THE JOURNAL OF EXPERIMENTAL ZOOLOGY 257:195-207 (1991)

Expression of the Cell Adhesion Molecules, L-CAM and N-CAM During Avian Scale Development ROSE B. SHAMES, ANITA G. JENNINGS, AND ROGER H. SAWYER Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208

ABSTRACT To examine the involvement of cell adhesion molecules in the inductive epithelial-mesenchymal interactions during avian scale development, a study of the spatiotemporal distribution of L-CAM and N-CAM was undertaken. During scutate scale development, L-CAM and N-CAM are expressed together in cells of the transient embryonic layers destined to be lost at hatching. The ongoing linkage of the cells of these layers by both CAMS sets them apart, early in development, as unique cell populations. L-CAM and N-CAM were also expressed simultaneously at the basal surface of the early germinative cells where signal transduction is presumed to occur. In spite of the differences in cell shape, adhesion, density and proliferative state between populations of epidermal placode and interplacode cells, the expression of L-CAM and N-CAM appeared to be uniform and nondiscriminating for these discrete cell lineages. The same pattern of L-CAM and N-CAM expression was observed during morphogenesis of reticulate scales that develop without placode formation. While L-CAM and N-CAM are present during the early stages of scale development and most likely function in cell adhesion, the data do not support a role for these adhesion molecules in the formation of the morphogenetically critical placode and interplacode cell populations. In both scale types, L-CAM became predominantly epithelial, and N-CAM became predominantly dermal as histogenesis occurred. Initially, N-CAM was concentrated near the basal lamina where it may be involved in the reciprocal epidermal-dermal interactions required for morphogenesis. However, as development of the scales progressed, N-CAM disappeared from the tissues. L-CAM expression continued in the epidermis and was intense on all suprabasal cells undergoing differentiation into either an a-stratum or p-stratum. However, L-CAM was more prevalent on the basal cells of a-keratinizing regions than on the basal cells of P-keratinizing regions.

The cell adhesion molecules, L-CAM (known as E-cadherin in the mouse system) and N-CAM, are cell surface glycoproteins believed t o be involved in regulating morphogenetic events by their local modulation at cell surfaces (Yoshida and Takeichi, '82; Edelman, '85a; Crossin et al., '85; Takeichi, '88). Modulation may involve changes in the chemical composition, prevalence, or distribution of the CAM (Rothbard et al., '82; Edelman and Chuong, '82; Hoffman and Edelman, '83; Chuong and Edelman, '84). It has been shown that these cell adhesion molecules are expressed on surfaces of tissues undergoing inductive interactions and therefore may be important in mediating control of inductive events (Thiery et al., '82; Edelman et al., '83; Thiery et al., '83; Edelman, '84, '85; Crossin et al., '85). The spatiotemporal distribution of L-CAM and N-CAM was followed during morphogenesis of embryonic and nestling feathers in the chick (Chuong and Edelman, '85a,b). Normal develop0 1991 WILEY-LISS, INC.

ment of these skin appendages occurs under the influence of inductive epithelial-mesenchymal interactions (Rawles, '63; Linsenmayer, '72; Dhouailly '75). It was observed that epidermal feather placodes linked by L-CAM were apposed to underlying dermal condensations linked by NCAM. While L-CAM was expressed on all epidermal cells, N-CAM expression followed the periodic appearance of placodes and condensations that produce the hexagonal array of feathers over much of the avian body. The importance of cell adhesion to feather pattern formation was further demonstrated by a study in which dorsal skin explants were treated with antibodies t o L-CAM (Gallin et al., '86). When L-CAM-mediated linkages between epidermal cells were disrupted by the antibodies, underlying N-CAM-positive conReceived January 31, 1990; revision accepted June 13, 1990. Address reprint requests to Dr. Rose B. Shames, Department of Biological Sciences, University of South Carolina, Columbia, SC 29208.

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densations were found in an abnormal nonhexagonal pattern. Thus, L-CAM linkages between feather epithelial cells are functionally important for normal inductive events that influence patterning of mesenchymal cells linked by N-CAM. Normal development of avian scutate and reticulate scales is also dependent on epithelialmesenchymal interactions (Rawles, '63; Linsenmayer, '72; Sengel, '76; Dhouailly et al., '78; Sawyer, '83; Sawyer et al., '86; Sawyer and Goetinck, '88). Scutate scales like feathers arise from epidermal placodes but, unlike feathers, the scutate scale placodes do not overlie discrete dermal condensations (Wessells, '65; Sengel and Rusaouen, '68; Dwyer, '71; Sawyer, '72a,b; Sawyer and Haake, '86). Reticulate scales arise without formation of either an epidermal placode or dermal condensation (Sawyer and Craig, '77). Both the scutate and reticulate scales produce the transient embryonic layers of periderm and subperiderm, while feathers form a different embryonic layer known as the sheath. Inductive tissue interactions give rise t o the different forms of these skin appendages and are also responsible for the different regional distributions of a- and pkeratins that constitute their final structures (Bell and Thathachari, '63; Kemp and Rogers, '72; Sawyer et al., '74; Dhouailly et al., '78; Sawyer and Borg, '79; O'Guin et al., '82; O'Guin and Sawyer, '82; O'Guin, '84; Haake et al., '84).Except for the sheath cells, feathers are composed of pkeratins. Reticulate scales contain only akeratins, except for the embryonic periderm and subperiderm, which contain both a- and pkeratins. The scutate scales also produce a- and pkeratins in their peridermal and subperidermal layers. In the mature scale, the inner surface and hinge region consist of only a-keratins, while the overlapping outer scale surface consists of both aand P-keratins. To further our knowledge of the role of cell adhesion molecules in the epithelial-mesenchyma1 interactions that control morphogenesis, histogenesis, and terminal differentiation, a study of the spatiotemporal distribution of L-CAM and NCAM in developing scutate and reticulate scales was undertaken. These cell adhesion molecules were found to be expressed together in cells of the transient embryonic layers and in germinative cells during the early stages of scale morphogenesis. Neither anti-L-CAM nor anti-NCAM staining discriminated between the distinctive cell populations of the placode and interplacode regions, indicating that L-CAM and

