Ephrin signalling controls brain size by regulating

Ephrin signalling controls brain size by regulating apoptosis of neural progenitorsVanessa Depaepe 1,Nathalie Suarez-Gonzalez 1,Audrey Dufour 1,Lara Passante 1,Jessica A Gorski 2,Kevin R.Jones 2,Catherine Ledent 1&Pierre Vanderhaeghen 1Mechanisms controlling brain size include the regulation of neural progenitor cell proliferation,differentiation,survival and migration 1,2.Here we show that ephrin-A/EphA receptor signal-ling plays a key role in controlling the size of the mouse cerebral cortex by regulating cortical progenitor cell apoptosis.In vivo gain of EphA receptor function,achieved through ectopic expression of ephrin-A5in early cortical progenitors expressing EphA7,caused a transient wave of neural progenitor cell apoptosis,resulting in premature depletion of progenitors and a subsequent dramatic decrease in cortical size.In vitro treatment with soluble ephrin-A ligands similarly induced the rapid death of cultured dissociated cortical progenitors in a caspase-3-dependent manner,thereby confirming a direct effect of ephrin/Eph signalling on apoptotic cascades.Conversely,in vivo loss of EphA function,achieved through EphA7gene disruption,caused a reduction in apoptosis occurring normally in forebrain neural progenitors,resulting in an increase in cortical size and,in extreme cases,exencephalic forebrain overgrowth.Together,these results identify ephrin/Eph signalling as a physiological trigger for apoptosis that can alter brain size and shape by regulating the number of neural progenitors.Ephrin and Eph receptor genes have been implicated in the control of cell and axon guidance in many neural systems 3–5.To identify additional roles for ephrin/Eph genes in the developing forebrain,we generated a mouse transgenic line that allows ectopic expression of ephrin-A5under the control of the EphA7receptor promoter,taking advantage of their complementary patterns of expression.Ephrin-A5and EphA7are expressed as early as embryonic day (E)10.5in the forebrain.EphA7is preferentially expressed in cortical progenitors,and ephrin-A5is expressed in progenitors of the ventral telencepha-lon and the medial dorsal telencephalon (Fig.1a).Perinatally,they are expressed in complementary gradients in the cerebral cortex (Fig.1a and ref.6).To faithfully reproduce the EphA7expression pattern of the ephrin-A5transgene without interfering with EphA7function,we used a transgenic bacterial artificial chromosome (BAC)approach,in which a conditional,Cre-recombinase-dependent,lox/enhanced green fluorescent protein (eGFP)/lox/ephrin-A5expression cassette was placed under the control of EphA7regulatory elements using BAC recombineering 7,8(Fig.1b).The resulting transgenic mice (TGA7A5)expressed the conditional cassette in a pattern very similar to the endogenous pattern of EphA7,both at early and later stages of cortical development,as assessed by expression of the eGFP transgene (Fig.1c,d).No expression of the Flag-ephrin-A5transgene was detected in the absence of Cre-recombinase.To drive expression of the ephrin-A5transgene specifically in the developing cortex,we crossed this line with an Emx1IREScre line (Emx1-Cre)that promotes efficient and specificCre-recombination in the dorsal telencephalon from E10.5(ref.9).Although TGA7A5mice and Emx1-Cre mice did not show any overt phenotype (ref.9and data not shown),mice doubly transgenic for TGA7A5and Emx1-Cre (TG/Emx1-Cre)died at birth and showed a severe reduction in cerebral hemisphere size (up to threefold by E16.5;Fig.2a),with the rest of the brain appearing normal (Fig.2a,b).This reduction in cortical size was clearly visible as early as E12.5(Fig.2a,b),and was accompanied by a decrease in the tangential extent and radial thickness of the post-mitotic cortical plate at later stages (Fig.2b),suggesting that the phenotype resulted from an impairment in early neurogenesis in the dorsal forebrain.Such a phenotype could result from dysregulation of cell prolifer-ation,migration,differentiation or death.Staining for nestin revealed no decrease in the density of cortical progenitor cells (Fig.2c).To detect abnormal patterns of proliferation or cell cycle progression among cortical progenitors,we used 5-bromodeoxyur-idine (BrdU)pulse-labellings combined with Ki67staining (to mark proliferating cells).Both long and short time courses of BrdU/Ki67labelling showed a normal labelling index (the proportion of BrdU-labelled cells among Ki67þprogenitors)for cortical progenitors,suggesting that their proliferative pattern and cell cycle were unim-paired in the mutant,even though