CORRECTION

Correction: Mice humanised for the EGF receptor displayhypomorphic phenotypes in skin, bone and heartMaria Sibilia, Bettina Wagner, Astrid Hoebertz, Candace Elliott, Silvia Marino, Wolfram Jochum andErwin F. Wagner

There was an error published in Development 130, 4515-4525.

In Fig. 6, owing to an error in figure assembly during preparation of the proofs, the incorrect image (a duplication of panel D) was displayedin panel G. The corrected figure appears below.

We apologise to the authors and readers for this mistake.

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© 2016. Published by The Company of Biologists Ltd | Development (2016) 143, 4755 doi:10.1242/dev.146209

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IntroductionThe epidermal growth factor receptor (EGFR/Erbb1) belongsto a family of structurally related tyrosine kinase receptors,including Erbb2/neu, Erbb3 and Erbb4, which upon ligandbinding can form homo- and heterodimers (Olayioye et al.,2000; Schlessinger, 2000; Yarden and Sliwkowski, 2001).Several growth factors such as epidermal growth factor (EGF),transforming growth factor α(TGFα), amphiregulin, heparin-binding EGF (HB-EGF), β-cellulin and epiregulin can bind theEGFR and induce receptor dimerisation. Subsequent activationof the intrinsic tyrosine kinase induces complex downstreamsignalling pathways, which can instruct cells either toproliferate, differentiate and/or survive. Little is known abouthow these cellular responses are regulated in different celltypes in vivo (Olayioye et al., 2000; Yarden and Sliwkowski,2001).

Most of the current knowledge about the function of theEGFR and its family members during normal developmentderives from the analysis of mutant mice. Mice lacking Erbb2,Erbb3 and Erbb4 die at midgestation, because of cardiacdysfunction associated with lack of ventricular trabeculation,and display abnormal development of the peripheral nervoussystem (Gassmann et al., 1995; Lee et al., 1995; Riethmacher

et al., 1997). Inactivation of the Egfr in mice reveals thatmutant mice die at midgestation (129/Sv), birth (C57BL/6) orcan live up to postnatal day 20 (MF1, C3H and CD1)depending on their genetic background. They exhibit epithelialand neural phenotypes as well as craniofacial malformations(Miettinen et al., 1995; Miettinen et al., 1999; Sibilia andWagner, 1995; Threadgill et al., 1995). Death in utero resultsfrom a placental defect, as the embryonic lethality can berescued by generating aggregation chimeras between Egfr–/–

and tetraploid wild-type embryos, the latter contributingexclusively to extra-embryonic tissues (Sibilia et al., 1998).Independent of the genetic background, surviving Egfr–/– micedevelop a progressive neurodegeneration in the olfactory bulbsand frontal cortex, which is characterised by massive apoptoticcell death affecting both neurones and astrocytes (Kornblum etal., 1998; Sibilia et al., 1998). Ectopic neurones are alwaysdetected in the white matter of the hippocampus, suggestingthat EGFR signalling might be important for neuronalmigration (Sibilia et al., 1998). The lack of EGFR does notseem to affect the self-renewing potential of neural progenitorsin response to FGF (Tropepe et al., 1999). However, EGFR iscrucial for astrocyte development, as cerebral cortices fromEgfr–/– mice contain lower numbers of astrocytes, which

Mice lacking the epidermal growth factor receptor (EGFR)develop epithelial defects and a neurodegenerative diseaseand die within the first month of birth. By employing aconditional knock-in approach using the human EGFRcDNA mice humanised for EGFR (hEGFRKI/KI ) weregenerated. Homozygous hEGFRKI/KI mice are viable andlive up to six months. However, these mice are growthretarded and show skin and hair defects similar to Egfr–/–

mutants. Interestingly, the neurodegeneration is fullyrescued in hEGFRKI/KI mice, however, they develop a severeheart hypertrophy with semilunar valve abnormalities.Moreover, hEGFRKI/KI mice display acceleratedchondrocyte and osteoblast differentiation, a phenotype

that is also present in Egfr–/– mice and has not beenpreviously described. The severity of the phenotypescorrelates with the expression levels of the hEGFRKI allele,which is not efficiently expressed in epithelial and bonecells, but is expressed at similar and even higher levels asthe endogenous Egfrin brain and heart. These resultsdemonstrate that mice humanised for EGFR display tissue-specific hypomorphic phenotypes and describe a novelfunction for EGFR in bone development.

Key words: Bone, Hair growth, Heart hypertrophy, HumanisedEGFRknock-in mice, Skin

Summary

Mice humanised for the EGF receptor display hypomorphicphenotypes in skin, bone and heartMaria Sibilia 1,2,*, Bettina Wagner 1, Astrid Hoebertz 2, Candace Elliott 2,†, Silvia Marino 3,‡, Wolfram Jochum 2,‡

and Erwin F. Wagner 2

1Department of Dermatology and Biomolecular Therapeutics (BMT), University of Vienna, Medical School, Brunnerstr. 59, A-1235Vienna, Austria2Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, A-1030 Vienna, Austria3Division of Molecular Genetics and Centre of Biomedical Genetics, The Netherlands Cancer Institute, 1066CX Amsterdam, TheNetherlands*Author for correspondence (e-mail: maria.sibilia@univie.ac.at)†Present address: School of Botany, University of Melbourne, Victoria 3010, Australia‡Present address: Institute of Clinical Pathology, University Hospital Zurich, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerland

Accepted 12 June 2003

Development 130, 4515-4525© 2003 The Company of Biologists Ltddoi:10.1242/dev.00664

Research article

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display a severe proliferation defect in vitro (Kornblum et al.,1998; Sibilia et al., 1998). Therefore, the EGFR is mostprobably required for the proliferation/differentiation ofastrocytes and for preserving brain integrity after birth.

In addition to the neural defects, Egfr–/– mice also displayseveral abnormalities in epithelial tissues. They are born withopen eyes and show impaired epidermal as well as hair follicledifferentiation and fail to develop a hairy coat most likelybecause EGFR signalling is necessary for maintenance of hairfollicle integrity (Miettinen et al., 1995; Sibilia and Wagner,1995; Threadgill et al., 1995). However, Egfr–/– mice do notsurvive beyond the termination of the first hair cycle and,therefore, a careful analysis of EGFR function during skin andhair follicle development could not be performed. Graftingexperiments of Egfr–/– skin explants on athymic nude micesuggest that Egfr–/– follicles cannot proceed from the anagento telogen phase of the hair cycle (Hansen et al., 1997). Thesealterations eventually lead to necrosis and disappearance of thefollicles, accompanied by strong infiltration of the skin withinflammatory cells (Hansen et al., 1997). A similar but moresevere skin and hair phenotype could be detected in transgenicmice expressing a dominant negative (DN) Egfr (CD533) fromthe keratin 5 (K5) promoter (Murillas et al., 1995). However,in these mice it could not be excluded that other Erbb receptorswere also inhibited by the DN EGFR, thereby exacerbating theskin defects. Milder phenotypes characterised by a ‘wavy’ coatand curly whiskers are observed in mice deficient for the geneencoding TGFαand in mice homozygous for the hypomorphicEgfr allele waved 2 (wa2), which carry a point mutation in thekinase domain of the EGFR resulting in a drastically reducedkinase activity (Fowler et al., 1995; Luetteke et al., 1994;Luetteke et al., 1993; Mann et al., 1993).

