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First published online August 18, 2003
doi: 10.1242/10.1242/dev.00664

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Mice humanised for the EGF receptor display hypomorphic phenotypes in skin, bone and heart

Maria Sibilia^1,2,*, Bettina Wagner¹, Astrid Hoebertz², Candace Elliott^2,, Silvia Marino^3,, Wolfram Jochum^2, and Erwin F. Wagner²

¹ Department of Dermatology and Biomolecular Therapeutics (BMT), University of Vienna, Medical School, Brunnerstr. 59, A-1235 Vienna, Austria
² Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, A-1030 Vienna, Austria
³ Division of Molecular Genetics and Centre of Biomedical Genetics, The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands

View larger version (33K): [in a new window]   Fig. 1. Generation of hEGFRKI/KI mice. (A) Schematic representation of the targeting strategy employed to insert a floxed human EGFR cDNA (hEGFR) into the mouse Egfr locus. The full-length hEGFR expression cassette (grey box) contains its own ATG and polyA (pA) site and is flanked by loxP sites (grey triangles). After homologous recombination, the hEGFR cDNA will be inserted into the first exon of the mouse Egfr gene (black box) and placed under the control of the endogenous mouse Egfr promoter. This correctly targeted allele will be referred to as the hEGFR knock-in allele (hEGFRKI). The neomycin resistance gene (RSV-neo) and the thymidine kinase gene (HSV-tk) are shown. The broken lines delineate the homology regions in the targeting vector, the horizontal bar indicates the Southern blot probe, and the black arrowheads indicate the PCR primers employed for genotype analysis. X, XbaI; BX, BstXI; N, NdeI. (B) PCR analysis of genomic DNA isolated from the progeny of hEGFRKI/+ intercrosses. (C) Photograph of 6-week-old hEGFRKI/KI and littermate control mice showing that hEGFRKI/KI mice are smaller and display short fur hair. (D) Weight representative of a hEGFRKI/KI and control littermate mouse at different postnatal ages. At all stages, hEGFRKI/KI mice are significantly smaller than their littermates. w, weeks; m, months

View larger version (55K): [in a new window]   Fig. 2. The hEGFRKI allele is not efficiently expressed in epithelial tissues. (A) Expression of the endogenous mouse Egfr and the hEGFRKI mRNAs measured by RNAse protection assay. The analysis was performed on total RNA isolated from various organs of a heterozygote hEGFRKI/+ male mouse employing an antisense Egfr riboprobe, which detects both the endogenous mouse Egfr and the hEGFRKI transcripts. In addition to 93 bp of nonspecific sequence (broken line), this riboprobe encompasses the region of the hEGFRKI allele bridging the Egfr mouse promoter, the loxP site and the 5' end of the hEGFR cDNA. The input probe and the protected fragments are depicted schematically and the black triangle indicates the loxP site. A mouse S16 riboprobe (lower panel) was used as an internal control for equal sample loading in each lane. (B) Western blot analysis showing EGFR protein expression in brain and liver of mice of different genotypes. Immunoblotting was performed on total protein extracts using an anti-EGFR antibody recognising human and mouse EGFR and anti-tubulin was used as a control for protein loading. (C) In vitro EGFR autophosphorylation assay measuring EGFR protein levels. Prior to the kinase assay, protein lysates from various organs were immunoprecipitated with an anti-EGFR antibody recognising both the mouse and the human EGFR protein. Equal loading of protein was verified by Coomassie Blue staining (data not shown)

View larger version (75K): [in a new window]   Fig. 3. Normal brain development in hEGFRKI/KI mice. Dorsal view of the brains of control (A) and hEGFRKI/KI mice (B) isolated 3 months after birth. Histological sections through the frontal cortex (C,E) and hippocampus (D,F) of control (C,D) and hEGFRKI/KI (E,F) brains showing normal architecture and morphology. (G,H) Sections of Egfr-/- brains show neuronal degeneration in the cortex (G), starting around postnatal day 5, and groups of ectopic neurones (arrows) in the white matter of the 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-Cre and Egfr-/- primary cortical astrocytes isolated from newborn brains showing that hEGFRKI/KI astrocytes display a normal proliferation capacity. Removal of the hEGFRKI allele in astrocytes of hEGFRKI/KI GFAP-cre mice results in severe proliferation defects as observed in Egfr-/- astrocytes. (J) Southern blot analysis of genomic DNA isolated from astrocytes shown in I and hybridised with the probe shown in Fig. 1A. The bands corresponding to the different alleles are indicated. EGFR: hEGFRKI allele after Cre-mediated deletion.