their total number was reduced (Fig.2c,e).Next,we measured the fraction of cells leaving the mitotic cycle (the quitting fraction,or the proportion of Ki672post-mitotic cells among BrdU-labelled cells)10.No difference was observed between the controls and mutants,suggesting that cortical neuro-genesis per se was occurring normally in the mutants (Fig.2e).In addition,analysis of pan-neural (MAP-2and Tuj1)and specific (reelin,calretinin,Tbr1,and Brn1)markers showed a normal proportion and position of neurons and progenitors at all stages examined (see Fig.2c,d and Supplementary Fig.1),confirming that neural differentiation and migration appeared to take place normally in TG/Emx1-Cre mutants.We next looked for dysregulation of cell death in the mutants (Fig.3).A large increase in the number of dying early cortical progenitors was observed at E11.5and E12.5,as labelled by terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL)(Fig.3a,b).A similar increase was observed for cells immunoreactive for activated caspase-3,one of the main effectors of developmental apoptotic death 11,12(Fig.3a,b).Co-labelling for these apoptosis markers and nestin showed that the bulk (more than 90%)of dying cells were cortical progenitors (Fig.3c).This apoptosis was restricted both spatially and temporally in a pattern similar to that observed for EphA7and the ephrin-A5transgene 6(Figs 1a,c,d and 3b).Specifically,apoptosis was restricted to the developing cerebral cortex (Fig.3a,b)and was transient,peaking at E11.5–E12.5,reduced by E14.5and essentially absent by E17.5(Fig.3d,e),except for theLETTERS1Institut de Recherches Interdisciplinaires en Biologie Humaine et Mole´culaire (IRIBHM),University of Brussels,Campus Erasme,808Route de Lennik,B-1070Brussels,Belgium.2Department of Molecular,Cellular,and Developmental Biology,University of Colorado,Boulder,Colorado 80309,USA.activation of caspase-3in a few post-mitotic neurons in the cortical plate(Fig.3d).The rapid and transient nature of the induction of apoptosis of cortical progenitors upon ectopic expression of ephrin-A5,together with the lack of evidence for significant disruption of cell migration or differentiation,strongly indicated a direct pro-apoptotic effect of ephrin/Eph signalling in these cells.To test this hypothesis,we turned to an in vitro assay in which cultured cortical progenitors (at E12–E13,a stage at which most cortical progenitors express EphA7(data not shown))were challenged with EphA agonists (consisting of clustered ephrin-A5–Fc fragment fusion proteins). Ephrin stimulation of early cortical progenitors resulted in a rapid induction of cell death,as measured by TUNEL staning(Fig.3f).This cell death was inhibited by the pan-caspase inhibitor z-VAD-fmk (Fig.3f),and was concurrent with an increase in caspase-3activity (Fig.3g),indicating that ephrins can directly trigger cortical progenitor cell death through a caspase-dependent pathway. These gain-of-function data suggested a potentially important function for ephrin/Eph signalling in the regulation of progenitor cell death in the developing forebrain.To test for a physiological role of Eph signalling in this context,we next turned to a loss-of-function analysis,using EphA7knockout(EphA72/2)mice13.We found an approximately twofold decrease in cortical progenitor cell death at mid-neurogenesis(E13.5,which corresponds to a peak of apoptosis during cortical neurogenesis14,15),as assessed by quantification of TUNEL-positive and activated caspase-3-positive cortical progeni-tors,double-stained for nestin(Fig.4a–c).Notably,analysis of the labelling index,quitting fraction,and the expression of specific neural markers at the same stage of corticogenesis indicated that neural proliferation and differentiation proceeded normally in EphA72/2mutants(see Fig.4g and Supplementary Fig.2).In addition,EphA72/2mutants showed a significant increase inFigure1|Generation of EphA7/ephrin-A5transgenic mice.a,In situ hybridization(ISH)on forebrain sections shows complementary expression patterns of ephrin-A5and EphA7at E10.5and P0.DT,dorsal telencephalon; VT,ventral telencephalon.Arrowheads show the approximate boundaries of the neocortex.b,A BAC centred on EphA7exon-1was targeted with a conditional expression cassette to generate a transgenic mouse line (TGA7A5)allowing expression of eGFP under the control of EphA7regulatory sequences.After crossing this line with Emx1-Cre mice(TG/ Emx1-Cre)the eGFP cassette is removed by Cre recombinase,allowing expression of ephrin-A5in the DT.c,d,ISH on E12.5(c)and P0(d)brain sections illustrates the distribution of ephrin-A5,EphA7and