Expression of a dominant negative mutant of epidermal growth factor ...

The EMBO Journal vol.14 no.21 pp.5216-5223, 1995

Expression of a dominant negative mutant of epidermal growth factor receptor in the epidermis of transgenic mice elicits striking alterations in hair follicle development and skin structure R.Murillas, FLarcher, C.J.Contil, M.Santos, A.U1Irich2 and J.L.Jorcano3 Department of Cell and Molecular Biology, Centro de Investigaciones Energeticas, Medioambientales y Tecnol6gicas (CIEMAT), 28040 Madrid, Spain, 'The University of Texas M.D.Anderson Cancer Center, Science Park-Research Division, Smithville, TX 78957, USA and 2Department of Molecular Biology, Max-Planck-Institut fiir Biochemie, 82152 Martinsried, Germany 3Corresponding author

Epidermal growth factor receptor (EGFR) is a key regulator of keratinocyte biology. However, the physiological role of EGFR in vivo has not been well established. To analyze the role of EGFR in skin, we have generated transgenic mice expressing an EGFR dominant negative mutant in the basal layer of epidermis and outer root sheath of hair follicles. Mice expressing the mutant receptor display short and waved pelage hair and curly whiskers during the first weeks of age, but subsequently pelage and vibrissa hairs become progressively sparser and atrophic. Eventually, most mice present severe alopecia. Histological examination of the skin of transgenic mice shows striking alterations in the development of hair follicles, which fail to enter into catagen stage. These alterations eventually lead to necrosis and disappearance of the follicles, accompanied by strong infiltration of the skin with inflammatory elements. The interfollicular epidermis of these mice shows marked hyperplasia, expression of hyperproliferation-associated keratin K6 and increased 5-bromo-2-deoxyuridine incorporation. EGFR function was inhibited in transgenic skin keratinocytes, since in vivo and in vitro autophosphorylation of EGFR was almost completely abolished on EGF stimulation. These results implicate EGFR in the control of hair cycle progression, and provide new information about its role in epidermal growth and differentiation. Keywords: EGFR/epidermis/hair cycle/mouse/skin structure

Introduction Signaling through the epidermal growth factor receptor (EGFR) is a fundamental event in the regulation of epidermal biology (Cohen, 1965). EGFR binds and is activated by a family of growth factors comprised of epidermal growth factor (EGF), transforming growth factor-a (TGF-a), amphiregulin, heparin-binding EGF (HB-EGF) and betacellulin. Some of these factors (TGF-a, amphiregulin and HB-EGF) are produced and secreted by keratinocytes, which suggests that they are autocrine growth factors in skin (Coffey et al., 1987; Hashimoto et al., 1994). Moreover, addition of EGFR ligands (EGF/

TGF-a) to keratinocytes in culture has a strong effect on growth promotion, which is dependent not only on mitogenesis, but also on cell migration (Barrandon and Green, 1987; Coffey et al., 1988). EGF injection provokes accelerated eye opening and tooth eruption in neonatal mice (Cohen, 1962), as well as hyperproliferation of the epidermis and delay in hair follicle development (Cohen and Elliot, 1963). In adult skin, EGF receptors are found mainly in the cells of the basal layer of epidermis, which have proliferative capacity. The number of receptors decreases as keratinocytes migrate to the suprabasal layer of epidermis, entering the pathway of terminal differentiatiation. EGF receptors are much more abundant in the rapidly growing epidermis of neonatal mice, undergoing a strong downregulation a few days after birth (Green et al., 1983). EGFR is also localized in tissues that do not undergo rapid proliferation (Nanney et al., 1984), which suggests a complex regulatory role for this receptor. Previous reports demonstrate the effect of EGFR activation in hair physiology (Moore et al., 1983; Philip et al., 1985; Vassar and Fuchs, 1991; Cros, 1993). These data, together with EGFR localization in the outer root sheath of hair follicles (Nanney et al., 1984), point to EGFR as a major regulator of hair biology (reviewed in Hardy, 1992). Recently characterized waved-i and waved-2 mutant mice present alterations in the EGFR signaling pathways. TGF-a null and waved-i mutant mice lack TGF-a protein (Luetteke et al., 1993; Mann et al., 1993). waved-2 mice have a point mutation in the tyrosine kinase domain of the EGFR gene (Luetteke et al., 1994); however, these mutations do not result in a complete loss of EGFR function. All three mutants show a similar, mild phenotype consisting mainly of a waved coat and curly whiskers in the first weeks after birth, becoming progressively milder in older animals. Activation of EGFR stimulates multiple pathways of signal transduction, leading to a wide array of cellular responses. EGFR binding to its ligands provokes receptor dimerization and autophosphorylation on tyrosine residues (reviewed in Ullrich and Schlessinger, 1990). As recent findings indicate, receptor autophosphorylation is the crucial event for signal transduction (reviewed in Pawson and Schlessinger, 1993). Phosphorylated tyrosine residues act as binding sites for molecules containing Src-homology 2 (SH2) domains. This recruitment mechanism links EGFR activation to fundamental cytoplasmic signaling pathways. Interaction of activated EGFR with Grb-2 and Shc proteins triggers a signaling cascade which leads to activation of the ras signaling pathway. In addition, phosphorylated EGFR binds and activates other SH2-containing molecules such as PLC-,y and Ras GTPase-activating protein (RasGap), also involved in signaling mechanisms. A truncated EGFR, lacking most of its cytoplasmic domain (HERCD533), has proved to act as a dominant negative mutant