Numerous studies have documented alterations in EGFRsignalling pathways in the development of human neoplasms(Olayioye et al., 2000; Yarden, 2001). Amplifications,rearrangements and overexpression of the Egfrgene have beenshown to occur at high frequency in human squamous cellcarcinomas and glioblastomas (Olayioye et al., 2000; Yarden,2001). The first in vivo evidence for a direct involvement ofthe EGFR in epithelial tumour development stems from theanalysis of transgenic mice expressing an activated form of theRas activator son of sevenless (Sos) from the K5 promoter(Sibilia et al., 2000). All K5-Sostransgenic mice develop skintumours only in the presence of a functional EGFR. K5-Sostransgenic skin papillomas in a EGFR mutant backgroundshow increased apoptosis and are more differentiatedindicating that signalling through the EGFR enhances thesurvival and inhibits differentiation of epidermal cells (Sibiliaet al., 2000). This is supported by the fact that Egfr is moststrongly expressed in the proliferating compartments of thebasal layer of the epidermis and in the outer root sheath of thehair follicles, and the number of receptors decreases askeratinocytes enter the pathway of terminal differentiation andmigrate to the suprabasal layers of the epidermis (Peus et al.,1997; Sibilia and Wagner, 1995; Stoll et al., 2001).

The function of the EGFR in adult mice could not beaddressed because of the early postnatal lethality of EGFR-deficient mice. Here, we employed a knock-in strategy togenerate mice in which the endogenous mouse Egfr is replacedby a human EGFRcDNA that is flanked by loxP sites(hEGFRKI allele). This approach enables the generation of

humanised EGFR mice (hEGFRKI/KI mice) to study thefunctional homologies between the mouse and human EGFR ina conditional way. In addition, the phenotypic consequences ofknocking-in well characterised human EGFR mutants can alsobe addressed. Homozygous hEGFRKI/KI mice are growthretarded but can survive for up to 6 months after birth. However,they display skin and hair growth defects similar to survivingEgfr–/– mice. Interestingly, the neurodegeneration is fullyrescued, although hEGFRKI/KI mice develop a severe hearthypertrophy with abnormalities in semilunar valvedevelopment. Accelerated chondrocyte and osteoblastdifferentiation are detected in both hEGFRKI/KI and Egfr–/–

mice, suggesting that EGFR signalling negatively regulatesbone cell differentiation. The severity of the phenotypescorrelates with the expression levels of the hEGFRKI allele invarious tissues. In bone cells and epithelial tissues theexpression of the hEGFRKI allele is severely reduced, whereasthe hEGFRKI allele is expressed at similar levels as theendogenous mouse gene in the brain thereby rescuing theneurodegeneration. Moreover, higher levels of expression of thehEGFRKI allele are detected in the heart and are likelyresponsible for the development of the heart hypertrophy. Theseresults demonstrate that mice humanised for EGFR displaytissue-specific hypomorphic phenotypes thereby uncoveringnovel functions of the EGFR in bone and heart development.

Materials and methodsGeneration of targeting construct and hEGFRKI/KI miceTo generate the EGFR knock-in vector the pGNA-based knock-outvector described previously (Sibilia and Wagner, 1995) was modifiedas follows. The lacZgene was removed and replaced by a multiplecloning site. A loxP-flanked human EGFR (hEGFR) cDNA wasinserted into the multiple cloning site and the vector was linearisedwith XbaI before electroporation into feeder-dependent GS-1 ES cells.Selection and screening of ES cell colonies by PCR and Southern blotanalysis was performed as previously described (Sibilia and Wagner,1995). Two correctly targeted ES cell clones were injected intoC57BL/6 blastocysts and germline male chimeric mice were obtainedwith both clones. Chimeras were mated to 129/Sv and/or C57BL/6females to obtain hEGFRKi/+ mice of inbred 129/Sv and mixed 129/Sv× C57BL/6 background, which were used for further breeding andgeneration of hEGFRKI/KI mice. Genotyping of the mice wasperformed by PCR as previously described (Sibilia and Wagner,1995).

HistologyMice were sacrificed and tissues were fixed in 4% PBS-bufferedformaldehyde and embedded in paraffin wax. Sections (5 µm) werecut and stained with Haematoxylin and Eosin (SigmaImmunochemicals) according to standard procedures.Immunohistochemical staining for Ki67 (Novocastra, NCL-Ki67p,1:1000) was performed using the ABC staining kit (VectorLaboratories) according to the manufacturer’s recommendations. Forhistomorphometric analysis, Haematoxylin and Eosin stained cross-sections of hearts from 2.5- to 3.5-month-old hEGFRKI/KI mice (n=4)and age-matched controls (n=4) were evaluated using an Axioskopmicroscope (Carl Zeiss, Oberkochen, Germany) with integratedmorphometric device at 400×magnification. The size ofperpendicularly cut cardiomyocytes was determined in thesubendocardial layers of the left ventricle.

RNAse protection assayTotal RNA was extracted and purified from various organs using

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TRIzol reagents (Gibco) and subjected to RNAseprotection analysis as previously described (Fleischmannet al., 2000). For the generation of the EGFR riboprobe,the region encompassing the 3′ end of the Egfrmousepromoter, the loxP site and the 5′ end of the hEGFRcDNA were cloned into SP64 plasmid, linearised andtranscribed in vitro using the in vitro translation Kit fromStratagene.

Western blot analysis and in vitro EGFRautophosphorylation assayProtein lysates were prepared from various tissues,cleared by centrifugation and either processed for westernblot analysis or subjected to immunoprecipitationemploying the rabbit anti EGFR antibody #1001 (SantaCruz) as previously described (Sibilia et al., 2000; Sibiliaand Wagner, 1995). Immunoprecipitated EGFRcomplexes were resuspended in 20 µl HNTG (20 mMHEPES pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10%Glycerol) containing 0.4 µl γ32PdATP (10 µC/µl,Amersham) and incubated for 15 minutes on ice. Kinasereactions were stopped and protein complexes separatedon 10% SDS-PAGE. Gels were dried at 80°C undervacuum and exposed to X-ray films (Amersham). Westernblot analysis was performed with the anti EGFR antibody#06-129 (Upstate, 1:1000).

Astrocyte culturesPrimary astrocyte cultures were prepared from brains ofnewborn mice as previously described (Sibilia et al.,1998). Cells were plated in DMEM/F12 (1:1, GIBCO)culture medium containing 10% foetal calf serum (PAA)and after reaching confluency passaged at a 1:3 split ratio.Before replating, viable cell numbers were determinedusing a Neubauer-type haemocytometer with Trypan Bluestaining (Sigma). The primary cultures consisted of >95%GFAP- (Sigma) positive astrocytes.

Osteoblast culturePrimary osteoblasts were isolated from calvariae ofneonatal hEGFRKI/KI, Egfr–/– and wild-type mice.Calvariae were sequentially digested for 10 minutes at37°C in α-MEM (GIBCO) containing 0.1% collagenaseand 0.2% dispase (Roche). Cells isolated in fractions 2-5were combined as an osteoblastic cell population,expanded twice for 2-3 days in α-MEM with 10% foetalcalf serum (PAA), and either replated in α-MEM forproliferation assays (105 cells/well in a 6-well plate), or inα-MEM supplemented with 5 mM β-glycerolphosphateand 100 µg/ml ascorbic acid (Sigma) (105 cells/well in a24-well plate) for bone nodule assays.