the eGFP transgene.Arrowheads indicate boundaries of expression for ephrin-A5 versus EphA7/eGFP.Medial(M)is to the left(a,c),caudal is to the left(d), dorsal(D)is at the top in(a,c,d).Scale bar,200m m.NATURE|Vol435|30June2005LETTERSFigure2|Reduction in cortical size in TG/Emx1-Cre mutants.a,FromE14.5to P0the size of the cortex(Cx,arrows)of TG/Emx1-Cre mice is severely reduced,but the rest of the brain(MB,midbrain;HB,hindbrain)is normal in size.Quantification of cortical size at E16.5reveals a threefold reduction in TG/Emx1-Cre mice(results represent mean corticalarea^s.e.m.;P¼0.0022,n¼3per genotype).b,Coronal brain sections stained with cresyl violet,illustrating the reduction of the cortical plate in TG/Emx1-Cre mice(arrows and arrowheads).Scale bar,500m m.c,d,Coronal brain sections of mice at E12.5(c),E14.5and E16.5(d)stained with nestin,BrdU,Tuj1and MAP2.Despite the size reduction(red arrowheads),the overall cellular organization in the cortical plate(CP), intermediate zone(IZ)and ventricular zone(VZ)is normal in TG/Emx1-Cre mutants.Arrows in c indicate nestin-and BrdU-stained cortical cells. Scale bar in c and d,250m m.e,BrdU labelling(green)and Ki67staining (red)reveal normal labelling index and quitting fraction in the cortex of TG/Emx1-Cre mutants.PP,cortical preplate.Results represent the mean number^s.e.m.of counted cells(detected by BrdU or Ki67)from three animals per genotype.P¼0.2and0.9for labelling index after24h and1h, respectively;P¼0.4for quitting fraction.LETTERS NATURE|Vol435|30June2005Figure3|Ephrin-A/EphA stimulation triggers cortical progenitor cell apoptosis in vivo and in vitro.a,b,At E11.5(a)and E12.5(b),the DT of TG/Emx1-Cre animals shows an increase in the number of apoptotic cells,as labelled by TUNEL(red)or immunostained for activated caspase-3(red; Hoechst stain in green).Levels of apoptotic cells correlate with the expression of the ephrin-A5transgene(Flag-ephrin-A5,detected by ISH). Arrowheads indicate the limits of DT versus VT.c,Dual labelling ofTG/Emx1-Cre cortex with nestin(green)and caspase-3(red)shows that most apoptotic cells are nestinþ(white arrows).d,At later stages(E17.5),no increase in apoptosis is detected in the VZ,but a few apoptotic cells are found in the CP.Scale bar in b and d,125m m.e,Quantification of TUNELþcells in the cortex.Results represent mean number^s.e.m.of TUNELþcells normalized to the number of brain sections used(n¼3animals per genotype).Single asterisk,P¼0.02;two asterisks,P¼0.005;three asterisks,P,0.0001.f,Treatment of dissociated cortical progenitors with clustered ephrin-A5–Fc(100nM)induces a rapid increase in cell death, which is inhibited by the pan-caspase inhibitor z-V AD-fmk.Results represent the mean value^s.e.m.from six independent experiments performed in duplicate.Single asterisk,P¼0.0112(n¼6);two asterisks, P¼0.0065(n¼6).g,Clustered ephrin-A5–Fc(100nM)also induces a rapid increase in caspase-3activity.Single asterisk,P¼0.0411(n¼11);two asterisks,P¼0.0021(n¼11).Results represent the mean value^s.e.m. from11independent experiments performed in duplicate.All values are normalized to apoptosis under control conditions(100nM clustered Fc fragments).NATURE|Vol435|30June2005LETTERSFigure4|Decrease in cortical progenitor cell apoptosis and increase in forebrain size in EphA72/2mutants.a,Coronal brain sections immunostained for activated caspase-3(red,Hoechst in green)illustrate the decrease in apoptotic cell numbers in mutant cortex.Scale bar,125m m. b,Co-staining with nestin(green)and TUNEL(red)shows that most apoptotic cells correspond to nestinþcortical progenitors(white arrows). c,Quantification of cell death in E13.5EphA72/2and EphA7þ/2littermates. Results show mean values^s.e.m.of counted cells normalized to the number of brain sections counted(n¼5–7animals of each genotype). Single asterisk,P¼0.030(n¼5per genotype);two asterisks,P¼0.031 (n¼7per genotype).d,Results represent the mean value^s.e.m.of the cortical area at E13.5in EphA72/2and EphA7þ/2littermates,measured at two rostro-caudal levels(posterior and anterior).Single asterisk,P¼0.030; two asterisks,P¼0.004;three asterisks,P¼0.006(n¼7per genotype). e,f,Forebrain overgrowth in some EphA72/2mice,with ectopic proliferation(arrows in f)and cortical foldings(arrowhead in f).Scale bar, 250m m.g,BrdU labelling(green,performed after1h at E11.5or24h at E13.5)and Ki67staining(red).Labelling index and quitting fraction were normal in the cortex of EphA72/2mutants.Results represent the mean value^s.e.m.of counted cells(n¼5animals per genotype).P¼0.3and 0.47for the labelling index after24h and1h,respectively;P¼0.4for