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EGFR, hair follicle development and skin structure

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Fig. 1. Transgene construct, expression and phenotype of K5-HERCD-533 transgenic mice. (A) Diagram of the pK5-HERCD-533 transgene construct. The functional elements include bovine K5 regulatory sequences (K5 promoter box), rabbit l-globin intron 2 ( GI box), coding sequence of the truncated human EGFR cDNA (HERCD-533 box) and l-globin and SV40 early gene poly(A) addition signal (PA box). (A) Asp718 restriction site. The arrow indicates the initiation of transcription. (B) Immunofluorescence detection of the transgenic protein in a skin section from a transgenic mouse tail. Note the continuous expression in the basal layer of epidermis and outer root sheath of hair follicles. (C) Strongly curved vibrissae in a 4-week-old homozygous T5 transgenic mouse. (D) Comparison of homozygous T5 mouse (top), hemizygous T5 littermate (middle) and control littermate (bottom) at 4 weeks of age. The hemizygous mouse shows a waved coat of normal length (mild phenotype); the homozygous mouse presents very short waved pelage (severe phenotype). (E) Representative example of a 7-month-old pK5-HERCD-533 transgenic mouse (line T3, top), relative to a control littermate (bottom).

because of its ability to form non-functional heterodimers with full-length EGFR upon EGF stimulation (Kashles et al., 1991). Overexpression of the truncated receptor inhibits wild-type receptor phosphorylation in a dosedependent manner and supresses its mitogenic and oncogenic signals (Redemann et al., 1992). Expression of dominant negative mutants of tyrosine kinase receptors other than EGFR in transgenic mice and other in vivo models has been a useful approach to understand the function of these receptors (Amaya et al., 1991; Werner et al., 1993, 1994; Millauer et al., 1994; Peters et al., 1994). To explore the in vivo role of EGFR in skin further, we have targeted the expression of the dominant negative HERCD-533 mutant to the basal layer of epidermis and outer root sheath of hair follicles of transgenic mice using the 5' regulatory region of the keratin K5 gene (Ramirez et al., 1994). Since the K5 expression pattern matches that of EGFR in skin, our approach allows the precise targeting of dominant negative EGFR to the appropriate localization.

Results The construct used for transgenic generation is outlined in Figure IA. To drive the expression of the human HERCD-533 cDNA, we have used the 5' regulatory fragment of keratin K5 gene. We have previously shown that a 5 kb fragment of the 5' upstream region of bovine keratin K5 gene is adequate to drive expression of reporter genes to the basal cell compartment of stratified squamous epithelia (Ramirez et al., 1994). Seven founder transgenic mice were identified by Southern blot analysis of tail DNA and transgene expression was detected by immunofluorescence analysis of tail skin sections in six transgenic founder mice. As expected, the transgene was expressed in the basal layer of interfollicular epidermis and in the outer root sheath of hair follicles (Figure iB), matching precisely the pattern of expression of keratin K5. Newborn transgenic mice were readily identified by their curly whiskers, already apparent in 2-day-old animals. Development of the first coat was 5217

