Characterization and distribution of epidermal growth factor receptors in the skin and wool follicles of the sheep fetus during development

r~ l E• I . II TN ETER WMO A RNT

H

N

CHARACTERIZATION AND DISTRIBUTION OF EPIDERMAL GROWTH FACTOR RECEPTORS IN THE SKIN AND WOOL FOLLICLES OF THE SHEEP FETUS DURING DEVELOPMENT 1 P.C. Wynn,? 2 G. Brown,* and G.P.M. Moore** tDepartment of Animal Science, University of Sydney, Camden NSW 2570, Australia *Biometrics Unit, Institute of Animal Production and Processing, CSlRO, P.O. Box 239, Blacktown NSW 2148, Australia **CSlRO, Division of Animal Production, P.O. Box 239, Blacktown, NSW, 2148, and Department of Biological Sciences, University of Western Sydney, Neplan Kingswood, NSW 2747, Australia Received August 4, 1994

ABSTRACT We have determined the binding affinity and capacity and relative distribution of epidermal growth factor (EGF) receptors in the skin of the Merino sheep fetus before and during the development of the wool follicle population. Autoradiography of tissue sections incubated with [125I]EGF revealed that label was confined predominantly to the epidermis and dermoepidermal junction before follicle formation, at 30 and 55 d of gestation. During follicle initiation (Days 60 to 65), receptor activity was distributed over the epidermis, including the epidermal aggregations of primordia at the dermoepidermal junction. However, receptor concentrations, as revealed by grain counts of autoradiographs, were reduced in these regions when compared with 55-d skin. The receptor distribution over the epidermis and its derivatives did not alter during subsequent follicle development, although the intensity of labeling increased as the follicles matured. Specific receptor binding was not observed above background levels in the dermis and dermal papillae during all stages of follicle development. At follicle maturation, EGF receptors were widely distributed over the cells of the epidermis and the epidermal derivatives of the cutaneous appendages but were particularly localized in the sebaceous glands and outer root sheath (see also Wynn et al. 1989j. EGF immunoreactive material has also been found at these sites (du Cros et al. 1992), suggesting an autocrine role for EGF in the regulation of cell function. It is likely that the differentiationpromoting activities of EGF may predominate over those of growth, because the receptor-bearing cells were not members of rapidly proliferating populations.

INTRODUCTION Epidermal growth factor (EGF) and other members of the EGF family have been implicated in the regulation of normal skin functions in mammals. EGF from mouse salivary glands has profound effects on the proliferation and differentiation of epithelial and dermal cell populations of the epidermis and hair follicles in vivo (I-5J and of cultures derived from these tissues (see Refs. 6-10 for reviews). Receptors for EGF have been identified in cells of a variety of tissues, including keratinocytes, dermal fibroblasts. and cells o f the cutaneous appendages (11-15). Furthermore, the expression of the EGFlike transforming growth factor-alpha (TGF-et) in the epidermis (16) and the detection of an EGF-like molecule in sheep hair and wool follicles (17,18) suggest local producuon for Domestic Animal Endocrinology 12:269-281, 1995 © Elsevier Science Inc. 1995 655 Avenue of the Americas, New York, NY 10010

0739-7240/95/$10.00 SSDI 0739-7240(95)00024-9

270

WYNN ET AL.

