EphA7-ephrin-A5 signaling in mouse somatosensory cortex: Developmental restriction of molecular...

EphA7-ephrin-A5 Signaling in MouseSomatosensory Cortex: DevelopmentalRestriction of Molecular Domains andPostnatal Maintenance of Functional

Compartments

KATHERINE MILLER, SHARON M. KOLK, AND MARIA J. DONOGHUE*

Department of Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06520

ABSTRACTMembers of the Eph family of receptor tyrosine kinases and their ligands, the ephrins, are

expressed in distinct patterns in the forming cortex. EphA7 is expressed early in cortical devel-opment, becoming concentrated in anterior and posterior domains, whereas ephrin-A5 is ex-pressed later in corticogenesis, highest in the middle region that has low levels of EphA7. TheEphA7 gene produces full-length and truncated isoforms, which are repulsive and adhesive,respectively. Analysis of cortical RNA expression demonstrates that proportions of these isoformschange with time, from a more repulsive mix during embryogenesis to a more permissive mixpostnatally. To examine how EphA7 and ephrin-A5 influence the formation of cortical regions,EphA7–/– mice were analyzed. Within the cortex of EphA7–/– mice, the distribution of ephrin-A5was more extensive, encompassing its usual medial domain but also extending more posteriorlytoward the occipital pole. Moreover, relative levels of ephrin-A5 along the cortex’s anatomicalaxes changed in EphA7–/– animals, creating less striking shifts in ligand abundance. Further-more, in vivo functional studies revealed that EphA7 exerts a repulsive influence on ephrin-A5-expressing cells during corticogenesis. In contrast, EphA7 appears to mediate permissive inter-actions in the postnatal cortex: the area of somatosensory cortex was significantly reduced inEphA7–/– mice. A similar reduction was present in ephrin-A5–/– animals and a more pronounceddecrease was observed in EphA7/ephrin-A5–/– cortex. Taken together, this study supports a rolefor EphA7 and ephrin-A5 in the establishment and maintenance of certain cortical domains andsuggests that the nature of their interactions changes with cortical maturity. J. Comp. Neurol.496:627–642, 2006. © 2006 Wiley-Liss, Inc.

Indexing terms: cortical areas; corticogenesis; barrel cortex; eph/ephrin

The mature cerebral cortex is divided into functionallydedicated, stereotypically connected, and cytoarchitectoni-cally distinct areas (Brodmann, 1909; Creutzfeldt, 1977).The proper delineation of these areas is a prerequisite forthe rapid, precise, and integrated responses of this neuralstructure (Goldman-Rakic, 1988). Understanding how dis-tinct cortical areas form during development has been agoal of neurobiological research for decades (O’Leary,1989; Rakic, 1988). Genetic determination of cortical cellidentity is an important factor in the formation of corticalcompartments; transcription factors, growth factors, celladhesion molecules, and intercellular signaling moleculesimpact cellular identity (Miyashita-Lin et al., 1999; Na-kagawa et al., 1999; Donoghue and Rakic, 1999; Bishop etal., 2000; Fukuchi-Shimogori and Grove, 2003). Subse-quently, the formation of reciprocal connections betweenthe cortex and thalamus influences the definition andrefinement of cortical areas (Roe et al., 1990; Schlaggarand O’Leary, 1991; Herrmann and Shatz, 1995; Sharma et

al., 2000). The relative contributions of intrinsic programsand extrinsic forces to the designation of cortical areas

Grant sponsor: National Institutes of Health; Grant number: R01-NS39979 (to M.J.D.); Grant sponsor: Netherlands Organization for Scien-tific Research (TALENT grant to S.M.K.).

Current address for Katherine J. Miller: Department of Molecular, Cel-lular, and Developmental Biology, Yale University, 266 Whitney Ave.,KBT-244, New Haven, CT 06511.

Sharon M. Kolk’s current address is Department of Pharmacology &Anatomy, Subsection Neurodevelopment, Rudolf Magnus Institute of Neu-roscience, University of Utrecht, UMC, Universiteitsweg 100, 3584 CGUtrecht, The Netherlands.

*Correspondence to: Maria Donoghue, 333 Cedar St., SHM/B301, NewHaven, CT 06520. E-mail: [email protected].

Received 12 August 2005; Revised 19 October 2005; Accepted 30 Novem-ber 2005

DOI 10.1002/cne.20926Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 496:627–642 (2006)

© 2006 WILEY-LISS, INC.

remain under debate (Pallas, 2001; Sur and Leamey,2001) and the molecular bases of each process are still notfully understood.

The EphA family of cell surface bound receptor tyrosinekinases (RTKs) are patterned within the forming cerebralcortex, marking presumptive cortical domains (Donoghueand Rakic, 1999a; Sestan et al., 2001; Yun et al., 2003).Interaction of Eph RTKs and their ligands, the ephrins, onneighboring cells results in both forward signaling withinthe receptor-expressing cell and reverse signaling withinthe ligand-containing cell (Friedman and O’Leary, 1996;Flanagan and Vanderhaeghen, 1998; Kullander et al.,2001; Davy et al., 2004). Engagement of Eph-ephrin sig-naling often results in negative cellular outcomes: retrac-tion of cell processes or movement of cells away from oneanother (Gale and Yancopoulos, 1997; Flanagan andVanderhaeghen, 1998; Xu et al., 2000). However, Eph–ephrin interactions can also result in positive, sustainedinteractions between participating cells (Castellani et al.,1998; Holmberg et al., 2000; Knoll, 2001; Hansen et al.,2004). Within the nervous system, Eph–ephrin signalingcan influence the migration, placement, and physiologicalproperties of cells (Ferri and Levitt, 1993; Torres et al.,1998; Conover et al., 2000; Dalva et al., 2000; Xu et al.,2000). Select subsets of EphA genes are expressed in cor-tical domains, both when areas are emerging in develop-ment and also when they are well defined postnatally(Mackarehtschian et al., 1999; Yun et al., 2003). Pertur-bation of EphA-mediated signaling in the forming cortexcan cause altered functional topography and connectivity(Zhang et al., 1996; Gao et al., 1998; Vanderhaeghen et al.,2000; Dufour et al., 2003).

We focus on EphA7 and ephrin-A5 in this study becauseof their patterned and reciprocal expression in the formingand postnatal cortex. EphA7 is expressed early in corticaldevelopment (embryonic day (E)12) and is patterned alongthe anteroposterior (AP) axis of the cortex. The EphA7gene produces two major isoforms, a full-length (FL) and atruncated (TR) form, which are coexpressed during thedevelopment of the nervous system (Maisonpierre et al.,1993; Ciossek et al., 1995, 1999; Rogers et al., 1999). Invitro and in vivo analyses demonstrated that binding ofephrin-A5 to the truncated EphA7 receptor causes cellularadhesion, while binding of ephrin-A5 to the full-lengthEphA7 receptor results in cellular repulsion (Holmberg etal., 2000). Expression of ephrin-A5 is activated later incorticogenesis (E15) in the domain with low levels ofEphA7 (Yun et al., 2003). We postulated that EphA7 in-fluences the parcellation of the cortex, perhaps by regu-lating the expression pattern of ephrin-A5. To test thishypothesis we examined the cerebral cortex of EphA7–/–,ephrin-A5–/–, and EphA7/ephrin-A5–/– mice. Our resultssuggest that within the cerebral cortex EphA7-mediatedsignaling defines certain areas as they form and main-tains functional domains in postnatal life.