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GAPDH Fig. 2. Northern blot analysis of transgene expression. Total RNA (15 jig) from whole skin of control and transgenic mice was hybridized using the HERCD-533 cDNA as a probe. Co, control mouse skin; T5, skin from T5 line mice (mild phenotype); TO, skin from TO line mice (severe phenotype).

delayed in all transgenic lines and hair presented a waved appearance (Figure 1D). Mice from lines TI, T3, T4 and TO displayed a more severe phenotype, in which hair appears atrophic and much shorter than in non-transgenic littermates. The animals are smaller (-25%) and their vibrissae appear completely tangled (Figure IC). The coat hair of transgenic animals of all lines undergoes a degenerative process during which it becomes progressively sparser and atrophic. Vibrissae suffer a similar destructive process. Hair degeneration is faster in mice with the severe phenotype, but also affects mice exhibiting the milder phenotype. After several months, most mice presented marked scarcity of hair (Figure IE). Although mice showing a mild phenotype (lines T2 and T5) resemble waved-i and waved-2 mutant mice during the first weeks of life, gradual degeneration and loss of pelage hair and vibrissae in K5-HERCD-533 transgenic mice contrast with the progressive disappearance of hair alterations observed in waved mutant mice. Some TI and some homozygous T5 transgenic mice presented open eyelids at birth. These mice developed comeal opacity with aging. Two founder mice (TO and T3) showed a patchy distribution of waved and normal hair. Only a small proportion of their offspring was transgenic, but in these mice the entire coat was affected, confirming that both founder mice were mosaics for the transgene. The severity of the phenotype correlated with the level of expression of the transgene, as detected by Northern blot analysis of total RNA from skins of mice of T5 and TO lines (Figure 2). In addition, homozygous T5 mice presented a more severe phenotype, similar to that observed in the most affected lines (Figure ID). Thus, a positive correlation exists between transgene expression and phenotype severity.

Histological changes in the skin of K5-HERCD-533 mice Histological examination of transgenic mouse skin showed striking alterations in the distribution and morphology of hair follicles, and in the progression of the follicle through the stages of the hair cycle. Signs of altered development can already be observed in 4- to 6-day-old animals, whose most characteristic alteration is a derangement of hair

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follicle orientation and the presence of abnormally longer follicles reaching the panniculum carnosum (Figure 3B and C). However, the more dramatic changes detected in K5-HERCD-533 mice were observed after the third week. At this time, follicles of control mice go through a telogen stage (see Figure 3D, showing a skin in telogen stage), while follicles of transgenic mice fail to enter into catagen and remain in an aberrant anagen stage (Figure 3E), demonstrating that EGFR activation is necessary for hair cycle progression. In addition to alterations in length and orientation, follicles in 3- to 4-week-old animals display structural abnormalities, mainly thinning or loss of the inner and outer root sheath (Figure 3E and F). Hair follicle degeneration leads to a strong alteration of skin architecture (Figure 3E and F). The interfollicular space in the panniculum adiposum is gradually replaced by granulomatous tissue composed essentially by macrophages and newly formed blood vessels, with moderate infiltration of neutrophils and lymphocytes. In some areas, the inflammatory cells totally replaced the adipose tissue and infiltrated the panniculum camosum. Later (6-8 weeks), hair follicles penetrating the panniculum carnosum are frequent (Figure 3G), increasing the disruption of skin structure. Eventually, most follicles undergo degeneration and destruction through an unknown mechanism (Figure 3H). The inflammatory reaction seems to be triggered by necrotic follicles and remaining hair shafts. A common phenomenon is the presence of naked hair shafts surrounded by macrophages and multinucleated giant cells, characteristic of the foreign body reaction. Although degenerative hair follicles are already observed during the fourth postnatal week, follicle destruction is not simultaneous, since growing follicles are still found later on. After several months, most follicles have degenerated and pigmented debris and necrotic follicles eliciting a strong foreign body reaction are observed (Figure 31). Although the mild early alterations in hair follicle development have also been observed in waved-i and -2 mice, the subsequent process of follicle degeneration and destruction is a hallmark of the severe phenotype exhibited by K5-HERCD-533 transgenic mice. The interfollicular epidermis of K5-HERCD-533 mice is markedly hyperplastic, becoming more apparent as mice age. In addition, hyperproliferation-associated keratin K6 is expressed in interfollicular epidermis (Figure 4C). To determine the proliferation rate in transgenic and control mice, we studied the incorporation of the thymidine analog 5-bromo-2-deoxyuridine (BrdU) in the skin of 8-weekold animals, and observed much higher incorporation into the epidermis (9-fold) and hair follicles of transgenic as compared with normal control mice (Figure 4B and D). BrdU incorporation was remarkably high in aberrant giant follicles found in transgenic mice, proving their permanence in anagen (Figure 4F), while hair follicles of control littermates are in telogen stage and show very low BrdU incorporation (Figure 4B).

