Arch Dermatol Res (1992) 284:159-166

9 Springer-Verlag 11992

Expression of cadherin cell adhesion molecules during human skin development: morphogenesis of epidermis, hair follicles and eccrine sweat ducts M. Fujita t, F. Furukawa 1, K. Fujii 1, y. Horiguchi 1, M. Takeichi 2, and S. Imamura 1 Department of Dermatology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan 2 Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606, Japan Received December 16, 1991

Summary. Expression of E (epithelia) and P (placental) cadherin cell adhesion molecules was examined immunohistochemically using human developing skin. In adult skin, E-cadherin was expressed on cell surfaces of whole epidermal layers including skin appendages, whereas P-cadherin was expressed only on those of basal layers and the outer layers of skin appendages, which was consistent with the compartment of proliferating cells. In fetal skin, while the patterns of E- and P-cadherin expression were generally similar to those in the adult, P-cadherin temporarily showed a unique spatiotemporal expression pattern in developing sweat ducts. During this stage, the expression of P-cadherin accumulated in the epidermal ridges and showed a discrepancy with the compartment of proliferating cells. These results suggest that the expression of P-cadherin is spatiotemporally controlled, and may be closely related to the segregation of basal layers as well as to the arrangement of epidermal cells into eccrine sweat ducts, but is not closely related to cell proliferation.

and non-proliferating regions. When cultured with a monoclonal antibody (MoAb) to P-cadherin, the mouse skin tissue shows perturbed morphogenesis, suggesting a substantial role of P-cadherin in skin morphogenesis [10]. However, the expression patterns and roles of cadherins in h u m a n skin morphogenesis have not been fully examined. Only a brief report is available on cadherin expression in h u m a n epithelial tissues [15] to the effect that E- and P-cadherins are detected in the epidermis and sweat gland duct of h u m a n skin. There appear to be no other data on the distribution of cadherins in h u m a n skin or their association with skin morphogenesis during development of the epidermis and skin appendages. In the present study, we examined the expression patterns of E- and P-cadherin in the h u m a n epidermis in order to clarify their roles in the morphogenesis of h u m a n skin, especially during skin appendage development. We also examined the relationship between the expression of P-cadherin and the proliferating activity of cells.

Key words: Cadherin cell adhesion molecules -

Skin morphogenesis - Epidermis - Hair follicles - Ecerine sweat ducts

Materials and methods Skin tissues

Embryonic morphogenesis proceeds with the organized arrangement of cells. These processes are thought to be governed by the selective affinity of the cells in recognizing particular cell types for adhesion [18]. The Ca2+-dependent cell-cell adhesion molecules, termed cadherins, are one of the molecules involved in such selective adhesion of cells [17]. Cadherins are subdivided into different subclasses with different binding specificities and tissue distributions [8]. Studies on the cadherin distribution in the mouse epidermis [10, 13] have demonstrated that P (placental) cadherin is expressed in proliferating regions such as the basal layer, the outer root sheath and the hair matrix, whereas E (epithelial) cadherin is expressed in both proliferating

Correspondence to: M. Fujita

A total of 15 aborted human embryos and fetuses were examined, ranging in estimated gestationai age (EGA) from 7 to 21 weeks. The tissue collection conformed to the recommendations of the Kyoto University Committee on Investigations Involving Human Subjects. Signed informed agreement was obtained. The EGA was determined from maternal history and crown-rump length. Samples of skin were obtained from multiple anatomic sites including head, trunk, palms and soles. Skin tissues were also obtained from an infant sole (n = 1, age 5 years), an adult palm (n = 1), and adult breast (n = 5).

Monoclonal antibodies

A mouse MoAb to human E-cadherin (HECD-I) was prepared as described previously [15]. A mouse MoAb to human P-cadherin (NCC-CAD-299) was a gift from Dr. Y. Shimoyama [15]. Antiproliferating cell MoAb (Ki-67) (DAKO, Copenhagen, Denmark), reacting with all proliferating cells during late G1, S, M and G2 phases of the cell cycle, was purchased [3, 4, 14].