MATERIALS AND METHODS

Maintenance of mutant mice

Yale animal protocol #2005-10098 guided our treatmentof mice in this study. All of the mice examined in thisstudy had been extensively bred into the C57BL/6 inbredbackground within our colony. To produce inbred lines,adult C57BL/6 mice were obtained from Charles River

Laboratories and these mice were backcrossed with mu-tant animals. Data presented are derived from mice thatcorresponded to generations 7–15 of these inbreedingschemes. In most cases, comparisons between mutant andcontrol mice were performed using littermates.

EphA7 mutant mice were produced by U. Drescher(King’s College, London, UK) and T. Ciossek (AltanaPharma, Konstanz, Germany). The stem cells used to cre-ate the targeted mutation were of the R1 line, derivedfrom the 129 strain. All generations of mice resulting fromthe original C57BL/6 chimera were maintained in aC57BL/6 background via backcrossing. Details of the ge-netic mutation have been thoroughly described elsewhere(Depaepe et al., 2005; Rashid et al., 2005). EphA7 mutantmice were maintained in the Yale Animal Research Cen-ter on an inbred background (C57BL/6). Wildtype (WT),heterozygous (EphA7�/–), and mutant (EphA7–/–) ani-mals were born in the expected ratios. For example,EphA7�/– � EphA7�/– breedings produced 354 mice, ofwhich 30% were WT, 43% were EphA7�/–, and 27% wereEphA7–/–. Similarly, EphA7�/– � EphA7–/– breedingsproduced 102 mice, of which 51% were EphA7�/– and49% were EphA7–/–. EphA7�/– and EphA7–/– mice sur-vived into adulthood and were capable of breeding. Incontrast with a recent report, we find no prenatal le-thality associated to the EphA7–/– genotype (Depaepeet al., 2005).

Ephrin-A5 mutant mice were obtained from J. Frisen(Karolinska Institute, Stockholm, Sweden). Again, thestem cells used to create the targeted mutation were of theR1 line, derived from the 129 strain, and all generations ofmice resulting from the original C57BL/6 chimera weremaintained in a C57BL/6 background via backcrossing.Details of the genetic mutation in these mice have beendescribed previously (Feldheim et al., 1998; Frisen et al.,1998). Ephrin-A5 mutant mice were maintained as ho-mozygotes and were genotyped as previously described(Frisen et al., 1998).

EphA7/ephrin-A5 double knockout mice were generatedfrom breedings of EphA7–/–/ephrin-A5�/–, animals, againin a C57BL/6 background, resulting in �25% EphA7–/–/ephrin-A5�/�, 50% EphA7–/–/ephrin-A5�/–, and 25%EphA7–/–/ephrin-A5–/–. All genotypes survived and brednormally.

In situ hybridization

In situ hybridization was performed as previously de-scribed (Donoghue et al., 1996; Donoghue and Rakic,1999a). Antisense probes corresponding to the EphA fam-ily members expressed within the cortex (EphA3, EphA4,EphA5, EphA6, EphA7, and ephrin-A5) and to unrelatedgenes that are patterned within the embryonic cortex [Id2,RZR-�, Cadherin-8 (Cad8), and Tbrain-1 (Tbr1)] wereexamined. None of the probes used contain motifs capableof cross-reacting with other genes under the conditions ofour experiments. The name of each probe, the nucleotide(nt) coordinates within the mouse gene that were used,and the reference(s) demonstrating specificity follow:EphA3, nt 49–406 (Brown et al., 2000; Feng et al., 2000;Kudo et al., 2005); EphA4, nt 1–1014 (Helmbacher et al.,2000); EphA5 nt 575–1549 (Zhou, 1997); EphA6, nt 610–1608 (Maisonpierre et al., 1993); EphA7, nt 191–1204;ephrin-A5, nt 865–1548 (Mackarehtschian et al., 1999;Fukuchi-Shimogori and Grove, 2001; Fukuchi-Shimogoriand Grove, 2003); Id-2, nt 153–566 (Miyashita-Lin et al.,

The Journal of Comparative Neurology. DOI 10.1002/cne

628 K.J. MILLER ET AL.

1999; Fukuchi-Shimogori and Grove, 2001); RZR-�, nt745–1249 (Miyashita-Lin et al., 1999; Mackarehtschianet al., 1999; Fukuchi-Shimogori and Grove, 2001); Cad8,nt 2–400 (Miyashita-Lin et al., 1999; Fukuchi-Shimogoriand Grove, 2001); Tbr1, nt 157–625 (Bulfone et al., 1995;Miyashita-Lin et al., 1999). Following exposure to film,slides were dipped in NTB2 nuclear track emulsion(Kodak, Rochester, NY), exposed for �1 month at 4°C,developed, counterstained with Nissl, and coverslipped.Images were obtained from either the autoradiographicfilms or the developed slides.

RNase protection assay

Total RNA was isolated from control and EphA7–/–cortex. RNase protection assays were performed using theMulti-Probe RNase Protection Assay System (BD Bio-sciences, San Jose, CA). cRNA probes, previously shown tobe selective for these gene products (Ciossek et al., 1995;Donoghue et al., 1996), which corresponded to either theextracellular region of mouse ephrin-A5 (nt 374–679) or aregion surrounding the transmembrane region of EphA7(nt 1740–2100), were transcribed from polymerase chainreaction (PCR) fragments containing a linked T7 RNApolymerase promoter. These probes were specific for theintended gene products, as demonstrated by examinationof mutant animals (Fig. 3A and data not shown). In vitrotranscribed products were purified on a Qiagen (Chats-worth, CA) RNA purification kit. Then 10–25 �g of eachRNA sample was hybridized with 4 � 105 counts of eachprobe and incubated at 56°C overnight. The hybridiza-tions were subjected to RNase A � T1 digestion, followedby inactivation of the RNases, phenol extraction, ethanolprecipitation, and electrophoresis on denaturing acryl-amide gels. After drying, the gels were exposed to autora-diographic film with intensifying screens and stored at–80°C. Films were developed following 24–96-hour expo-sures. Gel images were obtained using Adobe Photoshop(San Jose, CA) and analyzed using ImageJ (NIH, Be-thesda, MD) analysis software. For EphA7, each isoformwas presented as a proportion of the total amount ofEphA7 message. The number of samples quantified was:ephrin-A5 in WT (n � 5) and EphA7–/– (n � 4), EphA7 atE13 (n � 4), E14 (n � 3), E15 (n � 2), E16 (n � 4), E18 (n �4), postnatal day (P)0 (n � 4), P2 (n � 4), P5 (n � 4).Statistical analysis was performed and standard error isindicated.

Morphological and histological analyses

To evaluate the general morphology of control andEphA7–/– brains, mice were sacrificed at E15 or P0, theirbrains were fixed, and gross external structure of thebrains was analyzed. Tissue was also sectioned in thecoronal or parasagittal plane and Nissl stained. Briefly,paraffin sections of fixed tissue were incubated in 0.1%cresyl violet in 0.1 M acetate, pH 4.5, for 5 minutes, rinsedin water, passed through 70% EtOH, and then differenti-ated in 95% EtOH containing one to two drops of glacialacetic acid until the proper staining was achieved. Thetissue was then dehydrated, cleared in xylenes, andmounted in Permount.

Quantification of histological analysis

The anteroposterior (AP) cortical lengths at P0 (n � 7for control and n � 9 for EphA7–/–) and P7 (n � 7 forcontrol and n � 6 for EphA7–/–) and the thickness of the

cerebral wall at the AP midpoint at P0 (n � 4 for controland n � 5 for EphA7–/–) and P7 (n � 6 for control and n �6 for EphA7–/–) were measured on parasagittal sectionsusing ImageJ software. Mean values were calculated foreach animal and averaged within genotype. Statisticalsignificance was possessed by ANOVA (� � 5%) and ex-pressed as mean � SEM.