Inhibition of EGFR function To determine the extent of inhibition of endogenous EGFR function by the expression of the HERCD-533 dominant negative mutant, we examined EGFR autophosphorylation in the epidermis of K5-HERCD-533 mice (Donaldson and Cohen, 1992). Protein extracts of skins from 1- to 2-day old transgenic mice of the line T4 and control litermates

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Fig. 3. Histopathology of K5-HERCD-533 mouse skin. (A) Mid-dorsal section of back skin of a 10-day-old control mouse. (B) Skin section from a 6-day-old K5-HERCD-533 mouse. Note the deep implantation and proximity of follicles to the panniculum camosum. (C) Section from a 10-day-old K5-HERCD-533 mouse showing altered orientation of hair follicles. (D) Mid-dorsal back skin section of a 55-day-old control mouse with hairs at the telogen stage. (E) Skin section from a 21-day-old transgenic mouse showing necrotic follicles (arrowhead) and granulomatous tissue replacing the s.c. adipose layer. (F) Higher magnification showing another field of the section shown in (E). Note the degenerative follicles with altered root sheaths (arrow), abundant vascularization and very deep implantation of follicles. The muscular layer remains intact. (G) Skin section of a 55-dayold K5-HERCD-533 mouse showing follicles disrupting the panniculum carnosum (PC) indicated by an arrow; see also epidermal hyperplasia. (H) Higher magnification of a field from the same section as in (G) showing a necrotic follicle trapped in the muscular layer and inflammatory reaction around the follicle (arrowhead). (I) Skin section of a 70-day-old K5-HERCD-533 mouse showing debris of hair follicles, hair shafts and melanin. Magnification: (A-E) and (G), 40X; (F) and (H), 120X; (I), lOOX.

injected either with EGF or saline were resolved by SDSPAGE and immunoblots were probed with antiphosphotyrosine antibody PY-20. As reported previously (Donaldson and Cohen, 1992), several tyrosine-phosphorylated bands are detected in skin homogenates from control mice upon s.c. injection of EGF (Figure 5, lane 1). The intensity of a 170 kDa band corresponding to the

EGF receptor is strongly reduced in lane 3, corresponding to T4 mouse injected with EGF. Since we could not rule out that a part, albeit minor, of the EGF-induced autophosphorylation of the EGFR takes place in the dermis, where the transgene is not expressed, we studied EGFR tyrosine phophorylation in primary keratinocyte cultures. After 48 h of serum starva5219

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Fig. 4. Double-immunofluorescence analysis of expression of keratin K6 and cell proliferation in K5-EGFR-533 transgenic mice. (A) Back skin section of an 8-week-old control mouse labeled with anti-K6 antibody showing staining at bulbs of hair follicles in telogen phase and absence of staining in interfollicular epidermis. (B) Same field as in (A) stained with an anti-BrdU antibody. Rare positive nuclei are indicated by arrowheads. (C) K6 staining of a back skin section from a pK5-HERCD-533 transgenic littermate showing an intense reaction in interfollicular epidermis and hair follicles. (D) Same field as (C) stained with an anti-BrdU antibody showing abundant positive nuclei in the basal layer of epidermis and hair follicles (arrowheads). (E) and (F) Another field of the previous section showing numerous BrdUpositive nuclei in the bulb of an aberrant anagen follicle. Note also the abundance of positive nuclei in other hair follicles and interfollicular epidermis. (A), (B), (E) and (F), 75x; (C) and (D), 125x.