the regulation of cell functions by this family of growth factors. In addition, sequences homologous with the homeotic gene Notch of Drosophila, which encodes for multiple EGF-like repeats (19), have been detected in the hair follicle (20). The putative product is large, its sequence indicating that it may be an integral membrane protein rather than a diffusible effector. Mutations at the Notch locus are cell autonomous, suggesting that the protein exerts its effects via an autocrine mechanism or through direct cell-cell interactions, although it is not known whether signal transduction is mediated by a tyrosine kinase EGF receptor (EGF-R) or through the receptor-like characteristics of the Notch protein (21). There is also evidence of the involvement of EGF in mammalian development. The growth factor has been detected in embryonic and fetal tissues of the mouse (22-25) and sheep (26), and rat and mouse embryos synthesize EGF- and TGF-ct-specific mRNAs (27-29). Exogenously administered EGF affects the development of the palate (30) and lung in utero (31) and the skin during the perinatal period (32,33). Of more immediate interest are the observations that this factor induces morphogenetic changes in organs that develop as a consequence of epithelial and dermal interactions: the lung (34), mammary glands (35), and the eccrine sweat glands of the skin (36). EGF and related molecules have also been specifically localized in the cells of these tissues, which is consistent with the highly regionalized nature of the processes of differentiation and development. We have recently reported the presence and distribution of EGF-immunoreactive material in the skin and wool follicles of mature and fetal sheep (18,37). In order to examine relationships between the localization of this growth factor and its possible functions during follicle morphogenesis, we have determined the distribution, concentration, and binding affinity of EGF-R in the sheep skin during fetal development. A previous study of EGF-Rs in the skin of mature sheep has been reported (15). MATERIALS AND METHODS Animals. Fifty-five mature Merino ewes were used in these experiments. Estrus was synchronized with Repromap (Upjohn, Kalamazoo, MI) intravaginal sponges. The sponges were removed after 12 d, and the ewes were penned with Merino rams at the beginning of the second cycle, 19 d later. Each ram was harnessed with a crayon (38), and ewes were inspected daily for mating marks. The day on which a mark was detected was designated as Day 1 of gestation. Pregnancy was confirmed where appropriate with a real-time ultrasound scanner (ADR Model 2130, ADR Ultrasound, Tempe, AZ). Fetuses were recovered from the ewes at times that coincided with major developmental events, namely, before follicle initiation (30 to 55 d), during the first wave of initiation (55 to 80 d), and at the development of secondary follicles (80 to 140 d), in order to cover the principal developmental events associated with wool follicle morphogenesis (39). A group of mature Merino male castrate animals was used as a source of skin biopsies with which to compare the receptor-binding characteristics of the fetal tissues. The skin biopsies were obtained from the midside with a l-cm-diameter trephine and were placed in 2 methyl butane in dry ice before storage at - 8 0 ° C (15). Receptor-binding assay. EGF was prepared from the submaxillary glands of mice according to the procedure of Savage and Cohen (40), and its purity was assessed by electrophoretic analysis on cellulose gels and by high-performance liquid chromatography. The ~25I-labeled EGF was prepared as reported previously (15). Receptor-binding studies were conducted on crude membrane particles prepared from whole embryos (Days 31 to 32 embryos only) or on flank skin recovered from the older fetuses by a procedure described previously (15). In brief, skin was powdered in liquid N2 and dispersed by 10 strokes with a mechanical

EGF-R IN FETAL SHEEP SKIN

271

homogenizer in Tris-HCl buffer (50 mmol/1; pH 7.4) containing 5 mmol/1 of MgC12. Homogenates were centrifuged at 48,000 x g for 30 min at 4 ° C and resuspended in the same buffer containing 100 kallikrein inhibitor units (KIU) of aprotinin/ml. (Sigma Chemical Co., St. Louis, MO) to give a final concentration of 80 to 150 p~g of protein in 100 ~xl. For the assay, 100 Ixl of the membrane suspension was incubated with 850 Bq of [lZSI]EGF (100 Ixl) (specific activity, 22-36 MBq/ixg) and increasing concentrations of EGF in the membrane suspension buffer with the addition of bovine serum albumin (BSA) (0.1%, w/w; Sigma). In the case of the 30-d-old embryos, there was insufficient tissue to conduct a full displacement curve, and so, membrane particles were incubated with a subsaturating concentration of EGF (4 nmol/1). After incubation for 60 min at 23 ° C, the assay was terminated with the addition of phosphate-buffered saline containing 0.1% BSA, and receptor-bound radioactivity was separated by filtration through glass fiber filters (Whatman, Clifton, NJ) presoaked in 1% BSA. Filters were washed three times in the same buffer and counted in a gamma spectrometer (LKB, Uppsala, Sweden). Nonspecific binding was assessed in the presence of 100 nmol/1 EGF and did not exceed 20% of total binding. Receptor concentration and affinity were estimated by the use of computer analysis of the binding displacement curves with the analytical program, Ligand (41). These values were corrected for the maximum "bindability" of the radiolabel in the presence of excess skin membranes, which varied from 20 to 35%. The receptor concentration in 30-d embryo particles was estimated by equilibrating them with the radiolabeled tracer plus 4 nM unlabeled EGF (approximately 8 times the Kd) to give a total ligand concentration that achieves near-saturation (15). Estimates of receptor concentration by the two methods were found to be highly correlated (R = 0.77; n = 17). Localization of EGF-binding sites in skin. The distribution of receptor sites in skin was detected by autoradiography. By the use of a cryostat, longitudinal frozen sections ( - 18° C) were cut from skin samples at a thickness of 8 ixm, mounted on glass slides, and incubated with [125I]EGF by the procedure of Wynn et al. (15). In brief, the slidemounted sections were preincubated for 6 min at 4 ° C in sodium acetate (0.2 mol/1) containing sodium chloride (0.5 mol/l) and 0.1% BSA (pH 3.5) to dissociate endogenous ligand. After four washes, each of 2 min, in assay buffer, slides were incubated in assay buffer containing 1,700 Bq/ml [125I]EGF for 60 min (23 ° C) with or without the addition of unlabeled EGF (0.1 Ixmol/1) to determine nonspecific binding. Slides were sequentially washed in assay buffer (0 ° C) for 2 min on four occasions and air dried. The sections were fixed by incubation in the presence of paraformaldehyde vapor for 2 hr at 80 ° C under reduced pressure. All slides were coated with Ilford K2 nuclear emulsion (Ilford Ltd., Cheshire U.K.), air dried, and exposed at 4 ° C, in the dark. The relative densities of EGF-binding sites were quantified by grain counting. Skin sections were processed and autoradiographed together (42) after incubation with [125I]EGF at gestational ages of 55 and 70 d, which included the first wave of follicle initiation. Silver grain densities were monitored by developing test slides at intervals during the exposure period. When densities suitable for visual counting had been attained, the remaining slides were developed and fixed on the same day and stained with hematoxylin and eosin. The numbers of silver grains over areas of the epidermis, dermis, and the epidermal and dermal components of wool follicle primordia were estimated in skin sections from five animals, which were incubated with experimental or control media. The grains counted were those in a volume of emulsion contained within the boundaries of a microscope eyepiece graticule (dimensions: 15 × 60 Ixm) at a magnification of × 1,000. The numbers of cells within the grid boundaries were also counted.