Immunohistochemistry

E18.5 embryos (n � 3 control and n � 3 EphA7–/–) wereharvested. The brains of these embryos were fixed in 4%paraformaldehyde in phosphate-buffered saline (PBS, pH7.4) for 1 hour at 4°C, cryoprotected in 30% sucrose/PBSpH 7.4 overnight at 4°C, frozen, and sectioned sagittally at15 �m. Sections were incubated in blocking buffer (BB; 5%horse serum, 0.1% lysine, 0.1% glycine, 0.4% Triton X-100in PBS, pH 7.4) for 30 minutes at room temperature andthen incubated overnight at 4° with an anti-TAG1 mono-clonal supernatant, diluted 1:100. The anti-TAG-1 mono-clonal was generated by M. Yamamoto (Dodd et al., 1988)and obtained from the Developmental Studies HybridomaBank (University of Iowa, Department of Biological Sci-ences, Iowa City, IA a facility supported by the NationalInstitute of Child Health and Human Development(NICHD, Rockville, MD). The anti-TAG-1 monoclonal an-tibody resulted from the immunization of a mouse withfetal rat brain. Extensive characterization revealed thatthis antibody recognizes a 135-kDa glycoprotein that is amember of the Ig superfamily of molecules that arepresent throughout the nervous system, including on ax-ons. Staining patterns using this reagent are well de-scribed, as this is a common reagent in neurobiology re-search, and similar staining of corticofugal axons incontrol brain (Fig. 3G) has been described previously (Se-rafini et al., 1996; Kudo et al., 2005). No staining wasobserved when the primary antibody was absent from theexperiment. Following incubation with the diluted anti-body, sections were washed in PBS, pH 7.4, incubatedwith an Alexa-tagged secondary antibody and a fluores-cent Nissl stain (Molecular Probes, Eugene, OR; 1:400 and1:500, respectively) 40 minutes at room temperature,washed in PBS, and mounted in 90% glycerol/PBS. Imagesof anatomically matched sections were obtained by fluo-rescent microscopy (Zeiss Axioplan, Thornwood, NY, andOpenlab software, Improvision, Lexington, MA) and pat-terns of staining were compared.

Quantification of the limits and extent ofephrin-A5 expression

Images of in situ hybridization for ephrin-A5 RNA wereacquired (4–12 sagittal sections/animal) from P0 control(WT and EphA7�/–, n � 8) and EphA7–/– (n � 8) mice.Images were identically adjusted for resolution and con-trast using Adobe Photoshop, matched according to ana-tomical landmarks, and then imported into ImageJ. TheAP length of the cortex, the length of the ephrin-A5-positive portion of the cortex, and the length of the ephrin-A5-negative anterior cortex were measured. Mean valueswere calculated within each genotype. Statistical signifi-cance was tested by analysis of variance (ANOVA) (��5%)and expressed as �SEM.


629EphA7-ephrin-A5 IN SOMATOSENSORY CORTEX

Quantification of the profile of ephrin-A5expression

Digital files of in situ hybridizations for ephrin-A5 ex-pression were captured by brightfield microscopy of ana-tomically matched medial (control, n � 6; EphA7–/–, n �8) and lateral (control, n � 5; EphA7–/–, n � 6) parasag-ittal sections of P0 control and EphA7–/– mice. Theseimages were imported into IGOR and analyzed using acustom program designed to measure the mean gray valueat points along the AP axis of the cortex. A mean valuewas calculated for each 5% of the AP length (anterior �0% and posterior � 100%). These values were converted tothe percent of maximal expression within each section sothat relative values could be compared between animalsand across genotypes. Means, standard errors, and ante-rior and posterior slopes for medial and lateral sectionswithin each genotype were calculated. Statistical signifi-cance was tested using ANOVA (��5%) and expressed as�SEM.

In utero electroporation

Timed pregnant C57BL/6 (control) or EphA7–/– micewere anesthetized (100 mg/Kg ketamine, 10 mg/Kg xyla-zine), their abdomens were opened, and their uteruseswere exposed. Embryos were visualized and injected in-traventricularly with solutions containing either CMV-YFP (YFP) alone or a mixture of CMV-YFP and CMV-ephrin-A5 in Tris-buffered 0.02% Fast Green, pH 7.4.Upon observing ventricular filling, a current was passedacross the embryonic head using Tweezertrodes (BTX,Harvard Apparatus, Dover, MA). Four days postsurgerythe mothers were euthanized and the embryos were re-covered. Only embryos with YFP-expressing cells presentin the dorsal cortical ventricular zone that also containedtransfected cells within the cortical ventricular zone, ex-tending radially, were included in our analyses. Consis-tent with published data, cotransfection rates in our lab-oratory are �85% when the constructs are simultaneouslyintroduced (Bai et al., 2003). Survival rates for this pro-cedure in the laboratory are �80%, with �90% of recov-ered embryos expressing their DNA.

Analysis of cell position in electroporatedbrains

Images were obtained from several sections of eachtransfected animal in Openlab (Improvision), converted tograyscale in Adobe Photoshop, and imported into IGOR.Gray values were measured within a rectangle dividedinto 10 bins (200 pixels wide) that was placed perpendic-ular to the ventricle (bin 1) and pia (bin 10). To normalizefor interanimal variability, absolute levels were convertedto an “intensity ratio” by dividing each value by the max-imum fluorescence level in each section. Mean values wereobtained for each experimental group. Statistical signifi-cance between groups was tested by ANOVA. Values forthe number of animals and sections, respectively, were:YFP/control: 2, 9; YFP/EphA7–/–: 3, 11; ephrin-A5/control:3, 14; ephrin-A5/EphA7–/–: 7, 19.

Cytochrome oxidase staining

P7 control and mutant brains were hemisected, fixed byimmersion in 4% paraformaldehyde (4% PFA), and em-bedded in 4% suprasieve agarose/PBS, pH 7.4. Tissueintended for tangential sections was flattened between

glass slides and sectioned at 200 �m, whereas tissue in-tended for sagittal analysis was sectioned from the mid-line at 100 �m. The resulting sections were pretreated ina 10% sucrose/0.12 M phosphate buffer (PB) solution at37° and then incubated in the cytochrome oxidase (CO)reaction (30 mg Cytochrome C, 50 mg diaminobenzidine, 2mg Catalase, and 10 g sucrose per 100 ml of 0.12 M PB) for6–24 hours at 37°C. Stained sections were passed throughincreasingly dilute sucrose solutions (10%, 5%, 0% sucrosein 0.12 M PB), mounted on subbed slides, dried, dehy-drated through graded ethanols, cleared in xylenes, andcoverslipped. In most experiments, CO staining of mutantcortex was performed in parallel with CO staining of con-trol cortex.

Quantification of the limits and extent ofcytochrome oxidase activity

Tangential analysis. Brightfield images of CO-stained sections containing a complete map of somatosen-sory cortex, including a clearly delineated barrel cortex,were collected in Adobe Photoshop. The images weresized, cropped, and landmarks (such as the centers andthe outer bounds of all primary barrels (A1-4, B1-4, C1-7,D1-7, E1-7) were marked. These images were then im-ported into ImageJ and the areas of individual barrels, theentire barrel field (primary barrels and straddler barrels),and the entire cerebral cortex were measured. Means andstandard errors were calculated for each genotype andANOVA was used to test for significance between experi-mental and control groups. Numbers of mice were: control,10; EphA7–/–, 10; ephrin-A5–/–, 5; EphA7/ephrin-A5–/–,5.