ResultsGeneration of hEGFRKI/KI miceTo generate mice humanised for EGFR a gene-targeting vector was constructed, which afterhomologous recombination replaces parts of thefirst exon of the mouse Egfrgene with the loxP-flanked human EGFRcDNA (referred to ashEGFRKI allele, Fig. 1A). This vector is a modifiedversion of the one previously employed to generate Egfrknockout (Egfr–/–) mice (Sibilia and Wagner, 1995). Theconstruct was electroporated into ES cells and correctlytargeted clones were identified by PCR and Southern blot

analysis (data not shown). Two independent ES clones wereinjected into blastocysts and both formed germline chimeras.Heterozygous hEGFRKI/+ mice of inbred 129/Sv or mixed129/Sv×C57BL/6 background were intercrossed to generatehEGFRKI/KI mice, which were identified by PCR analysis

Fig. 1.Generation of hEGFRKI/KI mice. (A) Schematic representation of thetargeting strategy employed to insert a floxed human EGFRcDNA (hEGFR) intothe mouse Egfrlocus. The full-length hEGFRexpression cassette (grey box)contains its own ATG and polyA (pA) site and is flanked by loxP sites (greytriangles). After homologous recombination, the hEGFRcDNA will be insertedinto the first exon of the mouse Egfrgene (black box) and placed under thecontrol of the endogenous mouse Egfr promoter. This correctly targeted allelewill be referred to as the hEGFRknock-in allele (hEGFRKI). The neomycinresistance gene (RSV-neo) and the thymidine kinase gene (HSV-tk) are shown.The broken lines delineate the homology regions in the targeting vector, thehorizontal bar indicates the Southern blot probe, and the black arrowheadsindicate the PCR primers employed for genotype analysis. X, XbaI; BX, BstXI;N, NdeI. (B) PCR analysis of genomic DNA isolated from the progeny ofhEGFRKI/+ intercrosses. (C) Photograph of 6-week-old hEGFRKI/KI andlittermate control mice showing that hEGFRKI/KI mice are smaller and displayshort fur hair. (D) Weight representative of a hEGFRKI/KI and control littermatemouse at different postnatal ages. At all stages, hEGFRKI/KI mice aresignificantly smaller than their littermates. w, weeks; m, months

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(Fig. 1B). Although no viable hEGFRKI/KI mice wereobtained in a 129/Sv background (0/68), ~7% of hEGFRKI/KI

mice were born alive in a mixed 129/Sv×C57BL/6background (Fig. 1C). When bred with Egfr+/– mice, noviable hEGFRKI/– mice could be obtained, even in the mixed129/Sv×C57BL/6 background, indicating that one copy of thehEGFRKI allele is not sufficient to rescue the lethality ofEgfr–/– mice (data not shown). Therefore, the phenotypicanalysis was performed on surviving hEGFRKI/KI mice ofmixed 129/Sv×C57BL/6 background, which at birth could beidentified by their open eyes and curly whiskers (data notshown). The development of the first coat hair was delayedand the hair was generally much shorter and sparse (Fig. 1C).Some of the hEGFRKI/KI mice survived up to 7 months afterbirth, but were always significantly smaller than their controllittermates (Fig. 1C,D). These observations strongly suggestthat the hEGFRKI allele is most probably not efficientlyexpressed leading to a hypomorphic phenotype.

Expression analysis in hEGFRKI/KI miceTo compare the expression levels of the hEGFRKI allele to theendogenous mouse Egfr, total RNA was extracted from variousorgans of a hEGFRKI/+ heterozygous mouse. Ribonuclease(RNAse) protection assay was performed using a riboprobe,which recognises both the endogenous and the hEGFRKI

transcripts, but the respective protected bands are different insize. Surprisingly, the hEGFRKI allele was not expressed atlower levels than the endogenous Egfr in every tissue (Fig. 2A).Organs, which are mostly composed of epithelial cells such asliver, lung, skin and stomach expressed significantly lowerlevels of the hEGFRKI allele compared with the endogenousallele (Fig. 2A). By contrast, in brain regions such as cortexand hippocampus, in kidney and in thymus the hEGFRKI allelewas expressed at similar levels as the endogenous mouse allele(Fig. 2A). Interestingly, in the heart the hEGFRKI allele seemedto be expressed at even higher levels than the endogenous one(Fig. 2A). These results suggest that the hEGFRKI allelebehaves like a hypomorph particularly in epithelial tissues.

To investigate the expression of the hEGFRKI allele at theprotein level, lysates were prepared from brain, liver and heartof hEGFRKI/KI, hEGFRKI/+ , Egfr+/+ and Egfr–/– mice. Westernblot analysis employing an anti-EGFR antibody recognisingboth the mouse and the human EGFR protein showed thatEGFR protein of similar size was produced in the brain andliver of wild-type and hEGFRKI/KI mice (Fig. 2B). The levelsof EGFR protein expressed in the brain were similar tocontrols, but severely reduced in the liver of hEGFRKI/KI micecorrelating with the mRNA expression results (Fig. 2A,B). Anin vitro EGFR autophosphorylation assay, which is usuallymore sensitive than western blot analysis, was performed onimmunoprecipitated EGFR complexes. Consistent with theprevious results, EGFR protein levels were similar to controlin the brain of hEGFRKI/KI mice, whereas they were lower andalmost absent in the liver (Fig. 2C). Using both assays, EGFRprotein could not be detected in the hearts of all mice analysed,most probably owing to very low EGFR levels (Fig. 2C anddata not shown).

Rescue of the brain phenotypes in hEGFRKI/KI miceIn the brain, the hEGFRKI allele is expressed at similar levels

Development 130 (19) Research article

Fig. 2.The hEGFRKI allele is not efficiently expressed in epithelialtissues. (A) Expression of the endogenous mouse Egfr and thehEGFRKI mRNAs measured by RNAse protection assay. Theanalysis was performed on total RNA isolated from various organs ofa heterozygote hEGFRKI/+ male mouse employing an antisense Egfrriboprobe, which detects both the endogenous mouse Egfr and thehEGFRKI transcripts. In addition to 93 bp of nonspecific sequence(broken line), this riboprobe encompasses the region of the hEGFRKI

allele bridging the Egfrmouse promoter, the loxP site and the 5′ endof the hEGFRcDNA. The input probe and the protected fragmentsare depicted schematically and the black triangle indicates the loxPsite. A mouse S16 riboprobe (lower panel) was used as an internalcontrol for equal sample loading in each lane. (B) Western blotanalysis showing EGFR protein expression in brain and liver of miceof different genotypes. Immunoblotting was performed on totalprotein extracts using an anti-EGFR antibody recognising human andmouse EGFR and anti-tubulin was used as a control for proteinloading. (C) In vitro EGFR autophosphorylation assay measuringEGFR protein levels. Prior to the kinase assay, protein lysates fromvarious organs were immunoprecipitated with an anti-EGFRantibody recognising both the mouse and the human EGFR protein.Equal loading of protein was verified by Coomassie Blue staining(data not shown)

4519Skin, bone and heart defects in hEGFRKI/KI mice

than the endogenous Egfr (Fig. 2A). In order to investigatewhether in hEGFRKI/KI mice the brain phenotypes were rescued,brains were isolated at different stages after birth and comparedto Egfr–/– and controls. At all stages analysed, brains ofhEGFRKI/KI mice appeared structurally normal and comparableto controls (Fig. 3A,B). Histological sections through the frontalcortex of a 3-month-old hEGFRKI/KI mouse did not reveal anysigns of degeneration and apoptosis and the cortical tissuesappeared normal and comparable to controls (Fig. 3C,E). Bycontrast, in the cortex of Egfr–/– mice morphological changesconsisting of nuclear condensations and diminished celldensities could be observed already at postnatal day 7 (Fig. 3G)(Sibilia et al., 1998). Similar to controls, in hippocampal sectionsof hEGFRKI/KI mice no ectopic neurones could be observed atany stage, whereas in Egfr–/– mice nests of ectopic neuroneswere always present (Fig. 3D,F,H) (Sibilia et al., 1998). Inaddition, other brain regions such as the olfactory bulb, thalamusand cerebellum of hEGFRKI/KI mice appeared normal andcomparable with controls (data not shown).