272

WYNN ET AL.

Analysis of grain counts and of receptor-binding variables. Statistically, the data were regarded as two groups (Days 55 and 70) with several sources of nested random variation as the result of sheep, slides, and sections. The sheep were the major sources of variation in the data, but transformation to stabilize within-sheep variances did not affect the conclusions. The data were analyzed by a modified Welch two sample t-test. Three questions were addressed: 1) are there differences in grain counts/cell between days for either the epidermis or dermis? 2) Is there a relationship between the epidermis/dermis grain count ratios? 3) Are there differences in the grain counts/cell between the cells of the primordia and those from interfollicular regions of skin on Day 70? Receptor-binding variables were analyzed by a one-way analysis of variance. Probabilities of less than 0.05 were considered significant. RESULTS

Binding characteristics of mature Merino castrate male skin membrane particles for [12SI]EGF. The binding of [125I]EGF to skin particles was saturable, and Scatchard analyses of displacement binding data showed a single high-affinity component (Figure la) with a dissociation constant (Kd) of 0.42 -- 0.04 nM and a binding capacity of 140 +- 15 fmol/mg of protein (Table 1). EGF-R binding in embryonic and fetal skin. An association between the morphogenetic processes of wool follicle formation and the distribution and binding characteristics of EGF-R was initially sought by incubating [125I]EGF with membrane particles prepared from fetal skin at various stages of gestation. Binding was saturable, and Scatchard analysis demonstrated a single high-affinity binding site in skin particles at Day 70 (Figure lb). The influence of fetal age on EGF-R concentration and affinity is shown in Table 1. It was not possible to sample embryonic skin earlier than 40 d for EGF-binding studies because of the difficulty with physically separating the skin. The binding capacity of the intact embryo, collected at 30 d of age, was highly variable. When the data for both binding capacity and affinity were plotted against fetal age, there was no trend, i.e., the regression coefficients were not significant indicating no influence of gestational age on these parameters. Localization of [12SI]EGF-binding sites in embryonie and fetal skin sections. Frozen skin sections incubated in the presence of [125I]EGF show localized uptake of label by autoradiography. The epidermis of the sheep embryo at 30 d of gestation consisted of one to two layers of cuboidal cells, covered by a thin, flattened periderm (43). The underlying mesenchyme was densely populated with cells (Figure 4a) and had a welldeveloped vasculature, with vessels running parallel to the dermal-epidermal junction (DEJ) (44). Autoradiography revealed that binding of [125I]EGF was predominantly confined to the epidermis and the DEJ (Figure 2b). Labeling of the mesenchyme did not appear to be greater than the background. Later, in the 55-d fetus, just before the onset of follicle initiation in midside skin, the epidermis had thickened, consisting of a basal layer of cuboidal cells, an intermediate layer one to two cells thick, and a flattened periderm (Figure 3a). The dermis contained scattered fibroblasts that appeared more densely clustered near the DEJ. As before, [125I]EGF binding was localized to the epidermis and the DEJ at this stage (Figure 3b). There appeared to be a relative reduction in label density associated with the basal layer when compared with the upper epidermal layers. Labeling of the dermal cells was generally very low and did not appear to be related to their proximity to the DEJ. The distribution of label did not appear to change markedly with the onset of follicle initiation (Day 69 in midside skin, Figure 4a); the density of grains over the epidermal aggregations resembled the basal layer, and the density over the dermal aggregate was low

EGF-R IN FETAL SHEEP SKIN

273

100

i

°'21 ~ 50 ~aO. 1

60 Bound ( p m o l / L )

120

1

5.0

2.5

(a)

Total EGF added (nmol/L)

40

20

U l 2.5

(b)

20 Bound (pmol/L)

40 5.