tion, EGF was added and protein extracts were also analyzed. EGFR phosphorylation was detected in cells from control mice after EGF addition (lane 5), but was reduced in transgenic cells incubated with EGF (lane 7) to a level similar to that in non-stimulated cells (lanes 6 and 8). To assess the amount of EGFR protein, immunoblots were reprobed with an antiserum against the C-. terminal domain of EGFR which specifically recognizes endogenous EGFR (Luetteke et al., 1994). Interestingly, samples showing heavily phosphorylated EGFR both in tissue extracts and in cultured cell extracts possess a significantly lower amount of receptor protein. This decrease in EGFR may be explained by the rapid receptor downregulation which takes place upon receptor activation or by a decreased affinity of the antibody for the phosphorylated EGFR.

Discussion In this study, we have shown that expression of a dominant negative mutant of EGFR driven by the regulatory sequences of the bovine K5 gene induces striking alterations in hair follicle development, skin architecture and regulation of hair cycling. These changes progressively lead to hair degeneration and alopecia. 5220

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Fig. 5. Western immunoblotting analysis of in vivo and in vitro EGFR autophosphorylation. Proteins were extracted from whole skin of 1- to 2-day-old mice 20 min after s.c. injection of either 2 gg/g of EGF (lanes 1 and 3) or saline (lanes 2 and 4), or from primary epidermal cell cultures 3 min after addition of 5 ng/ml EGF (lanes 5 and 7) or bovine serum albumin (BSA) (lanes 6 and 8). Phosphotyrosinecontaining proteins were detected by immunoblotting with peroxidasecoupled PY20 antibody (top panels). Lanes 3, 4, 7 and 8, K5-HERCD533 mice; lanes 1, 2, 5 and 6, control mice. The bottom panels show the amount of EGFR protein detected using an anti-EGFR antiserum. Lanes 1-4, 150 jtg protein loaded; lanes 4-8, 50 jig protein loaded.

Our results are consistent with previous data demonstrating a fundamental role of EGFR function in hair follicle morphogenesis and development. Study of EGFR binding to labeled EGF during embryonic development showed that hair follicles originate from epidermal patches, above dermal mesenchymal condensations, where [I25]EGF binding cannot be detected (Green and Couchman, 1984). Immunolocalization studies demonstrate the absence of EGFR in the keratinocytes that develop into hair germs (Nanney et al., 1990). EGF administration to adult sheep causes follicles to enter catagen phase (Moore et al., 1982; Philip et al., 1985). These data point to EGFR signaling as a major regulator of the orderly epithelium/mesenchyme interactions which control hair follicle morphogenesis and development (Hardy, 1992). These interactions seem to be consistently disrupted in K5-HERCD-533 transgenic mice, in which hair follicles fail to enter into the catagen phase, remaining in aberrant anagen (Figure 4E and F). The absence of catagen, the phase in which a substantial remodeling of the hair follicle accompanied by proteolytic processes takes place, is consistent with the demonstrated upregulation of tissue metalloproteinases in response to EGFR stimulation in murine hair follicles (Weinberg et al., 1990). The alteration of hair cycle progression leads to follicle degeneration and destruction accompanied by a strong immune response, characterized by infiltration of the skin with inflammatory elements. This response is probably triggered by the remains of hair follicles and nude hair shafts that are recognized as foreign bodies by the immune system. Inflammation is stronger between weeks 3 and 7, gradually decreasing afterwards. Interfollicular epidermal hyperplasia and induction of keratin K6, a marker of epidermal hyperproliferation, are