Sagittal analysis. Sagittal sections, corresponding toall mediolateral (ML) positions within the cerebral cortex,were imaged and assigned a position along the ML axis.The extent of CO staining in the middle cortex, indicativeof somatosensory cortex, was measured for each sectionusing NIH Image software. The ML position of somato-sensory cortex in control (n � 7) and EphA7–/– (n � 8)mice was determined by calculating the mean sectionnumber. Images were imported into ImageJ and the totalAP cortical length, the length of the middle CO-staineddomain, and the length of the barrel-negative anteriorregion were measured for control and EphA7–/– cortex.Means and standard errors were calculated within eachanimal and then within genotype. Statistical significancebetween control and EphA7–/– mice was tested usingANOVA.

RESULTS

Expression of EphA7 and ephrin-A5 incortical development

Early in corticogenesis (E10–13), EphA7 is expressed atlow levels, diffusely spread across the cerebral wall (Yunet al., 2003). As cortical development proceeds (E14–16),levels of EphA7 increase and become concentrated withinparticular embryonic zones of the cerebral wall (Yun et al.,2003). At E15, an anterior domain expressed EphA7 in thesubplate zone and cortical plate and a posterior domainexpressed EphA7 in the intermediate and subplate zones(Fig. 1C, with an arrow indicating the transition zone,using Fig. 1A as a morphological guide). At birth, twoEphA7-positive cortical fields existed: an anterior domain



that spanned the thickness of the cortical plate and aposterior domain that was limited to the most superficialcortical plate (Fig. 1D, with arrows indicating transitionsin expression, using Fig. 1B as a structural guide). Themiddle cerebral wall was notable because of low levels ofEphA7 expression and elevated levels of one of its ligands,ephrin-A5 (Fig. 1B, with arrows indicating boundaries,Fig. 1F,H). In contrast to EphA7, ephrin-A5 expressionwas only detectable using in situ hybridization from E17onwards (Fig. 1E,G) (Yun et al., 2003) and was restrictedto cells of the cortical plate (Fig. 1F). Thus, increasinglypatterned expression of EphA7 in anterior and posteriorcortical regions during development preceded localizedcortical expression of ephrin-A5 within a restricted middlecortical domain.

EphA7 isoform diversity during corticaldevelopment

The EphA7 locus produces two prominent transcripts: afull-length (FL) and a truncated (TR) isoform (Ciossek etal., 1995). Functional studies, performed in vitro and invivo, demonstrated that the FL receptor promotes cellularrepulsion, while the TR receptor mediates cellular adhe-sion (Holmberg et al., 2000). Expression studies demon-

strate that these isoforms are largely colocalized withinthe brain (Ciossek et al., 1995, 1999). Our in situ hybrid-ization analyses were performed using a probe corre-sponding to the ectodomain of EphA7 that was capable ofdetecting both isoforms. Analyses using isoform-selectivereagents, however, detected no major differences in FLand TR distribution within the cerebral cortex or thala-mus (Ciossek et al., 1995, 1999) (data not shown). Todetermine whether EphA7 isoform usage changed duringcortical development, we performed RNase protection as-says using cortical samples of a variety of ages and a probecapable of distinguishing both FL and TR EphA7 RNA(Fig. 2A). Initial results revealed that both isoforms wereproduced throughout corticogenesis, that the overallabundance of EphA7 increased with maturity, and thatthe relative amounts of FL and TR varied with develop-mental age (Fig. 2B). More extensive analyses demon-strated that relative proportions of FL and TR were sim-ilar from E11–16 (Fig. 2C), but became imbalanced fromE18 onward, when the TR isoform began to predominate(Fig. 2C). Hence, the isoform composition of the EphA7RNA pool shifts during cortical development, from anembryonic population that contains similar levels of the

Fig. 1. EphA7 and ephrin-A5 expression in the WT cerebral cortex.Images of embryonic day 15 (E15) (A,C,E,G) and postnatal day 0 (P0)(B,D,F,H) tissue stained with Nissl (A,B), or subjected to in situhybridization using probes specific for EphA7 (C,D) or ephrin-A5 (E,F)and images with the two probes merged, with EphA7 in red andephrin-A5 in green (G,H). A,B: Representative sections stained withNissl. C: EphA7 expression is patterned within the E15 cerebralcortex, with anterior expression in superficial domains and posteriorexpression in deeper zones of the cerebral wall. The transition area ismarked with an arrow. D: At P0, EphA7 is broadly expressed within

the anterior cortex and more localized within posterior regions. Ar-rows indicate transition zones between high and low expression. E:Ephrin-A5 is not detectable at E15. F: Ephrin-A5 is expressed in amiddle domain at P0. Arrows indicate transition zones between cortexwith high and low expression levels. G: Only EphA7 expression isapparent at E15. H: EphA7 and ephrin-A5 expression are detected atP0 and are mutually exclusively expressed in the AP axis of thecortex. Anatomical coordinates in A correspond to all panels. A, an-terior; D, dorsal; LV, lateral ventricle. Scale bar in A � 100 �m inA,C,E,G; � 200 �m in B,D,F,H.



two isoforms, to a postnatal population with higher pro-portions of the TR form.

Characteristics of EphA7 mutant brains

To assess the role of EphA7 signaling in corticogenesis,mice mutant for the EphA7 gene were examined. Al-though these mice have been studied previously, no in-depth description of their cerebral cortex has been under-taken (Depaepe et al., 2005; Rashid et al., 2005). Analysisof RNA from wildtype (WT) and EphA7–/– cortices con-firmed that the two major transcripts generated from theEphA7 locus, FL and TR, were present in cortex of WTmice and absent in cortex of EphA7–/– mice (Fig. 3A). WT,EphA7�/–, and EphA7–/– mice were born in Mendelian

ratios (see Materials and Methods for values), were viable,grew into maturity, and were capable of breeding. Thebrains of EphA7–/– mice were indistinguishable in sizeand shape from control brains (Fig. 3B) (Dufour et al.,2003; Holmberg et al., 2005; Rashid et al., 2005). A recentstudy concluded that a proportion of EphA7–/– mice didnot survive embryogenesis because of abnormal apoptosisin progenitor cells (Depaepe et al., 2005); however, thisembryonic lethality was not reproduced in our analysis.Since the mice analyzed in both studies were derived fromthe same line, we propose that distinctions in strain back-grounds may account for the observed differences (seeMaterials and Methods for details). Analyses of E15, P0,and P7 brains revealed that cortical morphology, antero-posterior cortical length, and cerebral wall thickness weresimilar between control and EphA7–/– mice (Fig. 3C–Fand data not shown). The composition and placement ofcells in the cortex as well as the connections formed bycortical cells were also similar in control and EphA7–/–cortex (Figs. 3G,H, 5A–D and data not shown) (Dufour etal., 2003).

In addition to its robust expression within the cortex,EphA7 is present in the thalamus, highest in limbic-associated (cmt, adt, ldt), visual (dlg), and auditory (mg)nuclei (Fig. 4). EphA7 is also expressed at lower levels inparts of the ventrobasal thalamus (Fig. 4C,E, using 4A,Das structural guides) (Vanderhaeghen et al., 2000; Dufouret al., 2003), overlapping a similar, but stronger, patternof EphA4 expression (Fig. 4B,F). In addition, one ofEphA7’s ligands, ephrin-A5, is present in most sensorynuclei, including the somatosensory thalamus (Fig. 4H).No changes were observed in the expression of EphA4(Fig. 4F,G), Id2 (Fig. 4J,K), or RZR- (Fig. 4L,M) betweenEphA7–/– and control mice. Ephrin-A5 levels were ele-vated in the lateral geniculate of some EphA7–/– mice(Fig. 4H,I), but characteristics of ventrobasal expressionwere similar in EphA7–/– and control mice.