Total EGF added (nmol/L)

Figure 1. Saturation plot and Scatchard plot (insert) of [125 I]EGF binding to membrane particles prepared from the skin of (a) a mature Merino castrate male sheep and (b) a 70-d-old Merino fetus. Equilibrium dissociation constants and binding site concentrations are given in Table 1.

(Figure 4b). With further development, the epidermal components of the follicle formed a plug that penetrated the dermis (Figure 5a). These cells were strongly labeled, but grain densities over the dermal condensations were low and did not appear to differ from those associated with the cells o f the surrounding dermis (Figure 5b). By 92 days o f gestation, the epidermis consisted of a basal layer, an intermediate layer four to five cells thick, and a periderm (Figure 6a). Follicle density had increased, and

274

WYNN ET AL.

TABLE I . T H E INFLUENCE OF GESTATIONAL AGE ON E G F - R - B I N D I N G CAPACITY AND AFFINITY IN OVINE SKIN. a

Gestational Age (d)

n

30 40-56 70 90 101 140 Mature wether

6 4 5 4 7 7 8

Bmax (fmol/mg of protein) 405 + 127 ± 124 ± 159 ± 184 ± 184 ± 140 ±

167 19 18 7 16 16 15

Ka (nmol/l) 0.52 0.40 0.48 0.45 0.25 0.42

-+ 0.17 ± 0.04 ± 0.03 ± 0.03 ± 0.02 ± 0.04

a Values are mean -+ SE.

many stages of development were evident. The most advanced follicles appeared as compact columns of epidermal cells penetrating the dermis (Figure 6c). Flattened dermal cells enveloped the epidermal plug, and the dermal condensation was located at its proximal end. Developing accessory structures, such as apocrine sweat glands and sebaceous glands, were also evident. [125I]EGF was associated with all of these epidermally derived structures (Figure 6b and d). By contrast, labeling of the dermal condensations was low. Incubation of sections with [125I]EGF in the presence of an excess of unlabeled EGF (nonspecific binding) displaced most of the label (compare Fig. 6d and f). At 120 d of gestation, the epidermis appeared as four layers of cells; the distal layer had become cornified, and the periderm had been sloughed (Figure 7a). The distribution of EGF-R within the epidermis and epidermally derived elements of the follicles was similar to that observed in 90-d skin, although densities had increased. The sebaceous glands were heavily labeled (Figure 7b). G r a i n densities in 55- a n d 70-d fetal skin. The relative distributions of label associated with the skin during the events surrounding follicle initiation were estimated from grain counts (Table 2). After adjusting for background, grain densities over the epidermis and dermis were compared at 55 to 57 and 69 to 70 d of gestation and at 70 d between initiating follicles and interfollicular skin. All of the sections used in this analysis were

(a)

(b)

Figures. 2-7. Localizationof EGF-bindingsites in developingfetal sheep skin: histologicsections were stained with hematoxylin and eosin and photographed with brightfield optics. The autoradiographic localization of [~25I]EGFbinding in cryostat sections was photographed with darkfield optics. Figure 2. A 31- to 32-d fetus. (a) The epidermisconsists of one to two layers of cuboidalcells and a flattened periderm. The mesenchymeis relatively unstructured and contains numerous blood vessels running parallel to the epidermal-mesenchymal junction. (b) [125I]EGF-bindingsites are concentratedin the epidermis and DEJ. Labelingof the mesenchyme does not appear to differ from that of the background. (scale as for Figure 3)

EGF-R IN FETAL SHEEP SKIN

(a)

275

(b)

Figure 3. A 55-d fetus. (a) The epidermis has thickened, and the basal layer is composed of cuboidal ceils, Overall, cell density of the dermis has decreased but some clustering is evident adjacent to the DEJ. (b) [nESI]EGF binding is evident, predominantly in the epidermis and DEJ, although grain densities associated with the basal cells appear reduced. Labeling of the dennis is low. Bar = 30 t.Lm.