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Immunofluorescence procedures Tissue specimens were cut into small pieces, fixed immediately in periodate-lysine-paraformaldehyde solution at 4 ~ and embedded in optimal cutting temperature (OCT) compound Tissue-Tek II (Miles Laboratories, Naperville, Ill., USA). They were cryosectioned at a thickness of 6 gm and stained with the indirect immunofluorescence method. Briefy, sections were treated with 1% BSA in PBS, the primary MoAb, and fluorescein- or rhodamine-conjugated goat anti-mouse IgG (10 gg/ml), TAGO, Burlingame, Calif., USA). All the buffers contained 1 mM CaClz to protect cadherins against proteolysis. When possible, serial sections were used for each primary MoAb. Controls included the use of non-immune mouse IgG and the omission of primary MoAb to check for non-specific staining. No staining was observed with the controls. Photographs were taken on Kodak Tri-X films with an epifluorescence microscope (Nikon, Tokyo, Japan).

spinous cells and granular cells, but the expression was weaker in the basal cells (Fig. 1A, C). On the other hand, P-cadherin was expressed only in the basal cells. It was expressed on the upper and lateral surfaces o f basal cells (Fig. 1 B, D). The lower surfaces of basal cells, the upper surfaces o f granular cells and keratinized layers themselves expressed neither E- nor P-cadherin.

Expression of E- and P-cadherin from the embryonic stage to childhood

The epidermis of the breast skin consisted of several cell layers, whereas that of the adult palm showed acanthosis and hyperkeratosis. In both types of epidermis, E-cadherin was expressed on the surfaces of all epidermal cells including basal cells,

The embryonic skin (7-8 weeks EGA) consisted of the basal and periderm cell layers. The basal cells had both E- and P-cadherin on their surfaces, whereas some of the periderm cells had only E-cadherin (Fig. 2A, B). After 9 weeks EGA, a third layer of cells, called intermediate cell layers, was formed between the basal and the periderm cell layers. Primordium of skin appendages was not recognized before 12 weeks EGA. The basal cells had both E- and P-cadherin, the intermediate cells only E-cadherin, and the periderm cells neither of the cadherins (Fig. 2C, D).

Fig. 1A-D. Immunofluorescent localization of E- and P-cadherin in the adult skin. A, B Breast skin; C, D palmar skin; A, C stained

for E-cadherin; B, D stained for P-cadherin. Original magnifications: A, B, x 300; C, D, • 500. Bars = 40 gin.

Results

Distribution of E- and P-cadherin in adult human skin

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Fig. 2A-D. Immunofluorescentlocalization of E- and P-cadherln in the trunk of an 8-week-oldembryo (A, B) and in the palm of a 10-week-old fetus (C, D). A, C Stained for E-cadherin;B, D stained for P-cadherin. Original magnifications:A, B, x 600; C, D, x 400. Bars = 20 gm After 12 weeks EGA, hair follicles were formed in the head and trunk, at first as crowded basal cells (hair germs), then as elongated cellular cords (hair pegs and bulbous hair pegs, Fig. 3A-D). Immunohistochemical staining showed that the outer root sheath and the outermost layer of the hair matrix and sebaceous gland, which were continuous with the basal cell layer of the surface epidermis, expressed both E- and P-cadherin (Fig. 3 A J). The inner root sheath and the inner portion of the hair matrix and sebaceous gland expressed only E-cadherin. The pattern of distribution of both cadherins in the overlying epidermis was similar to that observed in younger fetuses, although expression of E-cadherin in the basal cells was weaker than in intermediate cells (Fig. 3 A, C). In the palms and soles, undulations at the dermalepidermal junction appeared at 12 weeks EGA (Fig. 4A, B). They penetrated the dermis to form regularly spaced primary epidermal ridges (PERs) within which the intradermal portion of the sweat ducts developed later. In a 14-week-old fetus, the primary dermal ridges (PDRs) became broader and flatter (Fig. 4C, D) and the sweat