Gene expression in the EphA7 mutantcortex

Expression patterns for several cortically expressedgenes were similar between control and EphA7–/– ani-mals. For example, positionally selective characteristics ofId2 and Cad8 expression were comparable in EphA7–/–and control cortex (Fig. 5A–D). Moreover, the EphA recep-tors expressed by the cortex had similar expression pat-terns in control and EphA7–/– mice (data not shown).

The expression pattern of ephrin-A5, however, waschanged in EphA7–/– cortex. Ephrin-A5 is stereotypicallyexpressed within a middle cortical domain that corre-sponds to somatosensory cortex in control mice (Figs. 1F,5E,G) (Mackarehtschian et al., 1999; Vanderhaeghen etal., 2000; Yun et al., 2003; Fukuchi-Shimogori and Grove,2003). In EphA7–/– mice, ephrin-A5 expression was de-tected in the middle domain, with the usual anterior bor-der, but also extended into more posterior cortex (Fig.5E–H,P,Q). The expansion of ephrin-A5 in EphA7–/– cor-tex was more pronounced in lateral than medial sections(Fig. 5F–N,P). Ephrin-A5 expression was similar in ante-rior somatosensory cortex of control and EphA7–/– mice(Fig. 5I–K); however, ephrin-A5 levels were more uniformalong the ML axis of posterior somatosensory cortex inEphA7–/– mice (Fig. 5L–N). Despite these changes in dis-tribution, no changes in abundance of ephrin-A5 wereobserved between control and EphA7–/– cortex (Fig. 5O).

Fig. 2. EphA7 isoform diversity during cortical development.A: Diagram of full-length (FL) and truncated (TR) EphA7 transcriptsand the strategy used to detect them via RNase protection assay(RPA). B: Representative RPA results with lanes corresponding toprobe alone and hybridizations containing E13, E14, E15, E16, E18,P0, P2, and P5 cortex RNA, and yeast RNA. FL and TR EphA7 aredetected throughout cortical development. C: Quantification of theproportions of FL and TR at each stage of cortical development. Earlyin development, levels of FL and TR EphA7 are present at similarlevels (E13–16). Later in cortical development, the TR isoform pre-dominates (E18–P5).



Thus, the same amount of ephrin-A5 was spread over agreater area in EphA7–/– cortex.

Profiles of ephrin-A5 expression within thedeveloping cerebral cortex

To understand the nature of the redistribution ofephrin-A5 within the cortex, the anteroposterior profiles ofephrin-A5 expression were examined in control andEphA7–/– mice. Ephrin-A5 expression formed a bell-shaped curve along the AP axis of the cortex in controlanimals (Fig. 6A). The shape of ephrin-A5’s AP profileremained constant but the location of the peak of expres-sion varied with ML position, corresponding to the posi-tion of somatosensory cortex (Fig. 6A) (Yun et al., 2003).No remarkable differences in the distribution of ephrin-A5were observed in medial sections of control and EphA7–/–mice (Fig. 6C), reflected in their comparable anterior andposterior slopes (Fig. 6B, left). In lateral cortex, however,peak levels of ephrin-A5 extended over a greater AP dis-tance in EphA7–/– mice (Fig. 6D). This change is mirroredin the decreased posterior slope in ephrin-A5’s profile inEphA7–/– cortex (Fig. 6B). Since ephrin-A5 abundancewas similar between EphA7–/– and control cortex (Fig.5O), posterior increases in ephrin-A5 in lateral cortexmust be accompanied by commensurate decreases inephrin-A5 in other parts of the cortex.

In vivo analysis of EphA7 function in thedeveloping cortex

An in vivo bioassay was used to examine the interac-tions between EphA7 and ephrin-A5 in the developingcortex. In utero electroporation (IUE) was used to intro-duce expression vectors into cells of the E14.5 cortexand the fate of transfected cells were characterized laterin development. Previous studies demonstrated thatventricular zone cells are transfected by IUE and followtheir usual developmental path, eventually dispersingwithin the cerebral wall (Fukuchi-Shimogori and Grove,2001, 2003; Bai et al., 2003). In our experiments, vectorsencoding either a biologically inert gene product (YFP)or a biologically active Eph ligand (ephrin-A5) wereintroduced into cortical cells in order to assess the char-acteristics of these cells in distinct cortical environ-ments. Four days after transfection, the location of ex-pressing cells was assessed and comparisons were madebetween YFP- and ephrin-A5-expressing cells in controland EphA7–/– embryos. In control cortex, YFP-expressing cells were well dispersed within the cerebralwall, including some occupancy of the most mature cor-tical plate (Fig. 7A). In contrast, ephrin-A5-expressingcells were not widely spread within the cerebral wall,but were most concentrated near the ventricle (Fig. 7B).

Fig. 3. Characteristics ofEphA7 mutant cortex. A: RNaseprotection assay with lanes corre-sponding to probe alone and hy-bridizations containing yeastRNA, P0 control cortex RNA, or P0EphA7–/– cortex RNA. Full-length(FL) and truncated (TR) EphA7are detected in control but notEphA7–/– RNA. B: Images of P0control and EphA7–/– brains re-veal the presence of all neural sub-divisions and no overt differencesin size. C,D: Parasagittal views ofNissl-stained P0 control (C) andEphA7–/– (D) cortex. E: Compari-son of the anteroposterior length ofthe cerebral cortex at P0 (E, left)and P7 (E, right) reveal no signifi-cant differences between control(white bars) and EphA7–/– (graybars) mice. F: Comparison of thethickness of the cerebral wall atthe AP axis midpoint demon-strates no differences between con-trol (white bars) and EphA7–/–(gray bars) mice at P0 (F, left) orP7 (F, right). H: TAG-1 immuno-histochemistry at E18.5 in control(G) or EphA7–/– (H) parasagittalsections demonstrates normal cor-ticofugal staining. Anatomical co-ordinates in C refer to C,D,G,H. A,anterior; D, dorsal. Scale bar �200 �m in C (applies to C,D); 300�m for G,H.



Indeed, in control mice 29% of YFP cells were located inthe most superficial cortex (bins 1–5), while only 5% ofephrin-A5-expressing cells were housed within this do-main (Fig. 7C). These data demonstrate that ephrin-A5-expressing cells were restricted in their ability to pop-ulate the control cerebral cortex compared to YFP-expressing cells.

To determine whether the lack of dispersion of ephrin-A5-expressing cells in the control cortex relied on inter-actions with EphA7, similar experiments were per-formed using EphA7–/– mice. Again, YFP-expressingcells spanned the cerebral wall of EphA7–/– embryos(Fig. 7D), but this time ephrin-A5-transfected cells alsospanned the cerebral wall of EphA7–/– mice (Fig. 7E),similar to their YFP-expressing counterparts in bothgenotypes (Fig. 7A,D,E). Approximately 20% of ephrin-A5-expressing cells were located in superficial corticalregions (bins 1–5) in EphA7–/– cortex, a value morereminiscent of YFP-expressing cells in either genotype(Fig. 7C,F) and dramatically different than ephrin-A5-expressing cells in the control cortex (Fig. 7G). Theincreased ability of ephrin-A5-expressing cells to popu-late the EphA7–/– versus control cortex demonstrates

that EphA7 mediates an inhibitory interaction withephrin-A5 in the forming cortex.