incubated with the radiolabel in the same assay. Analysis of the grain counts extended the foregoing qualitative observations. Silver grain numbers over the epidermal cells at Days 55 and 70 were significantly higher than those associated with dermal cells. With the onset of follicle initiation by Day 70, label densities over both epidermal and dermal cells were reduced when compared with Day 55 values, with a relatively greater effect being observed in the dermis. The differences were significant in the dermis (t = - 3.09, P = 0.02) but not in the epidermis (t = - 1.86, P = 0.11). Grain numbers associated with the epidermal and dermal cells of the primordia at Day 70 did not differ from those found with related regions of interfollicular skin (t = - 0.30, P = 0.79 and t = - 0.84, P = 0.46, respectively; Table 2).

DISCUSSION EGF-binding sites have been detected in embryos and in fetal tissues of various mammals (24,45). Adamson and Meek (46) reported an increase in receptor concentrations and a decreased affinity in fetal mouse tissues with advancing gestational age and suggested that the changes were developmentally programmed as the tissues passed from early proliferative to late differentiation and maturation phases. The results of this study indicate the presence of EGF-R in the surface epithelium of the 30-d sheep embryo and in the

O

(a)

(b)

Figure 4. A 69-d fetus. (a) The follicle primordium appears as aggregations of epidermal and dermal cells juxtaposed to the DEJ. (b) [125I]EGF binding to the epidermal aggregate and the basal layer of the epidermis is less than that bound to the distal epidermis and DEJ. The dermal condensation of the primordium does not appear to be labeled above the background. (scale as for Figure 3)

276

(a)

WYNN ET AL.

(b)

Figure 5. A 7 l-d fetus. (a) The follicle plug of epidermalcells has penetrated the dermis. (b) []25I]EGFlabeling is concentrated in the epidermis and epidermalcells of the follicle plug. The dermal condensation binds little label. (scale as for Figure 3) skin at all subsequent stages of development examined. Similar increases with the development of the ovine fetus were not apparent, with little change being observed between the gestational ages studied. Similarly, the binding affinity of the receptor sites did not vary markedly between stages of development. In fact, neither of these receptor characteristics differed greatly from those assessed in mature skin. The high receptor concentration in the 30-d embryo was variable, which may have been a reflection of the large differences in the low protein concentrations present in the plasma membranes. Green and Couchman (12) first reported the localization of EGF-binding sites in fetal rat skin with [125I]EGF. Label was associated with all of the epidermis before follicle initiation, but with the appearance of the primordia, receptors became more localized to the basal layer. In the vicinity of the dermal condensation, epidermal labeling was reduced, possibly indicating local receptor down-regulation by follicle-derived homologous ligands. At the follicle plug and later stages of growth, EGF-R were found on the epidermal components but not in the dermal sheath or the dermal condensations. In a complementary histochemical study of developing human skin using antibodies to EGFR, Nanney et al. (47) detected immunoreactive material in embryonic and fetal epidermis, but with the onset of follicle morphogenesis, the intensity of the reaction was reduced in the basal epidermal cells and in the follicles between the germ and hair peg stages. EGF-R immunoreactivity increased during later differentiation and was found in the follicle bulb, outer root sheath (ORS), and sebaceous glands. Quantitative analysis of receptor binding in this sheep study did not reflect these increases at later gestational ages. However, the studies of the morphologic distribution of grains showed that densities appeared to be reduced over the epidermal cells and the epidermal components of follicle primordia at this stage of follicle initiation. However, this apparent decline in epidermal labeling was not significant. Of further interest was the observation that binding to the epidermal aggregate and the basal layer of the epidermis was less than that bound to the distal epidermis. Radioactivity associated with the dermal condensations at initiation sites was much lower than that found over the epidermal cells but did not differ from that of the interfollicular dermis. These distributions did not alter with further development, although grain densities increased over epidermal components of the follicles. In a recent immunochemical study, we reported the presence and distribution of EGFlike molecules in fetal sheep skin (18), suggesting that they functioned in the processes of

EGF-R IN FETAL SHEEP SKIN

277

(a)

(b)

(c)

(d) Y_

(e)

(f)

Figure 6. A 92-d fetus. (a and c) A variety of growth stages is exhibited by the follicle population at this stage, generally appearing as compact columns of cells penetrating the dermis. (b and d) [t25I]EGF binding to the follicle cells has increased; the developing sebaceous glands are labeled. (e and f) Skin section incubated in the presence of an excess of unlabeled EGF showing that most of the label has been displaced. Bar = 50 v,m.

278

WYNN ET AL.