other hallmarks of the skin alterations found in K5HERCD-533 mice. Although signaling through EGFR is considered to elicit a mitogenic response in epidermal keratinocytes, inhibition of EGFR function in the epidermis of K5-HERCD-533 mice, assessed by decreased EGFR tyrosine autophosphorylation, does not impede strong proliferation of the basal cells of epidermis, as demonstrated by BrdU incorporation (Figure 4D). Hence, abrogation of EGFR function may in fact lead to increased proliferation of epidermal keratinocytes, perhaps allowing other growth factors such as KGF or bFGF to increase their action. However, the increased rate of proliferation may alternatively be due to the action of mitogenic stimuli non-dependent on EGFR function. The infiltration of the skin with inflammatory elements that takes place in K5-HERCD-533 mice may be a source of mitogenic cytokines. The surprisingly mild phenotype observed in waved-i and waved-2 mutants, as compared with the severe alterations displayed by K5-HERCD-533 mice, means that only partial inhibition of EGFR function occurs in the waved mice. The lack of TGF-a in waved-i mice is likely to be compensated for by the presence of other EGFR ligands such as EGF, HB-EGF, betacellulin or amphiregulin. In vivo EGFR phosphorylation is slightly inhibited in waved-2 mice as a consequence of a point mutation in the catalytic domain of the EGFR gene, determining a mild phenotype similar to that found in waved-i mice. The Western blot results demonstrate strong inhibition of EGFR phosphorylation in K5-HERCD-533 mouse epidermis. The alterations in hair follicle development and skin architecture found in K5-HERCD-533 mice are also much more severe than those in waved mutants, and implicate EGFR function in the regulation of hair cycle progression. At least one other member of the EGFR family, HER2/ neu, has been detected in the basal layer of epidermis (Kokai et al., 1987). Heterodimerization between different members of the EGFR family seems to be a means for increasing the diversity of signaling pathways and may also constitute an important signaling mechanism in epidermal cells (Wada et al., 1990). It has been demonstrated that a dominant negative mutant of the HER2/neu receptor is able to inhibit EGFR function (Qian et al., 1994). Transgenic dominant negative EGFR expressed in K5-HERCD-533 mice probably inhibits the function of other members of the EGFR family present in epidermis in addition to EGFR. Since the action of HER2/neu or additional members of the EGFR family may compensate EGFR inhibition, the potential extension of signaling inhibition to all members of the EGFR family may elicit the phenotype of K5HERCD-533 transgenic mice. Ongoing experiments will clarify this possibility. Overexpression of EGFR and its ligands has been detected in a variety of epithelial carcinomas, including squamous cell carcinomas (Yamamoto et al., 1986; Derynck et al., 1987). Antibody-mediated blockade of EGFR inhibits the growth of EGFR-overexpressing tumors derived from squamous carcinoma cells (Modjtahedi et al., 1994). Since EGFR activity is inhibited in the epidermis of K5-HERCD-533 mice, skin carcinogenesis experiments performed on these transgenic mice will be of great value

in determining the role of EGFR activation in the formation and progression of skin tumors. Truncated mutants of tyrosine kinase receptors lacking the cytoplasmic domain have proven to be effective in inhibiting signal transduction through their wild-type counterparts both in vitro (Redemann et al., 1992) and in vivo (Werner et al., 1993, 1994; Millauer et al., 1994). Using a dominant negative transgenic approach, we have achieved epidermal-specific inhibition of EGFR function to a very high extent, without compromising other tissues, thus avoiding possible deleterious effects derived from EGFR blockade in multiple organs. Since our mice are fertile and viable in spite of the striking alterations that occur in their skin, they constitute a very useful tool to explore the implication of EGFR in epidermal neoplasia and other skin diseases.

Materials and methods Plasmid construction The cDNA of the truncated EGFR was excised as a XhoIINheI fragment from plasmid pNTK-HERCD-533 (Kashles et al., 1991) and introduced in the polylinker of the vector pl63/7 (Woodroofe et al., 1992). The SalIVKpnI fragment containing the 5' P-globin intron 2, the cDNA and the 3' polyadenylation sequences was inserted 3' downstream of the 5 kb bovine keratin K5 regulatory sequences (Blessing et al., 1987; Ramfrez et al., 1994) cloned in pBluescript. This construct was designated as pK5-HERCD-533.

Generation of transgenic mice The transgene was excised from the plasmid vector with Asp 718 restriction enzyme, purified by low-melting-point agarose electrophoresis and Elutip columns (Schleicher and Schuell), adjusted to a final concentration of -2 ,ug/mI and microinjected into (C57BL/6XDBA/2) F2 mouse embryos as described previously (Hogan et al., 1986). Founder mice were identified by Southern blot analysis of tail DNA, using the 5 kb fragment corresponding to the bovine keratin K5 promoter as a probe.