Changes in barrel cortex in EphA7mutant mice

We next examined the functional organization of thecortex in postnatal EphA7–/– mice. We focused on thesomatosensory domain for this analysis because of thechanges observed in ephrin-A5 expression, a somatosen-sory marker. Tangential sections of flattened P7 mousecortex were stained for cytochrome oxidase (CO) activity,an indicator of primary sensory cortical domains. Ouranalysis demonstrated that all primary sensory domainswere present in mutant animals and that total corticalarea was unchanged (Fig. 8A–D). Furthermore, within thebarrel field of somatosensory cortex, all principal andstraddler barrels were present and their topographic ar-rangement was normal (Fig. 8C,D). However, the primarybarrel field was 22% smaller in EphA7–/– than in controlcortex (Fig. 8E, right), even though the area of the entirecortex was unchanged (Fig. 8E, left). While all barrelswere smaller in EphA7–/– cortex, lateral barrels weremost affected (Fig. 8F).

Fig. 4. Gene expression in the control and EphA7–/– thalamus.Horizontal sections of P0 thalamus (A–C) or parasagittal sections ofE15 thalamus (D–M) stained for Nissl (A,D) or hybridized with probesfor EphA4 (B,F,G), EphA7 (C,E), ephrin-A5 (H,I), Id2 (J,K), or RZR-b(L,M). B,C: EphA4 and EphA7 are expressed in anteromedial gradi-ents in the ventrobasal thalamus of control mice. No gross changeswere observed morphologically or molecularly in somatosensory nu-

clei of the EphA7–/– thalamus (G,I,K,M) compared with control thal-amus (F,H,J,L). A, anterior; L, lateral; D, dorsal; adt, anterior dorsalthalamus; cmt, centromedian thalamus; lt, lateral thalamus; vpm,ventromedial thalamus; vpl, ventrolateral thalamus; avt, anteriorventral thalamus; ldt, lateral dorsal thalamus; pot, posterior thala-mus; dlg, dorsolateral thalamus; mg, medial geniculate. Scale bar in B� 50 �m in A–C and � 25 �m in D–M.



Fig. 5. Gene expression in the control and EphA7–/– cortex. Para-sagittal sections of P0 control (A,C,E,G) and EphA7–/– (B,D,F,H)cortex subjected to in situ hybridization using probes specific for Id2(A,B), Cad8 (C,D), or ephrin-A5 on medial (E,F) and lateral (G,H)sections. A–D: No differences were observed in a set of genes, includ-ing Id2 and Cad8, between control and EphA7–/– mice. Arrowheadsindicate positionally selective aspects of each gene’s expression. E–H:Ephrin-A5 expression is normal in medial (E,F) and expanded inlateral (G,H) sections in EphA7–/– cortex. Arrowheads in E–H indi-cate the posterior edge of high ephrin-A5 expression and the occipitalpole in each image. Coronal sections of E18.5 control (I,J,L,M) orEphA7–/– (K,N) cortex hybridized with probes for EphA7 (I,L) orephrin-A5 (J,K,M,N) in either anterior (I,J,K) or posterior (L,M,N)somatosensory (S1) regions I. EphA7 is high lateral and low medial.J,K: Ephrin-A5 is high medial and lower lateral, opposing the patternof EphA7. L: EphA7 expression in posterior S1 is high medial and

lateral and low in the intervening cortex. M: Ephrin-A5 expression ishigh lateral and low medial. N: Ephrin-A5 expression in the EphA7–/–is altered: levels are uniform. O: RNase protection assay with lanescorresponding to probe alone and hybridizations containing yeastRNA, P0 control cortex RNA, or P0 EphA7–/– cortex RNA. Levels ofephrin-A5 are similar in control and EphA7–/– cortex. P: Comparisonof the proportion of AP cortical length expressing ephrin-A5.Ephrin-A5 expression encompasses more cortical length in EphA7–/–(filled line) than control (dashed line) cortex. Q: Comparison of theanterior-most boundary of ephrin-A5 expression along the cortical APaxis. The anterior boundary of ephrin-A5 expression is similar medi-ally and is more posterior laterally in EphA7–/– (filled line) comparedto control (dashed line) cortex. Anatomical coordinates in A refer toA–H. A, anterior; D, dorsal: *P 0.05 and ***P 0.001 in P and Q.Scale bar in A � 200 �m in A–H and � 100 �m for I–N.



Position of barrel cortex in EphA7mutant mice

Tangential sections are a flattened representation of aninitially curved structure. While the flattening processdoes not alter the relative positions of individual barrelswithin the primary barrel field, changes in placement ofcortical areas within the section can occur. To avoid thesecomplications and to extend our analysis to all of thesomatosensory cortex, we analyzed positional characteris-tics of somatosensory cortex in parasagittal sections (Fig.9). CO reactivity was present in layer IV in the middledomain of the AP axis in both control and EphA7 mutantanimals: diffuse in medial sections and columnar in morelateral sections, consistent with the composition of so-matosensory cortex (Fig. 9A–F). Analysis revealed thatsomatosensory cortex both decreased in size (Fig. 9G,right) and shifted posteriorly slightly (Fig. 9G, left) inEphA7 mutant mice. However, no differences in the me-diolateral midpoints of somatosensory cortex in controland EphA7–/– animals were observed (Fig. 9H). Thus,changes observed in barrel cortex were reflected in so-matosensory cortex as a whole.

Characteristics of somatosensory cortex inephrin-A5–/– and EphA7/ephrin-A5–/– mice

Characteristics of somatosensory cortex in additionalmutant mice were assessed in order to determinewhether EphA7-ephrin-A5 interactions mediate the

changes observed in EphA7–/– cortex. First, we foundthat the area of barrel cortex in ephrin-A5–/– mice was74% of area of control mice and that the region ofgreatest decrease was located medially within the so-matosensory cortex (Fig. 10A,C,F). These findings aredifferent from those reported in a previous study(Vanderhaeghen et al., 2000). An explanation for thisdifference may be the extensively more inbred popula-tion of mice we are examining compared with the pre-vious study. Interestingly, the magnitude of the de-crease in barrel field size was similar between ephrin-A5–/– and EphA7–/– mice, 26% and 22%, respectively(Fig. 10A–C,F).

The recapitulation of the EphA7–/– phenotype in theephrin-A5 mutant suggested that these Eph family mem-bers are acting together in the functional specification ofthe cortex. To examine this hypothesis, EphA7/ephrin-A5double mutant (EphA7/ephrin-A5–/–) mice were charac-terized. Topography of barrel cortex was normal inEphA7/ephrin-A5–/– mice (Fig. 10D); however, the area ofbarrel cortex was reduced in size, more than in eithersingle mutant alone (Fig. 10A–D,F). Indeed, barrel area inEphA7/ephrin-A5–/– mice was 64% of the control size,whereas EphA7 and ephrin-A5 mutant animals had barrelcortex that were 74–78% of the control field area (Fig.10F). This suggests that EphA7 and ephrin-A5 act coordi-nately and with other family members to shape somato-sensory cortical features.

Fig. 6. Ephrin-A5 expression profiles in EphA7 mutant cortex.A: Relative ephrin-A5 levels along the AP axis of the cortex in medial(dashed line) and lateral (solid line) sections. Expression of ephrin-A5forms a bell-shaped curve along the AP axis in control cortex. B: An-terior (left in each panel) and posterior (right in each panel) slopes ofephrin-A5 expression in control (white bars) and EphA7–/– (gray bars)in medial (left panel) and lateral (right panel) sections. No significantdifferences are observed in medial values or in lateral anterior slope;

however, posterior slope is dramatically changed in EphA7–/– ani-mals. C: Comparison of ephrin-A5 profiles in medial sections of control(dashed line) and EphA7–/– (solid line) samples. No difference isapparent. D: Comparison of ephrin-A5 profiles in lateral sections ofcontrol (dashed line) and EphA7–/– (solid line) sections. Theephrin-A5 profile is less graded and more extensive along the corticalAP axis in EphA7–/– cortex. *P 0.05, **P 0.01, and ***P 0.001.