(a)

(b)

Figure 7. A 120-d fetus. (a) The first formed follicles and their accessory structures have matured, and fiber production has begun. (b) [a25I]EGF binding is concentrated over the cells of the epidermis and all of the epidermal derivatives. Bar = 80 p,m. follicle morphogenesis. E G F was identified in the upper epidermis during follicle initiation but was absent from the cells o f the basal layer and those of the follicle primordia. Because the fetal sheep E G F appeared to have a higher molecular weight than did the E G F derived from mature mice (37) and was perhaps cell associated (48,49), its absence from the primordia made it seem unlikely that it would be directly involved in follicle induction in this form. However, the rate of processing of the larger precursor molecule to yield mature E G F peptides will influence the availability of diffusible effector molecules that could serve such a function. Certainly, ['25I]EGF binding in the epidermis and epidermal aggregations of follicle primordia at 70 d of fetal life was reduced when compared with that at 55 d. A similar observation was cited by (12) as evidence of local receptor down-regulation in the skin. These results indicate that this might be a consequence of endogenous E G F activity. If E G F from the upper epidermis of the sheep does act locally by these means, then the mechanism by which it is translocated from its site o f synthesis to the target cells must also be developmentally regulated. TABLE 2. AUTORADIOGRAPHIC GRAIN COUNTS OF THE DISTRIBUTION OF [125I]EGF-BINDING SITES ON EPIDERMAL AND DERMAL TISSUE OF FETAL SHEEP SKIN.

Gestational Age (d) 55-57 69-70 Significance of difference (P value)a Day 55/70 ratio a Using the Welch modified t-test.

Mean No. of Grains/Cell ± SE Epidermis

Dermis

25.1 ± 5.1 13.9 ± 3.2 0.11 1.80

4.5 ± 0.7 1.7 ± 0.6 0.02 2.58

Epidermal Aggregation

Dermal Condensation

15.3 ± 3.9

2.0 ± 0.4

EGF-R IN FETAL SHEEP SKIN

279

The distribution of EGF-R in the sheep skin at maturity was similar to that in the adult rat and human. Receptors were predominantly located in the epidermis, the follicle bulb matrix, the ORS, and the sebaceous and sweat glands, whereas they were at relatively low densities or absent from the dermal cells and the follicle dermal papillae (12-15,47). Green et al. (11) suggested that the distribution and number of EGF-R in skin cells were correlated with their proliferative activity. Further, a similarity between receptor distribution and the expression of factors that bind to the EGF-R (16) might indicate an autocrine mechanism to regulate cell turnover. In the sheep, EGF-like activity was detected in the ORS and sebaceous glands of mature follicles (18). These regions also have relatively high levels of EGF-R. However, the cell populations are not rapidly proliferating, at least when compared with the bulb matrix cells (5). Thus, if EGF is acting locally, other cellular activities must predominate over those that stimulate growth (10,13). In this context, another member of the EGF family, TGF-ct, also binds to the EGF-R but has a different distribution to EGF in the skin (16,50). This also supports the proposition that the individual functions of these factors, in their different forms, might be related to the sites of their expression. It should be noted, however, that the presence of receptors on a cell does not necessarily make it susceptible to activation by the appropriate ligand (51). The challenge remains to identify the biologic function transduced by this receptor-effector pathway and to determine its significance in the morphogenesis of the wool follicle.

AC KNOWLEI) GMENTS/FOOTNOTES Denise Stevens operated the ultrasound scanner, Pat Pisansarakit and Kathy Isaacs assisted with the collection of the skin samples, and Warren Ward with the receptor assay. Diana du Cros, David Hollis and Mark Jones critically reviewed the manuscript. This work was supported, in part, by the Australian Wool Corporation. Aspects of this work were presented at the New York Academy of Sciences Conference on the Molecular and Structural Biology of Hair, January, 1991. 2 Address all correspondence to: Dr. P.C. Wynn; tel.: 61 46 550232; fax.: 61 46 552374