We next isolated astrocytes from the frontal cortex ofnewborn hEGFRKI/KI mice and analysed their proliferationpotential in vitro. As shown in Fig. 3I, the proliferation rate ofhEGFRKI/KI primary astrocytes was comparable withhEGFRKI/+ and Egfr+/+ astrocytes. As the hEGFRKI allele isflanked by loxP sites, the hEGFR was deleted by breedinghEGFRKI/KI mice to GFAP-cretransgenic mice, which expressthe Cre recombinase in astrocytes (Marino et al., 2000).Removal of the EGFR severely compromised the proliferationcapacity of primary astrocytes and after 42 days in culture theircumulative cell number reached only about 15% of the valuesobtained with hEGFRKI/KI and control astrocytes (Fig. 3I).Overall, the proliferation rate of hEGFRKI/KI GFAP-creastrocytes was reduced to the same extent as in Egfr–/–

astrocytes (Fig. 3I) (Sibilia et al., 1998). Southern blot analysisconfirmed the absence of the Egfrgene in hEGFRKI/KI GFAP-cre astrocytes (Fig. 3J). These results indicate that expressionof the hEGFRKI allele is fully rescuing the brain and astrocytedefects of Egfr–/– mice.

Severe hair follicle and hair cycle defects inhEGFRKI/KI miceThe hEGFRKI allele is not efficiently expressed in theskin based on the RNAse protection analysis (Fig. 2A).Moreover, hEGFRKI/KI mice displayed curly whiskersand the development of the first coat hair was impaired(Fig. 1C). These phenotypes greatly resemble the onesobserved in Egfr–/– mice (Miettinen et al., 1995; Sibiliaand Wagner, 1995; Threadgill et al., 1995). However, incontrast to Egfr–/– mice, which do not survive longer thanpostnatal day 20, hEGFRKI/KI mice survive up to 6months after birth and, therefore, represent a usefulmodel to analyse how reduced EGFR expression affectshair development. The skin of hEGFRKI/KI mice wasisolated postnatally at different stages of the hair cycle,after 18 days (end of first cycle, catagen/early telogen),after 1 month (second cycle, end of anagen/earlycatagen) and after 3 months (resting phase, telogen).Histological examination of hEGFRKI/KI skins showed

Fig. 3.Normal brain development in hEGFRKI/KI mice. Dorsalview of the brains of control (A) and hEGFRKI/KI mice (B)isolated 3 months after birth. Histological sections through thefrontal cortex (C,E) and hippocampus (D,F) of control (C,D)and hEGFRKI/KI (E,F) brains showing normal architecture andmorphology. (G,H) Sections of Egfr–/– brains show neuronaldegeneration in the cortex (G), starting around postnatal day 5,and groups of ectopic neurones (arrows) in the white matter ofthe hippocampus (H, arrows) (Sibilia et al., 1998).(C-H) Sections are stained with Haematoxylin and Eosin.

(I) Cumulative cell number of Egfr+/+, hEGFRKI/+ ,hEGFRKI/KI, hEGFRKI/KI GFAP-Creand Egfr–/–

primary cortical astrocytes isolated from newbornbrains showing that hEGFRKI/KI astrocytes display anormal proliferation capacity. Removal of the hEGFRKI

allele in astrocytes of hEGFRKI/KI GFAP-cremiceresults in severe proliferation defects as observed inEgfr–/– astrocytes. (J) Southern blot analysis ofgenomic DNA isolated from astrocytes shown in I andhybridised with the probe shown in Fig. 1A. The bandscorresponding to the different alleles are indicated.EGFR∆: hEGFRKI allele after Cre-mediated deletion.

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striking alterations in the morphology and distribution of hairfollicles. Around day 18, hair follicles of control mouse skingo through a telogen stage and are confined to the upper layerof the dermis (Fig. 4A). By contrast, at this stage follicles ofhEGFRKI/KI mice were still much longer reaching deep into thesubdermal adipose tissue, suggesting that they had failed toenter into catagen and were still in anagen (Fig. 4B,G).hEGFRKI/KI follicles started to appear much larger andhyperplastic compared with control follicles and thisphenotype was even more pronounced at 1 month when theanagen of the second hair cycle had started (Fig. 4C,D). Aftercompletion of the second hair cycle, follicles remain for a longperiod (around 40 days) in telogen (Fig. 4G). During this stage(at the age of 3 months), control hair follicles are short andlocalised in the upper layers of the dermis (Fig. 4E). Bycontrast, in hEGFRKI/KI mice, only very few structurallyabnormal follicles were present in the upper dermis and mostof the follicles, which had remained stuck in the subdermal fatlayers, were degenerated (Fig. 4F). The cell layers composingthe hair follicles were very thin and partly destroyed (Fig. 4F).Severe fibrosis of the dermis and subdermal fat tissue withmassive infiltration of inflammatory cells was observed andhad most likely been triggered by the exposure of naked hairshafts from the degenerating follicles (Fig. 4F and data notshown). The inflammatory infiltrate mainly containedmacrophages, lymphocytes, neutrophils and multinucleatedgiant cells (data not shown). The progressive degeneration andinflammation led to the loss of most follicles over time and themajority of hEGFRKI/KI mice were completely bald at the ageof 5-7 months (data not shown). These results indicate thatEGFR signalling is absolutely required for hair cycleprogression. Moreover, EGFR is also essential for proper hairfollicle differentiation, orientation and migration, as well as topreserve hair follicle integrity during the hair cycle.

Bone cell defects in hEGFRKI/KI mice hEGFRKI/KI and Egfr–/– mice are significantly smaller than theircontrol littermates. Impaired skeletal development is one ofseveral defects that can be responsible for growth retardation.Therefore, we investigated whether bone development wasdefective in hEGFRKI/KI mice. Histological analysis of the longbones at birth revealed that the hypertrophic chondrocyte zone ofthe growth plate was significantly increased in hEGFRKI/KI mice(Fig. 5A,B). This phenotype might be caused either by increasedor reduced expression of the hEGFRKI allele in the bonecompartment. To discriminate between these two possibilities,bone sections from Egfr–/– newborn mice of mixed geneticbackground were analysed. Interestingly, in Egfr–/– long bonesthe zone of hypertrophic chondrocytes was similarly or evenmore increased as in hEGFRKI/KI bones, indicating that thisphenotype is most probably caused by the absence of EGFRexpression in cartilage (Fig. 5B,C). The proliferation ofchondrocytes measured by Ki67 staining on sections was notaffected and comparable in all three genotypes (Fig. 5E). Theexpression of EGFR in chondroblasts was analysed by X-galactosidase staining in Egfr+/– E14.5 foetuses, as the knockoutallele harbours an E. coli lacZgene inserted in-frame downstreamof the first exon of the mouse Egfr gene (Sibilia and Wagner,1995). In control foetuses, EGFR expression was detected inchondroblasts of all ossification centres (Fig. 5D). These resultsshow that EGFR is expressed in chondroblasts during

Development 130 (19) Research article

Fig. 4. Impaired hair cycle and follicle development in hEGFRKI/KI

mice. Histological sections through the skin of control (A,C,E) andhEGFRKI/KI mice (B,D,F) at different stages of the hair cycle. Incontrol skins at 18 days (end of catagen/telogen) the hair follicles areconfined to the upper dermal layers (A, arrows), whereas inhEGFRKI/KI skins, hair follicles accumulate in the subdermal fat tissue(brackets in B,D,F), as they do not progress from anagen to telogen(B). Arrows in B indicate hair follicles that start to be enlarged inmutant skins. (C) At 1 month (late anagen/early catagen) controlfollicles are distributed in the upper dermal and subdermal fat layersand display a normal structure (arrows). (D) Mutant follicles in thesubdermal fat tissue are severely hyperplastic (arrows) at this stage.(E) At 3 months (telogen), control follicles are confined to the upperdermal layers (arrows). (F) In mutant skins, only a few aberrantfollicles are present in the upper dermis (arrowheads) and most of thefollicles stuck in the subdermal fat tissue have degenerated (arrows).Severe fibrosis with infiltration of inflammatory cells can be detectedin the subdermal fat tissue of hEGFRKI/KI skins as evidenced by thestrong eosin (pink) staining. (G) A normal mouse hair cycle profilewith the position where the hair cycle is blocked in hEGFRKI/KI mice.A, anagen; C, catagen; T, telogen; d, days; m, months. All sections arestained with Haematoxylin and Eosin.

