedtotheventralordorsaltelencephalontransgresstheirusual
boundariesintheSey/Seymutant;thus,thedorsoventralidentity
oftheregionaroundtheantihembecomesambiguous(Stoykova
etal.,2000;Toressonetal.,2000;Yunetal.,2001).Third,ventral
to dorsal cell migration is enhanced in the mutant, suggesting
that incorrect cells are crossing the ventrodorsal telencephalic
boundary and that Pax6 is essential to allow the correct cells to
immigrate (Chapouton et al., 1999). We propose that Pax6 reg-
ulates development of the antihem and associated EGF ligand
gradients and that the patterning and migration defects in the
Sey/Sey mutant are at least in part mediated by the loss of the
antihem.Finally,micethatlackEmx2showasubtler,butconsis-
tent, abnormality in this region. The antihem is present but
shifted dorsally (this report), and LAMP expression, marking
limbic cortex, is dorsally displaced in parallel (Mallamaci et al.,
2000).
The antihem differs from other signaling centers by express-
ing signaling molecules later than other centers and in a more
graded manner. However, these features may be significant to its
potential cortical patterning function. As the cortical primor-
dium grows larger, it becomes more difficult to explain how pat-
terning could occur according to the classic model of a morpho-
gen diffusing over an embryonic field with a width of 0.5 mm or
less (Wolpert, 1969; Gurdon et al., 1994). Yet, cortical pattern
remains labile relatively late in corticogenesis, when the embry-
onic cortex is larger than a typical embryonic field (Ragsdale and
Grove,2001).Onewaytoprolongcorticalpatterningwouldbeto
introducesecondarysignalingsourcesthatgenerateagradientof
signalingproteindirectly.Thus,agradientofEGFmRNAexpres-
sion with a peak at the antihem may directly set up a patterning
gradient of EGF proteins in the older and larger cortical primor-
dium without a need for long-distance protein diffusion.
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