ducts had developed as pear-like projections below the level of the PERs (Fig. 4E, F). After 16 weeks EGA, the basal layer of the epidermis projected downward at the mid-apex of the PDRs to form secondary epidermal ridges (SERs) and secondary dermal ridges (SDRs) (Fig. 4G-J). Morphogenesis was more advanced in the palms than in the soles. Immunohistochemically, the basal cell layers including PERs expressed both E- and P-cadherin, the intermediate cell layers only E-cadherin, and the periderm neither (Fig. 4A-D). The basal cell layers and PERs had lower levels of E-cadherin than in intermediate cell layers when the primary ridges were formed (Fig. 4A-D). During the development of sweat ducts, the distribution pattern of the two types of cadherin changed with time. Sweat duct buds and sweat ducts expressed both E- and P-cadherin strongly, whereas basal cells above the PDRs had little P-cadherin and expressed E-cadherin weakly (Figl 4E-H). After the formation of the secondary ridges, P-cadherin was readily detected in the SERs and the basal layers above the SDRs, although at lower levels (Fig. 4I, J). The sole of the 5-year-old infant showed thick epidermis with hyperkeratosis and was composed of basal cells, spinous cells, granular cells, and keratinized layers. Immunohistochemical findings showed an E- and Pcadherin distribution pattern similar to that in adult epidermis (Fig. 5A, B). The basal cells expressed both cadherins, whereas the spinous cells and granular cells had only E-cadherin. The keratinized layers had neither E- nor P-cadherin. Throughout the development from the embryonic stage to childhood, the epidermal cells expressing either cadherin had the cadherin on the entire cell surface, whereas the surfaces of basal cells to basement membrane had neither of the cadherins.

Relationships between P-cadherin expression and proliferation activity Tissue sections were stained with NCC-CAD-299 and anti-proliferating cell MoAb (Ki-67) in order to clarify the relationships between the expression of P-cadherin and the proliferation activity of cells. In the adult head and trunk, the distribution patterns for P-cadherin and Ki-67+cells were similar. Basal cell layers (Fig. 6A, B), the outer root sheath (Fig. 6C, D), and the outermost layer of the hair matrix (Fig. 6E, F) expressed both P-cadherin and Ki-67 antigens. In contrast, the expression pattern of P-cadherin was the opposite to that of Ki-67 antigens in palms and soles during sweat gland development. The basal cell layers including PERs expressed both P-cadherin and Ki-67 antigens before 14 weeks EGA in a similar way to those in other regions (Fig. 6G, H). However, during the development of sweat ducts, Ki-67 antigens of the PERs were decreased first (Fig. 6I, J) followed by P-cadherin of the basal layers above the PDRs (Fig. 6K, L). That is, the basal cell layers of the PERs expressed only P-cadherin, while those above the PDRs had only Ki-67 antigens. The basal cells above the PDRs again expressed

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Fig. 3 A - J . Immunofluorescent localization of E- and P-cadherin in the head and trunk after 12 weeks EGA. A, B A hair peg; C, D a bulbous hair peg; E - J hair foIlicles; A, C, E, G, I stained for

E-cadherin; B, D, F, H, J stained for P-cadherin. hm, Hair matrix; is, inner root sheath; os, outer root sheath; sg, sebaceous gland. Original magnifications x 300. Bars = 40 ~m

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Fig. 4 A - J . Immunofluorescent localization of E- and P-cadherin in the palms and soles after 12 weeks EGA. A, B Epidermal undulations; C, D formation of PERs and PDRs; E, F formation of sweat duct buds and sweat ducts; G, H elongation of sweat ducts. SERs are beginning to be formed (arrows), I, J Formation

of SERs (arrows) and SDRs (asterisks). A, C, E, G, l Stained for E-cadherin; B, D, F, H, J stained for P-cadherin. per, Primary epidermal ridge;pdr, primary dermal ridge; sd, sweat duct. Original magnifications: A - H , • 300; I, J, • 200. Bars = 40 ~tm

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Fig. 5A, B. Immunofluorescent localization of E- and P-cadherin in the sole of a 5-year-oldinfant. A Stained for E-cadherin; B stained

for P-cadherin. per, Primary epidermal ridge; ser, secondaryepidermal ridge. Original magnifications x 150. Bars = 40 gm

detectable amounts of P-cadherin and some of the PERs had Ki-67 antigens after the development of the secondary ridges (Figs. 4J and 6M). In the adult palm, the basal cell layers including epidermal ridges expressed both P-cadherin and Ki-67 antigens (Fig. 6N).