4521Skin, bone and heart defects in hEGFRKI/KI mice

development and suggest that EGFR signalling negativelyregulates the maturation of hypertrophic chondrocytes, becausethe zone of hypertrophic chondrocytes is enlarged in its absence.

To test whether loss of the EGFR also influencesosteoblastic differentiation and proliferation, calvarialosteoblasts were prepared from newborn hEGFRKI/KI ,Egfr–/– and control mice. The proliferation potential ofhEGFRKI/KI and Egfr–/– primary calvarial osteoblastswas decreased towards the end of the culture period (Fig.5F,G). Moreover, calvarial osteoblasts derived fromhEGFRKI/KI and Egfr–/– mice showed increaseddifferentiation compared to controls, as assessed by anin vitro mineralisation assay. Compared with controls,hEGFRKI/KI and Egfr–/– osteoblast cultures formedapproximately twice and three times as manymineralised bone nodules, respectively (Fig. 5H,I).Western blot analysis revealed a severe reduction ofEGFR expression in calvarial osteoblasts of hEGFRKI/KI

mice (Fig. 5J). Although differentiation and proliferationdefects were observed in cultured osteoblasts, the bonethickness of newborn hEGFRKI/KI and Egfr–/– mice wascomparable with controls and no overt bone remodellingdefects could be detected (Fig. 5A-C). These resultssuggest that EGFR signalling normally supportsproliferation, but inhibits differentiation of osteoblasticcells, keeping them in an undifferentiated, pre-osteoblastic state.

hEGFRKI/KI mice develop heart hypertrophy hEGFRKI/KI mice can survive up to 6 months after birth,but often sudden death is observed at earlier times. Toinvestigate the possible cause of lethality, hEGFRKI/KI

mice were sacrificed and different organ systems wereanalysed. An increase in heart size was consistentlyobserved, which was already visible at 3 weeks after birthand became more pronounced with age (Fig. 6A).Comparison of heart weights at 3 months of age showedthat hEGFRKI/KI hearts were about twice as heavy ascontrol hEGFRKI/+ and EGFR+/+ hearts (Fig. 6B). This

difference was even more dramatic when the heart weightswere corrected for the body weights considering thathEGFRKI/KI mice are smaller and only about half of the sizeof control mice (Fig. 6B). Histological cross-sections throughthe hearts of hEGFRKI/KI mice revealed a severe hypertrophywith dramatically increased thickness of the left ventricularwall and the interventricular septum at 3 weeks after birth(Fig. 6D). These defects became more severe with increasingage (Fig. 6G). Cardiomyocytes of 2.5- to 3.5-month-oldhEGFRKI/KI mice were hypertrophic and measurements oftheir mean cross-sectional areas revealed a 1.9-fold increasecompared with controls (mean cross sectional area±s.d.:309.7±97.6 µm2 versus 166.3±16.3 µm2). None of thesedefects was ever observed in control hEGFRKI/+ and Egfr+/+

mice (Fig. 6C,F; data not shown). In 3-week-old Egfr–/– mice,the heart size was normal, the heart-to-body weight ratioswere comparable with controls and histological sections didnot reveal signs of hypertrophy (Fig. 6E; data not shown).Cardiac hypertrophies can occur as primary myocardialdiseases or as a consequence of other conditions such as valvedefects leading to valvular stenosis and/or regurgitation (Katz,1990). When compared with controls, histologicalexaminations of hEGFRKI/KI hearts revealed that the cusps ofthe pulmonary and aortic valves were thickened andhypercellular, a condition that most likely resulted from the

Fig. 5.Bone cell defects in the absence of EGFR. Histologicalsections through the tibiae of control (A), hEGFRKI/KI (B) andEgfr–/–mice (C) at postnatal day 1. The black bar marks the zone ofhypertrophic chondrocytes, which is markedly increased in bothhEGFRKI/KI and Egfr–/– long bones. (D) X-galactosidase (X-gal)staining of an E14.5 Egfr+/– foetus showing EGFR expression (bluestain) in the ossification center of the vertebrae. Inset shows a highermagnification of the region boxed in D with arrows indicating X-galstaining in chondroblasts. (A-C) Haematoxylin and Eosin staining;(D) X-gal and Eosin staining. (E) The proliferation of chondrocyteswas measured by Ki67 staining on bone sections of mice of theindicated genotypes. The data represent the mean±s.d. of the numberof Ki67-positive chondrocytes present on six different sections.(F,G) Cumulative cell number of hEGFRKI/KI (F) and Egfr–/–(G)primary osteoblasts isolated from newborn calvariae showingreduced proliferation of hEGFRKI/KI and Egfr–/–osteoblasts at theend of the culture period. (H,I) Primary osteoblasts derived fromhEGFRKI/KI (H) and Egfr–/–(I) neonatal calvariae showed enhancedbone nodule formation compared with controls. Data represent themean±s.e.m. (J) Western blot analysis showing EGFR (180 kDa)protein expression in hEGFRKI/KI and control osteoblasts. Anti-Actinimmunoblotting was used as an internal protein loading control.

4522

accumulation of mesenchymal cells (Fig. 6H,I). By contrast,the atrioventricular valves were not affected and appearednormal (data not shown). The same semilunar valve defects asin hEGFRKI/KI hearts were also observed in Egfr–/– mice (datanot shown) indicating that these defects result from theabsence of expression of the EGFR in valve structures. AsEgfr–/– mice do not display severe myocardial hypertrophy,these results suggest that in hEGFRKI/KI mice the semilunarvalve defects together with the increased expression of thehEGFRKI allele in the myocardium (Fig. 2A) exacerbate theheart hypertrophy thereby contributing to increased lethalityof the mice.

DiscussionIn this study, a knock-in approach was employed togenerate humanised EGFR mice, which harbour a humanEGFR cDNA flanked by loxP sites. hEGFRKI/KI micedisplay tissue-specific hypomorphic phenotypes, whichcorrelate with the expression levels of the hEGFRKI allelein various tissues. This is surprising because othergenetically engineered alleles that displayed hypomorphicbehaviour showed reduced expression in most of the tissuesanalysed (Vivian et al., 1999). Often hypomorphic allelesstill harbour the neomycin resistance cassette with itsregulatory elements in the locus and it has been shown forseveral genes that this can interfere with their properexpression in many tissues (Meyers et al., 1998; Partanenet al., 1996). This is a likely explanation for the lowexpression of the hEGFRKI allele as it also contains aneomycin cassette in the first intron downstream of thehuman EGFR cDNA. The surprising finding, however, isthat the hEGFRKI allele is not hypomorphic in every tissue,but only in bone and epithelial cells. In other organs suchas the brain the expression levels of the hEGFRKI allelewere comparable with the endogenous mouse Egfrgene.This suggests that there might be elements regulating Egfrexpression in a tissue-specific manner. It is possible that thecontrolling elements of the neomycin gene interfere andcompete with transcription factors, which would otherwisedirect correct EGFR expression in the affected organs.Alternatively, the insertion of the human EGFRcDNA intothe mouse locus might alter its chromatin conformation,thereby rendering it inaccessible to tissue-specifictranscription factors.