DISCUSSION

Our results demonstrate that EphA7-ephrin-A5-mediatedsignaling influences the characteristics of somatosensorycortex throughout its development. The molecular expansionin EphA7–/– cortex and the cellular inhibition betweenephrin-A5 and EphA7 that we observe in embryonic cortexsupport a repulsive influence of EphA7 during the formationof the cortex. The postnatal decrease in barrel size we ob-serve when EphA7-ephrin-A5 signaling is altered supports apermissive role in the postnatal cortex. Thus, Eph signalingappears to promote distinct functions in the somatosensorycortex at distinct developmental stages (Fig. 11A).

EphA7-ephrin-A5 signaling in theestablishment of molecular landscapes in

early cortical development

Patterned expression of regulatory genes, such as tran-scription factors and morphogens, are essential for estab-

lishing the early positional framework upon which corticalparcellation depends (Bishop et al., 2000; Fukuchi-Shimogori and Grove, 2001, 2003; Muzio et al., 2002;Garel et al., 2003; Hamasaki et al., 2004; Huffman et al.,2004). Differential expression of cell adhesion and signal-ing molecules suggests that intercellular signals aretransduced between cortical cells, eventually establishingdistinct cortical identities that are instructive in estab-lishing proper neural connections (Nakagawa et al., 1999;Fukuchi-Shimogori and Grove, 2001, 2003).

The distribution of EphA7 and ephrin-A5 in the devel-oping cortex supports a model in which the engagement ofEphA7 and ephrin-A5 at compartment borders may con-tribute to the division of the cerebral cortex into distinctcellular domains. Consistent with this idea, changes inboth the absolute level and the dynamic range ofephrin-A5 were observed in the EphA7–/– cortex. Intrigu-ingly, changes of similar magnitude in Eph signaling in

Fig. 7. In vivo analysis of YFP and ephrin-A5 expressing cells incontrol and EphA7–/– cortex. In utero electroporated control (A,B)and EphA7–/– (D,E) cerebral walls transfected with either CMV-YFP(A,D) or CMV-ephrin-A5 and CMV-YFP (B,E) expression vectors.Both the sections (A,B,D,E) and the graphs (C,F,G) are oriented sothat the bottom corresponds to the ventricle and top represents thepial surface. A: Control cortex transfected with CMV-YFP. Trans-fected cells span the cerebral wall from ventricular zone (bottom) topial surface (top). B: Control cortex transfected with CMV-ephrin-A5and CMV-YFP. Transfected cells remain close to the lateral ventricle.C: Comparison of the distribution of YFP-transfected (dashed, gray)and ephrin-A5-transfected (solid, gray) cells in control cortex. Asmaller proportion of ephrin-A5-expressing cells populate the upper

cortical plate compared with YFP-expressing cells. D: EphA7–/– cor-tex transfected with CMV-YFP. Transfected cells span the cerebralwall from ventricular zone to pial surface. E: EphA7–/– cortex trans-fected with CMV-ephrin-A5 and CMV-YFP. Transfected cells span thecerebral wall from ventricular zone to pial surface. F: Comparison ofthe distribution of YFP-transfected (dashed, black) and ephrin-A5-transfected (solid, black) cells in EphA7–/– cortex. Distributions ofYFP- and ephrin-A5-expressing cells are more similar in EphA7–/–. G:Comparison of the distribution of ephrin-A5-transfected cells in con-trol (solid, gray) and EphA7–/– (solid, black) cortex. Transfected cellsare more dispersed in EphA7–/– than control cortex. *P 0.05, **P 0.01. Scale bar in B � 20 �m in A,B,D,E.



other experimental paradigms have resulted in pro-nounced functional consequences (Brown et al., 2000;Hansen et al., 2004). The composition of EphA7 tran-scripts during corticogenesis consists of a mix that in-

cludes relatively equal parts of FL and TR isoforms. Theexpansion we observe in ephrin-A5 expression in theEphA7–/– cortex as well as the reduced cell dispersion inour in vivo bioassay of cellular interactions within thecortex supports an inhibitory role for this embryonic mix-ture of EphA7.

The mechanism by which ephrin-A5 becomes ectopicallyexpressed in EphA7–/– cortex remains unclear. Possibili-ties include the ectopic activation of ephrin-A5 in corticalcells that are normally silent for ephrin-A5 or the abnor-mal movement of cells that normally express ephrin-A5 inthe EphA7–/– cortex. We favor the second hypothesis,since changes in overall abundance of ephrin-A5, a conse-quence of the first explanation, were not observed. More-over, there is precedent for a role of Eph signaling in therestriction of cell movement in other parts of the nervoussystem (Mellitzer et al., 1999; Xu et al., 1999). Whateverthe explanation, there is a posterior bias in the spread ofephrin-A5 in the cortex. This selective expansion mayreflect the presence of additional Eph receptors in anterior(EphA5) and posterior (EphA6) cortex (Yun et al., 2003)that exert differential effects (Gao et al., 1998; Knoll,2001). For example, in the absence of EphA7, EphA5 inanterior cortex may maintain the repulsive environmentfound in control cortex, thus limiting ephrin-A5 spread,while EphA6 in the posterior cortex may be more permis-sive.

The absence of EphA7 resulted in a more gradual andshallower profile of ephrin-A5 for an extended AP distancein the cortex. Such a change is intriguing in light of resultsthat demonstrated that relative, rather than absolute,levels of Eph signaling determine a cellular response(Brown et al., 2000). In addition, the finding that the samecells can respond positively or negatively to different lev-els of the same Eph-ephrin stimulation (Hansen et al.,2004) suggests that alterations in both absolute and rela-tive levels of ephrin-A5 have the potential to change thenature of cellular responses within the cortex. We hypoth-esize that the redistribution of ephrin-A5 in the EphA7–/–cortex alters the nature of interactions between incomingthalamic axons and somatosensory cortex (Fig. 11B).

Fig. 8. Characteristics of somatosensory cortex in EphA7–/– mice.Tangential sections from flattened P7 cortices stained for cytochromeoxidase (CO) (A,C,D) and quantification of the decreases observed(E,F). A: CO staining of a tangential section of cortex. B: Schematicrepresentation of somatosensory representations in CO stained tis-sue. C: Representative somatosensory region of a control animal. D:Representative somatosensory region of an EphA7–/– animal. E: Com-parison of areas of the entire cortex (E, left) or entire primary barrelfield (E, right) in control and EphA7–/– animals. EphA7–/– animalshave smaller barrel fields (gray bar indicates P 0.001) but similarcortical areas. F: Relative values within control and EphA7–/– cortex,with decreases in EphA7–/– mice represented above the line. Barrelareas are decreased in EphA7–/– compared to control barrel field,affected most in lateral regions. For panel F, white bars are notstatistically significant, dark bars are significant to P 0.01, and graybars are significant to P 0.001. M, motor cortex; S, somatosensorycortex; A, auditory cortex; V, visual cortex; HL, hind limb; FL, fore-limb; TR, trunk; LL, lower lip; UL, upper lip; SW, short whiskers;PBF, primary barrel field; T, tongue; SII, secondary somatosensorycortex. Scale bar in A � 400 �m for A and � 150 �m for C,D.