REFERENCES 1. Campbell AJ, Adams SS, Davey MW, Titchen DA. Effects of Lys-13-urogastronein vivo. Aust J Biol Sci 41:463-474, 1988. 2. Hollis DE, Chapman RE, Panaretto BA, Moore GPM. Morphological changes in the skin and wool fibres of Merino sheep infused with mouse epidermal growth factor. Aust J Biol Sci 36:419--434, 1983. 3. Moore GPM, Panaretto BA, Robertson D. Effects of epidermal growth factor on hair growth in the mouse. J Endocrinol 88:293-299, 1981. 4. Moore GPM, Panaretto BA, Robertson D. Inhibition of wool growth in Merino sheep following administration of epidermal growth factor and a derivative. Aust J Biol Sci 35:163-172, 1982. 5. Moore GPM, Panaretto BA, Carter NB. Epidermal hyperplasia and wool follicle regression in sheep infused with epidermal growth factor. J Invest Dermatol 84:172-175, 1985. 6. Hollenberg MD, Cuatrecasas P. Epidermal growth factor: Receptors in human fibroblasts and modulation of action by cholera toxin. Proc Natl Acad Sci USA 70:2964-2968, 1973. 7. Rheinwald JG, Green H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 265:421-424, 1977. 8. Katsuoka K, Schell H, Wessel B, Homstein OP. Effects of epidermal growth factor, fibroblast growth factor, minoxidil and hydrocortisone on growth kinetics in human hair bulb papilla cells and root sheath fibroblasts cultured in vitro. Arch Dermatol Res 279:247-250, 1987. 9. Moore GPM. Growth factors, cell-cell and cell-matrix interactions in skin during follicle development and growth. In: The Biology of Wool and Hair, Rogers GE, Reis PJ, Ward KA, and Marshall RC (eds). Chapman and Hall, London, p. 351-364, 1989. 10. Moore GPM, du Cros DL, Isaacs K, Pisansarakit P, Wynn PC. Hair growth induction by growth factors. In: The Molecular and Structural biology of Hair, Stenn KS, Messenger AG, and Baden HP (eds) Ann NY Acad Sci 642:308-325, 1991. 11. Green MR, Basketter DA, Couchman JR, Rees DA. Distribution and number of epidermal growth factor receptors in skin is related to epithelial cell growth. Dev Biol 100:506--512, 1983.

280

WYNN ET AL.

12. Green MR, Couchman JR. Distribution of epidermal growth factor receptors in rat tissues during embryonic skin development, hair formation and the adult hair growth cycle. J Invest Dermatol 83:118-123, 1984. 13. Nanney LB, Magid M, Stoscheck CM, King LE. Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages. J Invest Dermatol 83:385-93, 1984a. 14. Nanney LB, McKanna JA, Stoscheck CM, Carpenter G, King LE. Visualization of epidermal growth factor receptors in human epidermis. J Invest Dermatol 82:165-169, 1984h. 15. Wynn PC, Maddocks IG, Moore GPM, Panaretto BA, Djura P, Ward WG, Fleck E, Chapman RE. Characterisation and localisation of receptors for epidermal growth factor in ovine skin. J Endocrinol 121:81-90, 1989. 16. Coffey RJ, Derynk R, Wilcox JN, Bringman TS, Goustin AS, Moses HL, Pittelkow MR. Production and auto-induction of transforming growth factor-a in human keratinocytes. Nature 328:817-20, 1987. 17. Pisansarakit P, du Cros DL, Moore GPM. Cultivation of keratinocytes derived from epidermal explants of sheep skin and the roles of growth factors in the regulation of proliferation. Arch Dermatol Res 281:530-535, 1990. 18. du Cros DL, Isaacs K, Moore GPM. Localization of epidermal growth factor immunoreactivity in sheep skin during wool follicle development. J Invest Dermatol 98:109-115, 1992. 19. Wharton KA, Johansen KM, Xu T, Artavanis-Tsakonas S. Nucleotide sequence from the neurogenic locus Notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell 43: 567-581, 1985. 20. Weinmaster G, Roberts VJ, Lemke G. A homolog of Drosophila notch expressed during mammalian development. Development 113:199-205, 1991. 21. Hoppe PE, Greenspan RJ. Local function of the Notch gene for embryonic ectodermal pathway choice in Drosophila. Cell 46:773-783, 1986. 22. Adamson ED. Oncogenes in development. Development 99:449-471, 1987. 23. Matrisian LM, Pathak M, Magun BE. Identification of an epidermal growth factor-related transforming growth factor from rat fetuses. Biochem Biophys Res Commun 107:761-769, 1982. 24. Nexo E, Hollenberg MD, Figueroa A, Pratt RM. Detection of epidermal growth factor-urogastrone and its receptor during fetal mouse development. Proc Natl Acad Sci USA 77:2782-2785, 1980. 25. Proper JA, Bjornson CL, Moses HL. Mouse embryos contain polypeptide growth factor(s) capable of inducing a reversible neoplastic phenotype in nontransformed cells in culture. J Cell Physiol 110:169-174, 1982. 26. Freemark M, Comer M. Epidermal growth factor (EGF)-like transforming growth factor (TGF) activity and EGF receptors in ovine fetal tissues: Possible role for TGF in ovine fetal development. Pediatr Res 22:609-615, 1987. 27. Lee DC, Rochford R, Todaro GJ, Villarreal LP. Developmental expression of rat transforming growth factor-a mRNA. Mol Cell Biol 5:3644-3646, 1985. 28. Popliker M, Shatz A, Avivi A, Ullrich A, Schlessinger J, Webb CG. Onset of endogenous synthesis of epidermal growth factor in neonatal mice. Dev Biol 119:38-44, 1987. 29. Warburton D, Seth R, Shum L, Horcher PG, Hall FL, Werb Z, Slavkin HC. Epigenetic role of epidermal growth factor expression and signalling in embryonic mouse lung morphogenesis. Dev Biol 149:123-133, 1992. 30. Bedrick AD, Ladda RI. Epidermal growth factor potentiates cortisone-induced cleft palate in the mouse. Teratology 17:13-18, 1978. 31. Sundell HW, Gray ME, Serenius FS, Escobedo MB, Stahlman MT. Effects of epidermal growth factor on lung maturation in fetal lambs. Am J Pathol 100:707-726, 1980. 32. Cohen S, Elliott GA. The stimulation of epidermal keratinization by a protein isolated from the submaxillary gland of the mouse. J Invest Dermatol 40:1-5, 1963. 33. Moore GPM, Panaretto BA, Robertson D. Epidermal growth factor delays the development of the epidermis and hair follicles of mice during growth of the first coat. Anat Rec 205:47-55, 1983. 34. Goldin GV, Opperman LA. Induction of supernumerary tracheal buds and the stimulation of DNA synthesis in the embryonic chick lung and trachea by epidermal growth factor. J Embryol Exp Morph 60:235-243, 1980. 35. Coleman S, Silberstein GB, Daniel CW. Ductal morphogenesis in the mouse mammary gland: Evidence supporting a role for epidermal growth factor. Dev Biol 127:304-315, 1988. 36. Blecher SR, Kapalanga J, Lalonde D. Induction of sweat glands by epidermal growth factor in murine X-linked anhidrotic ectodermal dysplasia. Nature 345:542-544, 1990. 37. Pisansarakit P, du Cros DL, Moore GPM. Cultivation of mesenchymal cells derived from the skin and hair