In all the brain regions analysed, the hEGFRKI allele wasexpressed at similar levels to the endogenous mouse gene.In fact, in brains of homozygote hEGFRKI/KI mice none ofthe defects observed in Egfr–/– mice could be detected.hEGFRKI/KI mice do not develop the corticalneurodegeneration and astrocyte proliferation in vitro iscomparable with controls. Only after Cre-mediated deletionof the floxed hEGFRKI allele a severe proliferation defectwas observed in astrocytes cultured in vitro, demonstratingthat the EGFR is required for proper proliferation of thesecells. hEGFRKI/KI mice did also not contain ectopic neuronesin the hippocampus, a phenotype that is always observed inEgfr–/– mice (Sibilia et al., 1998). Because all the braindefects observed in Egfr–/– mice were rescued in hEGFRKI/KI

mice, it can be assumed that the amount of EGFR proteinpresent in brain cells of hEGFRKI/KI mice is similar to thatin wild-type controls. Moreover, it appears that the bindingaffinity of the mouse EGFR ligands to the human EGFR is

not significantly altered, suggesting that impaired receptoractivation is not contributing to the hypomorphic defects seenin other tissues.

The observed phenotypes are probably determined by thelevel of transcription of the hEGFRKI allele in various tissues.In epithelial tissues such as the skin and hair follicles wherethe hEGFRKI allele is poorly transcribed, severe defects wereobserved that resemble those observed in Egfr–/– mice(Miettinen et al., 1995; Sibilia and Wagner, 1995; Threadgillet al., 1995). Because Egfr–/– mice do not survive longer thanP20, the skin and hair phenotypes could not be properlyanalysed directly in mutant mice. The hEGFRKI/KI mice proved

Development 130 (19) Research article

Fig. 6.Heart hypertrophy and semilunar valve defects in hEGFRKI/KI

mice. (A) Appearance of control and hEGFRKI/KI hearts at 3 weeks ofage. (B) Heart weights and relative heart weights corrected for bodyweight of control and hEGFRKI/KI mice. Data represent the mean±s.d. offive 3-month-old mice and hearts. Cross-sections through the heart ofcontrol (C,F), hEGFRKI/KI (D,G) and Egfr–/– (E) mice at differentpostnatal stages, showing severe myocardial hypertrophy in hEGFRKI/KI

mice. lv, left ventricle; se, septum; rv, right ventricle. Histologicalsections of semilunar valves from control (H) and hEGFRKI/KI (I) hearts.The arrows in H and I indicate the aortic valve leaflets, which arethickened and hypercellular in hEGFRKI/KI hearts. w, weeks; m, months.All sections are stained with Haematoxylin and Eosin.

4523Skin, bone and heart defects in hEGFRKI/KI mice

to be extremely useful to analyse how the absence of the EGFRaffects hair follicle differentiation, migration and cycling. Afterthe first hair cycle, hair follicles of hEGFRKI/KI mice fail toenter into catagen and remain in aberrant anagen, indicatingthat EGFR signalling is needed to regulate hair cycleprogression. The EGFR also seems to be required to preservehair follicle integrity over time, because in its absence thefollicles are degraded probably by the infiltrating inflammatorycells. The aberrant localisation of hEGFRKI/KI hair follicles inthe skin might have triggered an immunological response,which results in the destruction of the follicles. Alternatively,as the EGFR has been shown to inhibit differentiation andpromote survival of epithelial cells, it is possible that in theabsence of EGFR expression, hair follicles might be impairedin their survival capacity and/or undergo prematuredifferentiation. With time, this would lead to hair follicledestruction and release of follicle material into the dermis,which in turn would trigger the immunological response.Similar hair abnormalities have also been observed in graftingexperiments with Egfr–/– skin onto immunodeficient mice andin a skin targeted DN Egfrtransgenic mouse model (Hansenet al., 1997; Murillas et al., 1995). However, in the latter modelit could not be excluded that the severity of the phenotypesobserved was determined not only by inhibition of the EGFRitself but also by the inhibition of the other Erbb familymembers which are also expressed in the epidermis (Stoll etal., 2001). As the skin and hair follicle defects observed inhEGFRKI/KI and the DN EGFRtransgenic mice are verysimilar, it can be concluded that these phenotypes mainly resultfrom direct EGFR inhibition.

EGFR signalling does not only seem to inhibit epithelial celldifferentiation, but also the differentiation of bone cells. Bonedevelopment starts with the formation of mesenchymalcondensations that first differentiate into chondrocytes, whichbecome hypertrophic and are then invaded by blood vessels,bone-forming osteoblasts and bone-resorbing osteoclasts(Karsenty and Wagner, 2002). Active remodelling ultimatelygives rise to a bone with growth plate and a central marrowcavity (Karsenty and Wagner, 2002). Numerous extracellularfactors, such as hormones, growth factors and cytokines,modulate bone remodelling via differentiation andproliferation of bone cells, and this process has to be tightlyregulated in order to prevent bone diseases (Karsenty andWagner, 2002). The EGFR is expressed in chondroblasts of thedeveloping ossification centres and the EGFR has also beenshown to be expressed in osteoblasts and osteocytes in vivo(Davideau et al., 1995). However, no bone defects have beenreported so far for mice defective in EGFR signalling. A recentreport has shown that transgenic mice expressing EGFubiquitously are growth retarded, display defectivechondrocyte development in the growth plate and osteoblastsaccumulate in the endosteum and periosteum (Chan and Wong,2000). Controversial in vitro data regarding the role of EGFRsignalling in bone cells have been reported, although twostudies suggest that EGFR might stimulate osteogenic cellproliferation and suppress the differentiation into matureosteoblasts and chondrocytes (Chien et al., 2000; Yoon et al.,2000). In Egfr–/– and hEGFRKI/KI mice both osteoblasts andchondrocytes display increased differentiation in vitro and invivo, respectively. As the hEGFRKI allele was poorly expressedin the affected cell types, it is likely that these defects result

from the lack of Egfrexpression. In the long bones ofhEGFRKI/KI mice, the region of hypertrophic chondrocytes inthe growth plate was significantly increased, suggesting thatchondrocytes had undergone premature differentiation. Thisphenotype was even more severe in Egfr–/– mice, where Egfrexpression is completely absent, indicating that EGFRsignalling prevents differentiation of chondrocytes. Althoughno overt differences in bone mass could be detected in newbornEgfr–/– and hEGFRKI/KI mice, calvarial osteoblasts from thesemice proliferate more slowly and display an increaseddifferentiation capacity in vitro. It is likely that EGFRsignalling is important to keep osteoblasts and chondrocytes ina proliferative state and inhibit their differentiation, thusallowing proper development of long bones. In the absence ofEGFR expression, accelerated differentiation of chondrocytesand possibly decreased proliferation of osteoblasts can disturblongitudinal growth of long bones leading to severe growthretardation in the mice.

hEGFRKI/KI mice develop a severe heart hypertrophy withdramatically increased thickness of the ventricular walls andthe interventricular septum, which was visible already at 3weeks after birth and became more pronounced as ageproceeded. Interestingly, Egfr–/– mice did not display signs ofhypertrophy at this age, suggesting that this phenotype wasmost probably not due to the lack of EGFR expression. Cardiachypertrophy is an adaptive response to many forms of disorderssuch as hypertension, myocardial infarction and valve defectsaimed to augment the cardiac output (Katz, 1990). However,sustained hypertrophy usually leads to ventricular dilatationwith consequent heart failure and sudden death (Katz, 1990).The response of cardiomyocytes to hypertrophic signalsinvolves an increase in cell size and protein synthesis,induction of immediate-early genes such as AP-1 and re-expression of several foetal myocardial structural proteins(Sadoshima and Izumo, 1997). It has been shown thattreatment of rat cardiomyocytes with EGF can trigger ahypertrophic response resulting in increased protein synthesisand transcriptional activation of Fosand Jun(Rebsamen et al.,2000). Similarly, G-protein-coupled receptor (GPCR) agonistswere shown to transactivate the EGFR via shedding of HB-EGF by the metalloproteinase ADAM12 thereby inducingheart hypertrophy (Asakura et al., 2002; Prenzel et al., 1999).Moreover, EGF increases adenyl cyclase activity and cAMPaccumulation, thus enhancing the heart contractility andbeating rate (Nair et al., 1993). As heart-specific expression ofthe hEGFRKI allele seemed to be slightly higher than theendogenous wild-type allele, we speculate that increasedEGFR signalling in cardiomyocytes is contributing to thedevelopment of the heart hypertrophy.