including skin appendages, except for the horny keratinized layers. These results indicale two possible roles for P-cadhefin. First, as proposed previously [10, 15], P-cadherin may be essential for segregation of the basal proliferating layer from the upper non-proliferating layers, although E-cadherin is important for connecting these layers. This expression pattern of P-cadherin suggests an association of P-cadherin with the proliferating compartment. However, the present study showed the compartment of P-cadherin-expressing cells to be temporarily the opposite of that of proliferating cells during development of eccrine sweat glands, suggesting that it is not closely associated with cell proliferation. The second possible role for P-cadherin may be in the arrangement of epidermal cells into sweat ducts by its disappearance and reappearance. This expression pattern was observed only during the development of sweat ducts but not during that of hair follicles. Two possible explanations for these differences suggest themselves. One is found in the association of dermal cells with the epidermal basal cells. Because condensed dermal cells, which are important for the morphogenesis of hair follicles [5, 10], are not induced around sweat ducts, only epidermal cell-cell interaction may be associated with the morphogenesis of sweat ducts, and cadherin-mediated junctions may be more essential for this development. The other possible explanation lies in the structural difference between hair follicles and sweat ducts. During hair follicle development, both the outer basal cells and the central core cells elongate to form hair follicles [6, 11] whereas during sweat duct development, intradermal ducts are composed of one layer of outer basal cells and one layer of inner cells which arise from the outer basal cells [7]. The presence of keratinization in hair canals may also be related to these differences. Although we examined the expression patterns of E- and P-cadherin in the human epidermis, we cannot

Discussion

The expression of cadherins has been observed to be associated with various morphogenetic processes, such as arrangement, segregation and association of cells [9, 16]. Several studies suggest the correlation of expression of E- and P-cadherin with the mechanism of organogenesis and carcinogenesis [10, 12, 13, 15]. The present study showed the distribution of the two types of cadherin in the morphogenesis of human skin, with special emphasis on the development of skin appendages. The findings concerning the development of fetal hair follicles are consistent with those obtained for mouse skin [10]. The central portion of the hair pegs and follicles, called core cells, were intruded from the intermediate layers [6] and expressed only E-cadherin, while the outer portion, which was continuous with the basal layers, expressed P-cadherin together with E-cadherin. This expression pattern of E- and P-cadherin did not change during the development of fetal hair follicles. In contrast, P-cadherin showed a unique spatiotemporal pattern of expression during the development of sweat ducts. P-cadherin was distributed at first evently on the surfaces of basal cells, but later tended to accumulate in the epidermal ridges as the sweat ducts developed. By the time the secondary ridges had been formed after 16 weeks EGA, and when the elongated sweat ducts began to form a coil [7], the distribution of P-cadherin had become even again. Throughout the course of development, E-cadherin was distributed in all epidermal cells,

Fig. 6A-N. Immunofluorescent localization of P-cadherin and Ki-67 antigens. A, B The epidermis of an adult palm; C, D hair follicle; E, F hair matrix; G, H sweat duct buds; I, J elongation of PERs; K, L development of sweat ducts; M the palm of a 21-week-old fetus (adjacent to Fig. 5I and J). SERs (arrows) and SDRs (asterisks) have been formed; N the adult palm (adjacent to

Fig. 1C and D). A, C, E, G, I, K Stained for P-cadherin; B, D, F, H, J, L, M, N stained for Ki-67. hm, Hair matrix; per, primary epidermal ridge; pdr, primary dermal ridge; sd, sweat duct. Original magnifications: A-D, G - M , x 300; E, F, x 350; N, x 150. Bar = 40 gm

t66 rule o u t the c o o r d i n a t i o n a n d a s s o c i a t i o n o f o t h e r cell-adhesion mechanisms with cadherin expression and skin m o r p h o g e n e s i s . T h e s p a t i o t e m p o r a l e x p r e s s i o n p a t tern o f n e u r a l cell a d h e s i o n m o l e c u l e ( N - C A M ) h a s also b e e n o b s e r v e d in c h i c k e n f e a t h e r d e v e l o p m e n t [1, 2]. F u r t h e r m o r e , the d i s t r i b u t i o n p a t t e r n o f N (neural) c a d h e r i n h a s b e e n s h o w n to be g e n e r a l l y c o m p l e m e n t a r y to t h a t o f liver cell a d h e s i o n m o l e c u l e ( L - C A M ) a n d gener a l l y s i m i l a r to t h a t o f N - C A M in the m o r p h o g e n e t i c processes o f c h i c k e n e m b r y o s [9]. T h u s , c a d h e r i n s a n d o t h e r c e l l - c e l l a d h e s i o n m o l e c u l e s m a y be c o n t r o l l e d b y s o m e r e g u l a t o r y m e c h a n i s m w h i c h c o o r d i n a t e s their spatiotemporal expression during development.

Acknowledgments. We would like to thank Dr. H. Kodama for providing surgical materials. We are also grateful to Ms. F. Owatari for typing the manuscript. References

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