View larger version (67K): [in a new window]   Fig. 4. Impaired hair cycle and follicle development in hEGFRKI/KI mice. Histological sections through the skin of control (A,C,E) and hEGFRKI/KI mice (B,D,F) at different stages of the hair cycle. In control skins at 18 days (end of catagen/telogen) the hair follicles are confined to the upper dermal layers (A, arrows), whereas in hEGFRKI/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 in mutant skins. (C) At 1 month (late anagen/early catagen) control follicles are distributed in the upper dermal and subdermal fat layers and display a normal structure (arrows). (D) Mutant follicles in the subdermal fat tissue are severely hyperplastic (arrows) at this stage. (E) At 3 months (telogen), control follicles are confined to the upper dermal layers (arrows). (F) In mutant skins, only a few aberrant follicles are present in the upper dermis (arrowheads) and most of the follicles stuck in the subdermal fat tissue have degenerated (arrows). Severe fibrosis with infiltration of inflammatory cells can be detected in the subdermal fat tissue of hEGFRKI/KI skins as evidenced by the strong eosin (pink) staining. (G) A normal mouse hair cycle profile with the position where the hair cycle is blocked in hEGFRKI/KI mice. A, anagen; C, catagen; T, telogen; d, days; m, months. All sections are stained with Haematoxylin and Eosin.

View larger version (82K): [in a new window]   Fig. 5. Bone cell defects in the absence of EGFR. Histological sections through the tibiae of control (A), hEGFRKI/KI (B) and Egfr-/- mice (C) at postnatal day 1. The black bar marks the zone of hypertrophic chondrocytes, which is markedly increased in both hEGFRKI/KI and Egfr-/- long bones. (D) X-galactosidase (X-gal) staining of an E14.5 Egfr+/- foetus showing EGFR expression (blue stain) in the ossification center of the vertebrae. Inset shows a higher magnification of the region boxed in D with arrows indicating X-gal staining in chondroblasts. (A-C) Haematoxylin and Eosin staining; (D) X-gal and Eosin staining. (E) The proliferation of chondrocytes was measured by Ki67 staining on bone sections of mice of the indicated genotypes. The data represent the mean±s.d. of the number of 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 showing reduced proliferation of hEGFRKI/KI and Egfr-/- osteoblasts at the end of the culture period. (H,I) Primary osteoblasts derived from hEGFRKI/KI (H) and Egfr-/- (I) neonatal calvariae showed enhanced bone nodule formation compared with controls. Data represent the mean±s.e.m. (J) Western blot analysis showing EGFR (180 kDa) protein expression in hEGFRKI/KI and control osteoblasts. Anti-Actin immunoblotting was used as an internal protein loading control.

View larger version (100K): [in a new window]   Fig. 6. Heart hypertrophy and semilunar valve defects in hEGFRKI/KI mice. (A) Appearance of control and hEGFRKI/KI hearts at 3 weeks of age. (B) Heart weights and relative heart weights corrected for body weight of control and hEGFRKI/KI mice. Data represent the mean±s.d. of five 3-month-old mice and hearts. Cross-sections through the heart of control (C,F), hEGFRKI/KI (D,G) and Egfr-/- (E) mice at different postnatal stages, showing severe myocardial hypertrophy in hEGFRKI/KI mice. lv, left ventricle; se, septum; rv, right ventricle. Histological sections of semilunar valves from control (H) and hEGFRKI/KI (I) hearts. The arrows in H and I indicate the aortic valve leaflets, which are thickened and hypercellular in hEGFRKI/KI hearts. w, weeks; m, months. All sections are stained with Haematoxylin and Eosin.

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