the quitting fraction.LETTERS NATURE|Vol435|30June2005cortical size(,20%,at different rostro-caudal levels at E13.5) (Fig.4d).Moreover,in a small proportion of cases(10%), EphA72/2embryos showed exencephalic overgrowth of forebrain tissue(Fig.4e)both in the ventral forebrain and cerebral cortex, characterized by heterotopic foci of proliferation and aberrant cortical foldings(Fig.4f).Together,the data indicate that ephrin/Eph signalling triggers pro-apoptotic pathways in early cortical progenitors,thereby controlling their number,and hence thefinal size of the forebrain.Although the prominent role of programmed cell death of post-mitotic neurons in brain development has been well established,the physiological meaning of apoptosis in neural progenitors has remained less clear11.Its influence on brain size was revealed by the analysis of mice in which apoptosis effectors and regulators have been knocked out:caspase-3–9and Apaf1mutants all display a severe exencephalic overgrowth of brain structures and reduction in neural progenitor apoptosis11,12,strikingly reminiscent of our observations in some EphA72/2mutants(Fig.4e,f).Although the low penetrance of this extreme phenotype did not allow us to establish a strict correlation with abnormal apoptosis rates,it probably results from a more severe dysregulation of cell death at earlier stages of forebrain development, as previously described for caspase mutants.A recent study identified caspases-3–9among genes that underwent positive selection during primate evolution16,suggesting that regulation of cell death might be one of the mechanisms underlying the expansion of the human cerebral cortex.Region-specific apoptosis of neural progenitors has also recently been proposed as a mechanism to restrict in time and space the size of distinct neural stem cell lineages in the Drosophila central nervous system17.In this context,future work will be needed to determine which ephrin ligand(s)interact physiologically with EphA7during early cortical neurogenesis.Ephrin-A5is present in the ventral telencephalon(see Fig.1and Supplementary Fig.3), suggesting that tangentially migrating ventral neurons could display ephrin-A5in the developing cortex.Ephrin-A3,expressed early in the cortical plate,and ephrin-A2,expressed in the ventricular zone (Supplementary Fig.3),could also act synergistically as cortical EphA7-ligands to provide spatial control of neural progenitor apoptosis.Ephrins have previously been proposed to control cell survival in vitro18,19,but with no physiological implication of ephrins in programmed cell death.Recent data have implicated apoptosis effectors and regulators in the control of axon or cell guidance20,21. Conversely,other cell and axon guidance factors have been shown to be involved in the control of cell death during development and in oncogenesis22–26.In most cases,apoptosis was shown to result from lack of activation of the receptors by their ligand,leading to the concept of dependence receptors.However,in the case of ephrins,apoptosis seems to result from direct stimulation by the ligand,similarly to Fas-ligand or neurotrophin-induced cell death27,28.Ephrin/Eph signalling was recently reported to mediate an anti-proliferative effect on adult neural progenitors,and thereby to influence neurogenesis in the adult olfactory bulb29.However,our study demonstrates a direct pro-apoptotic effect of ephrins on embryonic cortical progenitors,with no influence on their prolifer-ation.This further illustrates that ephrins,like neurotrophins,have evolved as pleiotropic factors that can control very different func-tions depending on the cellular context.Although the mechanisms allowing ephrin signalling to control guidance,apoptosis or prolifer-ation in a cell-specific manner remain unknown,the identification of ephrin/Eph receptor genes as positive regulators of apoptosis during forebrain neurogenesis uncovers a novel signalling pathway that could potentially be involved in other aspects of developmental or stem cell biology,and in oncogenesis.METHODSTransgenic mice.Generation and genotyping of Emx1IREScre(Emx1-Cre)knock-in and EphA7knockout mice were described previously9,13.For the generation of TGA7A5mice,a BAC homologous recombination method was used7,8(see Supplementary Information for details).A BAC clone of200 kilobases(kb),centred on the Epha7gene,was targeted with a conditional cassette containing an eGFP expression cassetteflanked by loxP sites,followed by Flag-tagged human ephrin-A5complementary DNA and a kanamycin-resistance cassette(Fig.1b).Homologous recombinant BACs,in which the