Preparation of tissue and cell extracts For in vivo EGFR phosphorylation analysis, control and K5-HERCD533 transgenic pups (1-2 days old) were injected s.c. with phosphate-buffered saline (PBS) or EGF (2 ig/g body weight) in PBS. After 20 min, mice were killed and back skins were harvested and homogenized in a Tritoncontaining buffer as previously described (Donaldson and Cohen, 1992). For in vitro phosphorylation, primary keratinocyte cultures from 3-dayold control or K5-HERCD533 mice were performed as described previously (Hennings et al., 1980). Cells were incubated for 48 h in low-calcium (0.05 mM calcium) medium containing 0.5% fetal calf serum after 3 days in standard low-calcium, 8% serum-containing medium. Cells were harvested 3 min after EGF addition (5 ng/ml) to the medium, and lysed in the same buffer used for the tissue extracts. Tissue and cell extracts were heated in sample buffer at 95°C for 5 min prior to SDS-PAGE.

Detection of phosphorylated proteins by Western blot

analysis Equal amounts of protein extracts (150 jg of protein for tissues and 50 ig for cell extrcts), determined by the BioRad protein assay, from transgenic and control mice were separated by 7.5% SDS-polyacrylamide gels and transferred to immobilon-P membranes (Millipore). The membrane was probed with anti-phosphotyrosine antibody (1:5000 dilution of PY20-HRP, ICN) or with 1:1000 dilution of rabbit anti-ERCT antiserum (Luetteke et al., 1994), followed by a peroxidase-coupled anti-rabbit IgG (Jackson Immunolaboratories). Immunoreactive proteins were detected by chemiluminiscence (ECL, Amersham, UK).

RNA blot analysis Total RNA was extracted from skin samples by the acid-guanidinium method (Chomczynski and Sacchi, 1987). RNA (15 ,ig/lane) was electrophoresed through a 1% agarose gel containing 6% formaldehyde and transferred to a nylon membrane (Gene Screen Plus, DuPont) by capillarity in lOX standard saline citrate (SSC) (lx SSC = 0.15 M

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R.Murillas et aL NaCl and 0.015 M sodium citrate). The filter was UV irradiated for 2 min, baked for 2 h at 80°C and hybridized to 32P random primerlabeled probes specific for human EGF receptor (2.4 kb XholINheI fragment of human EGFR cDNA) or GAPDH (1.3 kb PstI fragment of rat GAPDH cDNA).

Histological analysis Skin samples were fixed in formaldehyde (10% in PBS) and embedded in paraffin. Sections were cut at 5 gm and stained with hematoxylin-eosin.

BrdU labeling experiments Eight-week-old K5-HERCD-533 transgenic mice and control littermates were injected i.p. with BrdU (Boehringer Mannhein, 0.25 mg/g in 0.9% NaCl ). Animals were killed 2 h after the injection. Tissue samples were fixed in 96% ethanol and embedded in paraffin.

Immunofluorescence analysis Keratin K6 and BrdU detections were performed on paraffin sections (4 gm). Tissue sections were dewaxed, treated with 1 N HCI for denaturation of DNA, washed twice with 70% ethanol and once with 30% ethanol and PBS (pH 7.5). Sections were then incubated with RK6 rabbit antiserum to keratin K6 (1:400 dilution) mixed with a rat monoclonal anti-BrdU antibody for 1 h. The secondary antibodies were Texas red-labeled anti-rabbit IgG and fluorescein isothiocyanate (FITC)labeled anti-rat IgG (Jackson Immunolaboratories). Truncated human EGFR was detected in frozen tissue sections. The sections were air dried, fixed in 1:1 methanol/acetone for 10 min at -20°C and incubated with mouse monoclonal antibody 108 that specifically recognizes human EGFR extracellular domain (Fendly etal., 1990). The secondary antibody was FITC-labeled anti-mouse IgG (Jackson Immunolaboratories). Slides were examined in a Zeiss Axiophot microscope using appropriate filters.

Acknowledgements We thank A.Dlugosz for helpful comments. We are grateful to D.R.Roop for K6 antiserum (RK6), H.Shelton Earp for anti-EGFR antiserum (ERCT) and Sybille Mittnacht for rat monoclonal anti-BrdU antibody. Recombinant human EGF was kindly provided by Serono, Spain. R.M. is a fellow of the Ministerio de Educaci6n y Ciencia of Spain. This work was partially funded by grants PM92-0203 and PB90-0390 of the Direcci6n General de Investigaci6n, Ciencia y Tecnologia (DGICYT) of Spain.

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