hEGFRKI/KI mice also display semilunar valve defects, whichare known to induce aortic stenosis and regurgitation, and as aconsequence can lead to heart hypertrophy. Heart valvesdevelop from the endocardium, a specialised endothelium thatundergoes complex epithelial-mesenchymal transformationsleading to the formation of mesenchymal outgrowths (alsocalled the cardiac cushions), from which the mature valveleaflets originate (Towbin and Belmont, 2000). The pulmonaryand aortic, but not the atrioventricular valves of hEGFRKI/KI

hearts were thickened and hypercellular, which most probablyresults from the accumulation of mesenchymal cells. As similarvalve defects were also present in Egfr–/– hearts, it is likely that

4524

the hEGFRKI allele is not expressed in the developing valves.Although we were unable to detect EGFR expression in wild-type valve structures by immunohistochemistry, we believe thatthe valve defects of hEGFRKI/KI and Egfr–/– hearts result fromlack of EGFR expression in these structures. The other EGFRfamily members Erbb2 and Erbb4 are expressed in theendocardium and have been shown to be involved in theformation of the mesenchymal cushions (Camenisch et al.,2002; Erickson et al., 1997). It still remains to be determinedhow EGFR alone or in combination with these receptorsregulates mesenchyme development and its differentiation intomature valve structures.

By studying the genetic interaction between EGFR and theprotein-tyrosine phosphatase Shp2, it was shown that EGFR isrequired for semilunar valve development (Chen et al., 2000).Compound mutants between Shp2 and the hypomorphicEGFRwa2/wa2mouse strains showed signs of aortic stenosis andregurgitation with subsequent development of myocardialhypertrophy (Chen et al., 2000). Although these defects weremost severe in compound mutants, thickened semilunar valveswere also observed in EGFRwa2/wa2and Egfr–/– mice, whereasno cardiac dilatation were reported for the single EGFRmutants (Chen et al., 2000). Similarly, we could not detect anysigns of myocardial hypertrophy in 3-week-old Egfr–/– mice,although semilunar valve thickening was observed. Bycontrast, the heart of hEGFRKI/KI mice at this stage was alreadyenlarged and displayed a severe thickening of the myocardialwalls. Therefore, it is likely that the severe hypertrophyobserved in hEGFRKI/KI mice results from the malformationsof the valves and from the enhanced hypertrophic response ofcardiomyocytes to increased EGFR signalling, as the hEGFRKI

allele is expressed at higher levels in the myocardium. Thesedefects can lead to a severe heart condition, which is probablyresponsible for the lethality of hEGFRKI/KI mice, whereas thevalve defects alone, as seen in Egfrwa2/wa2mice do not seem toincrease the mortality of these mice. Moreover, as hEGFRKI/+

mice do not develop heart hypertrophy, it seems unlikely thatenhanced expression of the EGFR in the myocardium alonecan lead to the development of this heart condition. Theconditional hEGFRKI/KI mice may provide a useful model notonly to study aortic valve diseases and developmental aspectsof bone differentiation, but also to test novel therapies aimedto inhibit EGFR signalling for the treatment of cardiachypertrophies and hyperproliferative bone diseases.

We are grateful to S. Pekez and M. Hammer for maintaining ourmouse colonies at IMP and at the Department of Dermatology,respectively. We thank Romeo Ricci for critical reading of themanuscript. B.W. was recipient of a DOC-Fellowship of the AustrianAcademy of Sciences. A.H. was funded by EMBO and Marie CurieIndividual fellowships. Work in E.F.W.’s laboratory at the IMP issupported by Boehringer Ingelheim and the Austrian IndustrialResearch Promotion Fund (FFF). Work in M.S.’s laboratory issupported by the Competence Center for Biomolecular Therapeutics(BMT, funded by the Austrian Ministry of Science and Technology,City of Vienna and Industrial Partners) and by the EC program QLG1-CT-2001-00869.

ReferencesAsakura, M., Kitakaze, M., Takashima, S., Liao, Y., Ishikura, F.,

Yoshinaka, T., Ohmoto, H., Node, K., Yoshino, K., Ishiguro, H. et al.

(2002). Cardiac hypertrophy is inhibited by antagonism of ADAM12processing of HB-EGF: metalloproteinase inhibitors as a new therapy. Nat.Med.8, 35-40.

Camenisch, T. D., Schroeder, J. A., Bradley, J., Klewer, S. E. andMcDonald, J. A. (2002). Heart-valve mesenchyme formation is dependenton hyaluronan-augmented activation of ErbB2-ErbB3 receptors. Nat. Med.8, 850-855.

Chan, S. Y. and Wong, R. W.(2000). Expression of epidermal growth factorin transgenic mice causes growth retardation. J. Biol. Chem.275, 38693-38698.

Chen, B., Bronson, R. T., Klaman, L. D., Hampton, T. G., Wang, J. F.,Green, P. J., Magnuson, T., Douglas, P. S., Morgan, J. P. and Neel, B.G. (2000). Mice mutant for Egfr and Shp2 have defective cardiac semilunarvalvulogenesis. Nat. Genet.24, 296-299.

Chien, H. H., Lin, W. L. and Cho, M. I. (2000). Down-regulation ofosteoblastic cell differentiation by epidermal growth factor receptor. Calcif.Tiss. Int.67, 141-150.

Davideau, J. L., Sahlberg, C., Thesleff, I. and Berdal, A.(1995). EGFreceptor expression in mineralized tissues: an in situ hybridization andimmunocytochemical investigation in rat and human mandibles. ConnectTiss. Res.32, 47-53.

Erickson, S. L., O’Shea, K. S., Ghaboosi, N., Loverro, L., Frantz, G.,Bauer, M., Lu, L. H. and Moore, M. W. (1997). ErbB3 is required fornormal cerebellar and cardiac development: a comparison with ErbB2-andheregulin-deficient mice. Development124, 4999-5011.

Fleischmann, A., Hafezi, F., Elliott, C., Reme, C. E., Ruther, U. andWagner, E. F. (2000). Fra-1 replaces c-Fos-dependent functions in mice.Genes Dev.14, 2695-2700.

Fowler, K. J., Walker, F., Alexander, W., Hibbs, M. L., Nice, E. C., Bohmer,R. M., Mann, G. B., Thumwood, C., Maglitto, R., Danks, J. A. et al.(1995). A mutation in the epidermal growth factor receptor in waved-2 micehas a profound effect on receptor biochemistry that results in impairedlactation. Proc. Natl. Acad. Sci. USA92, 1465-1469.

Gassmann, M., Casagranda, F., Orioli, D., Simon, H., Lai, C., Klein, R.and Lemke, G. (1995). Aberrant neural and cardiac development in micelacking the ErbB4 neuregulin receptor. Nature378, 390-394.

Hansen, L. A., Alexander, N., Hogan, M. E., Sundberg, J. P., Dlugosz, A.,Threadgill, D. W., Magnuson, T. and Yuspa, S. H.(1997). Geneticallynull mice reveal a central role for epidermal growth factor receptor in thedifferentiation of the hair follicle and normal hair development. Am. J.Pathol.150, 1959-1975.

Karsenty, G. and Wagner, E. F.(2002). Reaching a genetic and molecularunderstanding of skeletal development. Dev. Cell2, 389-406.