EphA7-ephrin-A5 control of postnatalsomatosensory field size

Examination of the area of somatosensory cortex re-vealed a 22% reduction in the area of barrel cortex inEphA7–/– mice, a change that was most pronounced inlateral cortex. The decrease in barrel size in EphA7–/–cortex appears to reflect the loss of a permissive interac-tion between EphA7 and its ligands in the postnatal cor-tex. We interpret the alterations in barrel cortex inEphA7–/– mice to reflect cortical rather than thalamicchanges, since the thalamus appeared unaffected inEphA7–/– mice (Fig. 4F–M) (Dufour et al., 2003). We de-tected a similarly sized decrease (26%) in the size of barrelcortex in ephrin-A5–/– animals. Our results are differentfrom the initial report of these mice (Vanderhaeghen etal., 2000). We suspect that this difference representsstrain differences: the ephrin-A5–/– mice analyzed in ourstudy are considerably more inbred (up to 10 generations)than the original mice examined which were outbred.

Such extensive backcrossing can result in more pro-nounced phenotypes. Interestingly, the change in barrelcortex described here is similar in magnitude but oppositein direction to shifts observed in another genetic alter-ation (McIlvain et al., 2003), raising the possibility ofhomeostatic regulation of areal size. EphA7/ephrin-A5–/–mice were also examined. A larger decrease was observedin the EphA7/ephrin-A5–/– than in either single mutantanimal, demonstrating that while phenotypes are overlap-ping, signaling with other molecules also contributes tothe maintenance of somatosensory size. Together, ourdata demonstrate that interactions of EphA7 andephrin-A5 influence the size of somatosensory cortex.

The mechanisms by which EphA7-ephrin-A5 interac-tions influence somatosensory size are currently unclear,however, our data suggest distinct effects at particularstages, perhaps guided by differential isoform usage (Fig.11A). Within the early cortex, ephrin-A5 expression insomatosensory cortex was expanded but was less graded

Fig. 9. Placement of somatosensory cortex in EphA7–/– mice.Parasagittal sections from P7 control (A–C) and EphA7–/– (D–F) miceassayed for cytochrome oxidase (CO) activity. Medial (A,D), interme-diate (B,E), and lateral (C,F) sections are shown. Arrowheads in A–Findicate the anterior and posterior limits of CO staining within layerIV. The length of the anterior (CO-negative) domain (G, left) and themiddle (CO-positive) somatosensory domain (G, right) were measured

in control (white bars) and EphA7–/– (gray bars) cortex and expressedas a percent of the entire AP axis. G: Somatosensory cortex has aslightly more posterior anterior border (left side) and is less extensive(right side) in EphA7–/– than control cortex. H: Mediolateral positionof somatosensory cortex does not vary between EphA7–/– and controlmice. *P 0.05; ***P 0.001. Scale bar in A � 200 �m in A–F.



in EphA7–/– mice than in control mice. These changesmay alter relative cellular assignments within the cortex,designating a smaller cortical domain that is attractive toincoming ventrobasal thalamic axons, which themselves

Fig. 10. Somatosensory cortex in ephrin-A5 and EphA7/ephrin-A5mutant cortex. Tangential sections from flattened P7 cortex stainedfor cytochrome oxidase (CO) activity (A–D) in control (A), EphA7–/–(B), ephrin-A5–/– (C), and EphA7/ephrin-A5–/– (D) mice and quanti-fication of the observed decreases (E,F). A: CO staining of a tangentialsection of control cortex. B: CO staining of a tangential section ofEphA7–/– cortex. C: CO staining of a tangential section of ephrin-A5–/– cortex. D: CO staining of a tangential section of EphA7/ephrin-A5–/– cortex. E: Relative barrel areas in ephrin-A5–/– compared tocontrol cortex, with decreases in ephrin-A5–/– mice represented in theupward direction. Barrel areas are less in the ephrin-A5–/– than thecontrol barrel field and changes are more prominent medially. Darkbars are more significant than light bars. F: Comparison of areas ofprimary barrel field in control, EphA7–/–, ephrin-A5–/–, and EphA7/ephrin-A5–/– cortex. Barrel areas are 78% of control values inEphA7–/–, 74% of control values in ephrin-A5, and 64% of controlvalues in EphA7/ephrin-A5–/– mice. Scale bar in A � 150 �m in A–D.

Fig. 11. Model of EphA7-ephrin-A5 signaling in somatosensorycortex. Roles of EphA7 in the parcellation of the cortex from molecular(A) and functional (B) perspectives. Red correspond to EphA7 expres-sion, blue to EphA4 expression, purple to EphA4 and EphA7 expres-sion, and green to ephrin-A5 expression. A: EphA7-ephrin-A5 inter-actions are repulsive embryonically, when the isoform pool containsequal amounts of truncated (TR) and full-length (FL) receptor, butwere permissive postnatally, when the receptor pool consists of highlevels of TR EphA7. B: When EphA7 is absent, the distribution ofephrin-A5 expands within the cortex and relative levels within thecortex become less graded. Under these circumstances, thalamic ax-ons, which themselves have more restricted receptor expression, in-nervate a smaller area of the cortex (indicated by the circle).


have reduced receptor-mediated signaling capacity. It ap-pears that ventrobasal axons innervate the cortical do-mains with the highest levels of ephrin-A5, but that agradient of ligand concentration guides synapse forma-tion, supporting a model in which attractive interactionbetween thalamic axons and barrel cortex are mediated byEph/ephrin signaling. Since patterns of connections be-tween the cortex and thalamus were unchanged inEphA7–/– and ephrin-A5–/– animals (Vanderhaeghen etal., 2000; Dufour et al., 2003), the decreases in somatosen-sory size described here are unlikely to reflect topographyof connections. Instead, we hypothesize that less corticalarea is attractive for input from the ventrobasal complex,leading to a smaller area of barrel cortex in EphA7–/–mice (Fig. 11B).

Our results demonstrate that signaling through thesame receptor-ligand pairing leads to distinct cellular re-sponses at particular times in development. This finding isnovel, both in the study of the cortex and in our under-standing of Eph signaling. Within the cortex, we demon-strate that the differential actions of a receptor-ligandpairings influences molecular profiles and somatosensoryfield size. In terms of Eph signaling, this work demon-strates that the same receptor-ligand pairing can lead todiscrete responses, consistent with previous studies (Cas-tellani et al., 1998; Eberhart et al., 2004; Hansen et al.,2004). Importantly, changes in Eph signaling does notalter global characteristics of cortical organization, as oc-curs when other gene products are perturbed (Bishop etal., 2000; Fukuchi-Shimogori and Grove, 2001; Fukuchi-Shimogori and Grove, 2003). Rather, alterations inEphA7-ephrin-A5 interactions lead to subtle reorganiza-tion of the cortex. Thus, the actions of this family ofintercellular signaling molecules appear to act in the re-finement, rather than the specification, of cortical do-mains. While our results address the characteristics ofsomatosensory cortex, we predict that Eph signaling re-fines other functional areas within the cortex also.

ACKNOWLEDGMENTS

We thank Uwe Drescher and Thomas Ciossek forEphA7 mutant mice, Jonas Frisen for ephrin-A5 mutantmice, John Fitzpatrick for creating a custom program forquantitative analyses performed in this study, and PierreVanderhaeghen for the ephrin-A5 expression vector. Fortechnical help, we thank Alice Walker and Andrea Gocke(animal husbandry) and James Salzano, Ncogquynh Chu,and Janice Wong (tissue preparation). We thank StephenStrittmatter, Charles Greer, Elke Stein, and Mary Whit-man for critically reviewing the article.

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EphA7-ephrin-A5 signaling in mouse somatosensory cortex: Developmental restriction of molecular...

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