EGF-R IN FETAL SHEEP SKIN

38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51.

281

follicles of the sheep; the involvement of peptide factors in growth regulation. Arch Dermatol Res 283: 321-327, 1991. Radford HM, Watson RH, Wood GF. A crayon and associated harness for the detection of mating under field conditions. Aust Vet J 36:57-66, 1960. Hardy MH, Lyne AG. The prenatal development of wool follicles in Merino sheep. Aust J Biol Sci 9:423-441, 1956. Savage CR, Cohen S. Epidermal growth factor and a new derivative. J Biol Chem 247:7609-7611, 1972. Munson PM, Rodbard D. LIGAND. A versatile computerized approach for characterization of ligandbinding systems. Anal Biochem 107:220-239, 1980. Moore GPM. RNA synthesis in fixed cells by endogenous RNA polymerases. Exp Cell Res 111:317-326, 1978. Lyne AG, Hollis DE. The structure and development of the epidermis in sheep fetuses. J Ultrastruct Res 38:444--458, 1972. Johnson CL, Holbrook KA. Development of human embryonic and fetal vasculature. J Invest Dermatol 93:10S-17S, 1989. Hortsch M, Schlessinger J, Gootwine E, Webb CG. Appearance of functional EGF receptor kinase during rodent embryogenesis. EMBO J 2:1937-1941, 1983. Adamson ED, Meek J. The ontogeny of epidermal growth factor receptors during mouse development. Dev Biol 103:62-70, 1984. Nanney LB, Stoscheck CM, King LE, Underwood RA, Holbrook KA. Immunolocalisation of epidermal growth factor receptors in normal developing human skin. J Invest Dermatol 94:742-748, 1990. Rail LB, Scott J, Bell GI, Crawford RJ, Penschow JD, Niall HD, Coghlan JP. Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues. Nature 313:228-231, 1985. Teixido J, Gilmore R, Lee DC, Massague J. Integral membrane glycoprotein properties of the prohormone pro-transforming growth factor-a. Nature 326:883-885, 1987. Finzi E, Harkins R, Horn T. TGF-a is widely expressed in differentiated as well as hyperproliferative skin epithelium. J Invest Dermatol 96:328-332, 1991. Greenwald I, Rubin GM. Making a difference: The role of cell-cell interactions in establishing separate identities for equivalent cells. Cell 68:271-281, 1992.