first exon was replaced by the conditional cassette,were obtained using established methods7.TGA7A5mice were generated using microinjection of the recombinant BAC in C57BL/6£CBA F1zygotes and were screened using Southern blotting and polymerase chain reaction(PCR).In situ RNA hybridization and immunohistochemistry.In situ RNA hybrid-ization using digoxigenin-labelled RNA probes onfixed brain cryosections was performed as described30.Immunodetection was performed using standard methods(antibodies used are described in the Supplementary Methods).All stainings were performed on at least three embryos from each genotype tested.Quantitative studies of cell proliferation,cell cycle exit and cortical size.For BrdU labelling,timed-pregnant female mice were injected intraperitoneally with a single pulse(50mg kg21body weight)of BrdU,killed after1h or24h,and embryosfixed by perfusion with4%paraformaldehyde.Simultaneous staining of Ki67and BrdU,and quantification of labelling index and quitting fraction were performed according to the methods in ref.10.For each brain analysed,at least400cells,distributed across four sections,were counted.To quantify cortical size,cortical area was measured on two sets of six sections for each animal, corresponding to definedfiduciary marks(rostral pole of the thalamus,caudal pole of the internal caspule).Statistical analyses were performed using Student’s t test.Culture of dissociated cortical progenitors.Cortex was dissected from E12.5 embryos and dissociated mechanically in L15medium(Gibco)containing 30mM glucose.The suspension was centrifuged at2,000g for4min at208C and the pellet resuspended in culture medium(70%basal Eagle medium,25% Hanks’balanced salt solution,25mM glucose,1mM glutamine,50U ml21 penicillin,50m g ml21streptomycin,5%horse serum).Dissociated cortical cells were plated at a density of6–8£105cells per well in6-well plates pre-coated with poly-D-lysine(33m g ml21)and laminin(3m g ml21).Cultures were main-tained for24h in a humidified atmosphere at5%CO2,378C,then stimulated with100nM preclustered ephrin-A5–Fc or control Fc fragment,with or without 40m M z-V AD-fmk(R&D Systems).After treatment,cells were eitherfixed in4% paraformaldehyde in PBS for15min at room temperature(for TUNEL assays) or trypsinized(for caspase-3activity assays).TUNEL detection and caspase activity assays.TUNEL assays were performed using a commercially available kit and following to manufacturer’s instructions (TMR red,Roche Diagnostics).The number of apoptotic cells(detected by TUNEL assay and activated caspase-3immunoreactivity)was counted through-out the cerebral cortex in EphA72/2and EphA7þ/2littermates,and normalized to the number of brain sections analysed.For in vitro assays,the percentage of apoptotic cells was determined by counting the number of TUNEL-positive cells in1,000cells(visualized by Hoechst staining)for each duplicate from each independent experiment.Enzymatic assays for caspase-3activity were per-formed using a commercially available kit according to manufacturer’s 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Supplementary Information is linked to the online version of the paper at /nature.Acknowledgements We thank G.Vassart for continuous support and interest, and members of IRIBHM for help and advice,in particular muni,N.Gaspard,mbot,S.Pietri,J.Seibt and F.Pauwels.We also thank the Development Studies Hybridoma Bank;T.Ciossek for sharing the EphA7mutant mice;C.d’Enfert and J.-M.Ghigo for the gift of the pKOBEGA plasmid;A.de Kerchove for discussions about BAC recombineering;A.Deplano for help with pulse-field electrophoresis;S.Tajbakhsh for the gift of thefloxed eGFP cassette;F.Stewart for the gift of the frt-Neo cassette and the294-Flp bacteria;A.Goffinet for the gift of mouse reelin plasmid;R.Hevner for the gift of Tbr1 antibody;and F.Polleux for critically reading the manuscript.This work was funded by grants from the Belgian Funds for Scientific Research(FNRS and FRSM),the Belgian Queen Elizabeth Medical Foundation,the Belgian Interuniversity Attraction Poles Programme(to P.V.),and grants from the American Cancer Society and National Institutes of Health(to K.R.J).P.V.and C.L.are Research Associates,and L.P.is a Research Fellow,of the FNRS.A.D. and V.D.were supported by the Fonds pour la Recherche en Industrie et Agriculture(FRIA).Author Information Reprints and permissions information is available at/reprintsandpermissions.The authors declare no competingfinancial interests.Correspondence and requests for materials should be addressed to P.V.(pvdhaegh@ulb.ac.be).LETTERS NATURE|Vol435|30June2005。