Katz, A. M. (1990). Cardiomyopathy of overload. A major determinant ofprognosis in congestive heart failure. New Engl. J. Med.322, 100-110.

Kornblum, H. I., Hussain, R., Wiesen, J., Miettinen, P., Zurcher, S. D.,Chow, K., Derynck, R. and Werb, Z. (1998). Abnormal astrocytedevelopment and neuronal death in mice lacking the epidermal growth factorreceptor. J. Neurosci. Res.53, 697-717.

Lee, K. F., Simon, H., Chen, H., Bates, B., Hung, M. C. and Hauser, C.(1995). Requirement for neuregulin receptor erbB2 in neural and cardiacdevelopment. Nature378, 394-398.

Luetteke, N. C., Phillips, H. K., Qiu, T. H., Copeland, N. G., Earp, H. S.,Jenkins, N. A. and Lee, D. C.(1994). The mouse waved-2 phenotyperesults from a point mutation in the EGF receptor tyrosine kinase. GenesDev.8, 399-413.

Luetteke, N. C., Qiu, T. H., Peiffer, R. L., Oliver, P., Smithies, O. and Lee,D. C. (1993). TGF alpha deficiency results in hair follicle and eyeabnormalities in targeted and waved-1 mice. Cell73, 263-278.

Mann, G. B., Fowler, K. J., Gabriel, A., Nice, E. C., Williams, R. L. andDunn, A. R. (1993). Mice with a null mutation of the TGF alpha gene haveabnormal skin architecture, wavy hair, and curly whiskers and often developcorneal inflammation. Cell73, 249-261.

Marino, S., Vooijs, M., van Der Gulden, H., Jonkers, J. and Berns, A.(2000). Induction of medulloblastomas in p53-null mutant mice by somaticinactivation of Rb in the external granular layer cells of the cerebellum.Genes Dev.14, 994-1004.

Meyers, E. N., Lewandoski, M. and Martin, G. R.(1998). An Fgf8 mutantallelic series generated by Cre- and Flp-mediated recombination. Nat.Genet.18, 136-141.

Miettinen, P. J., Berger, J. E., Meneses, J., Phung, Y., Pedersen, R. A.,Werb, Z. and Derynck, R. (1995). Epithelial immaturity and multiorganfailure in mice lacking epidermal growth factor receptor. Nature376, 337-341.

Development 130 (19) Research article

4525Skin, bone and heart defects in hEGFRKI/KI mice

Miettinen, P. J., Chin, J. R., Shum, L., Slavkin, H. C., Shuler, C. F.,Derynck, R. and Werb, Z. (1999). Epidermal growth factor receptorfunction is necessary for normal craniofacial development and palateclosure. Nat. Genet.22, 69-73.

Murillas, R., Larcher, F., Conti, C. J., Santos, M., Ullrich, A. and Jorcano,J. L. (1995). Expression of a dominant negative mutant of epidermal growthfactor receptor in the epidermis of transgenic mice elicits striking alterationsin hair follicle development and skin structure. EMBO J.14, 5216-5223.

Nair, B. G., Rashed, H. M. and Patel, T. B.(1993). Epidermal growth factorproduces inotropic and chronotropic effects in rat hearts by increasing cyclicAMP accumulation. Growth Factors8, 41-48.

Olayioye, M. A., Neve, R. M., Lane, H. A. and Hynes, N. E.(2000). TheErbB signaling network: receptor heterodimerization in development andcancer. EMBO J.19, 3159-3167.

Partanen, J., Puri, M. C., Schwartz, L., Fischer, K. D., Bernstein, A.and Rossant, J. (1996). Cell autonomous functions of the receptortyrosine kinase TIE in a late phase of angiogenic capillary growth andendothelial cell survival during murine development. Development122,3013-3021.

Peus, D., Hamacher, L. and Pittelkow, M. R.(1997). EGF-receptor tyrosinekinase inhibition induces keratinocyte growth arrest and terminaldifferentiation. J. Invest. Dermatol.109, 751-756.

Prenzel, N., Zwick, E., Daub, H., Leserer, M., Abraham, R., Wallasch, C.and Ullrich, A. (1999). EGF receptor transactivation by G-protein-coupledreceptors requires metalloproteinase cleavage of proHB-EGF. Nature402,884-888.

Rebsamen, M. C., Arrighi, J. F., Juge-Aubry, C. E., Vallotton, M. B. andLang, U. (2000). Epidermal growth factor induces hypertrophic responsesand Stat5 activation in rat ventricular cardiomyocytes. J. Mol. Cell Cardiol.32, 599-610.

Riethmacher, D., Sonnenberg-Riethmacher, E., Brinkmann, V., Yamaai,T., Lewin, G. R. and Birchmeier, C.(1997). Severe neuropathies in micewith targeted mutations in the ErbB3 receptor. Nature389, 725-730.

Sadoshima, J. and Izumo, S.(1997). The cellular and molecular responseof cardiac myocytes to mechanical stress. Annu. Rev. Physiol.59, 551-571.

Schlessinger, J.(2000). Cell signaling by receptor tyrosine kinases. Cell 103,211-225.

Sibilia, M. and Wagner, E. F.(1995). Strain-dependent epithelial defects inmice lacking the EGF receptor. Science269, 234-238.

Sibilia, M., Steinbach, J. P., Stingl, L., Aguzzi, A. and Wagner, E. F.(1998).A strain-independent postnatal neurodegeneration in mice lacking the EGFreceptor. EMBO J.17, 719-731.

Sibilia, M., Fleischmann, A., Behrens, A., Stingl, L., Carroll, J., Watt, F.M., Schlessinger, J. and Wagner, E. F.(2000). The EGF receptor providesan essential survival signal for SOS-dependent skin tumor development. Cell102, 211-220.

Stoll, S. W., Kansra, S., Peshick, S., Fry, D. W., Leopold, W. R., Wiesen,J. F., Sibilia, M., Zhang, T., Werb, Z., Derynck, R. et al. (2001).Differential utilization and localization of ErbB receptor tyrosine kinases inskin compared to normal and malignant keratinocytes. Neoplasia3, 339-350.

Threadgill, D. W., Dlugosz, A. A., Hansen, L. A., Tennenbaum, T., Lichti,U., Yee, D., LaMantia, C., Mourton, T., Herrup, K., Harris, R. C. et al.(1995). Targeted disruption of mouse EGF receptor: effect of geneticbackground on mutant phenotype. Science269, 230-234.

Towbin, J. A. and Belmont, J.(2000). Molecular determinants of left andright outflow tract obstruction. Am. J. Med. Genet.97, 297-303.

Tropepe, V., Sibilia, M., Ciruna, B. G., Rossant, J., Wagner, E. F. and vander Kooy, D. (1999). Distinct neural stem cells proliferate in response toEGF and FGF in the developing mouse telencephalon. Dev. Biol.208, 166-188.

Vivian, J. L., Gan, L., Olson, E. N. and Klein, W. H.(1999). A hypomorphicmyogenin allele reveals distinct myogenin expression levels required forviability, skeletal muscle development, and sternum formation. Dev. Biol.208, 44-55.

Yarden, Y. (2001). The EGFR family and its ligands in human cancer.signalling mechanisms and therapeutic opportunities. Eur. J. Cancer Suppl.37, S3-S8.

Yarden, Y. and Sliwkowski, M. X. (2001). Untangling the ErbB signallingnetwork. Nat. Rev. Mol. Cell Biol.2, 127-137.

Yoon, Y. M., Oh, C. D., Kim, D. Y., Lee, Y. S., Park, J. W., Huh, T. L.,Kang, S. S. and Chun, J. S.(2000). Epidermal growth factor negativelyregulates chondrogenesis of mesenchymal cells by modulating the proteinkinase C-alpha, Erk-1, and p38 MAPK signaling pathways. J. Biol. Chem.275, 12353-12359.