N-CAM may not be responsible for the formation of these morphogenetically important cell collectives. As development proceeded, L-CAM was expressed predominantly in the epidermis, and NCAM was expressed predominantly in the dermis. Finally, N-CAM disappeared from dermis of the differentiating scales while L-CAM continued to be expressed by epidermal cells undergoing terminal differentiation.

MATERIALS AND METHODS Fertile chicken eggs were obtained from a commercial White Leghorn stock (ISE America, Newberry, SC) and incubated at 37"C, 70% humidity in a Favorite Incubator (Leaky Manufacturing Company, Higginsville, MO). The embryos were staged according t o Hamburger and Hamilton (1951). Im m unohistochem is try The anterior metatarsal and footpad regions of the shank were dissected and fixed in a solution of ethanol-glacial acetic acid (95 :5 ) . The tissues were dehydrated in three changes of 100% ethanol, cleared in three changes of toluene and then embedded in paraffin. Tissues were sectioned to a 7-km thickness, deparaffinized in toluene, hydrated through a series of ethanols (loo%, 95%, 85%, 70%), and finally equilibrated in Sorenson's buffer (80% dibasic sodium phosphate-20% monobasic potassium phosphate, pH 7.4) containing 1%bovine serum albumin (BSA). The tissues were treated with primary antisera for 15 h at 20°C in a moist chamber. Following two rinses in Sorenson's buffer/l% BSA, the tissues were treated with fluorescein-conjugated goat antirabbit IgG (ICN Immunologicals) for 1h at room temperature. The tissues were rinsed as before and examined with a Zeiss Universal microscope equipped for epifluorescence. Controls were run as described but preimmune rabbit serum was used in place of primary antiserum. The control-treated tissues were entirely negative. Antibodies Polyclonal antisera against L-CAM and NCAM were generously provided by Dr. Warren Gallin, Department of Zoology, University of Alberta, Edmonton, Canada. Immunoblots of NP-40 extracts of 14-day anterior metatarsal skin were performed t o determine the size of molecules recognized by the antibodies used in immunofluorescent studies. Anti N-CAM was reactive with a

L-CAM AND N-CAM IN AVIAN SCALE DEVELOPMENT

broad band of - 140,000 M,. Anti L-CAM was reactive with two polypeptides of - 110,000 and 70,000 M,. These sizes are consistent with previously reported values for L-CAM and N-CAM in avian backskin (Chuong and Edelman, '85a).

RESULTS Expression of L-CAM and N-CAM during scutate scale development Before the onset of scale morphogenesis, the shank is covered by a simple epidermis consisting of a germinative layer of cuboidal-shaped cells and an overlying peridermal layer of flattened cells. Underlying the epidermis is a ridge of mesodermal cells. As shown in Figure 1, when a section of this early preplacode stage tissue is reacted with anti-L-CAM antibody, the fluorescent staining is localized to the peridermal cells and to the basal surface of the germinative cells. There is also a low but detectable level of staining of the underlying mesodermal cells. Reaction of tissue at this stage with anti-N-CAM results in a pattern of staining similar to that observed with anti-L-CAM. However, the mesodermal cells are stained more intensely by anti-N-CAM than by anti-L-CAM. Morphogenesis of scutate scales begins by formation of oval shaped epidermal placodes along the anterior surface of the shank. These placodes arise as groups of basal cells elongate downward into the superficial dermis. These newly formed columnar cells, the placodes, are surrounded by cuboidal cells that comprise the interplacode regions. Figure 2 shows the pattern of staining with anti-L-CAM and anti-N-CAM in tissue sections of the scutate scale at the epidermal placode stage. The intensity of fluorescent staining with both antibodies is greater than in the earlier preplacode stage, but the tissue distribution is essentially the same. Anti-L-CAM weakly stains the superficial dermal cells but brightly stains the epidermal cells. Anti-N-CAM stains the cells of dermis and epidermis with similar intensity. The peridermal cells are evenly stained by both antibodies but the germinative cells display a polar distribution of L-CAM and N-CAM at the basal surface of the cells. These staining patterns are uniformly present over placode and interplacode regions. Figure 3 shows the appearance of the developing scutate scale at the definitive scale ridge stage. This structure forms by the asymmetric

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elongation and subsequent elevation of the placode. Epidermal basal cells are now all columnar and have given rise t o a new intermediate layer of cells beneath the periderm. The superficial dermis has thickened under the elevated epidermal ridge but remains thin under the inturned region of the scale. At this stage, anti-NCAM stains the dermis brightly while epidermal staining is diminished relative to earlier stages. The lamellar corpuscles, which are avian touch and pressure receptors, are also stained with antiN-CAM. In the superficial dermis, the cells closest to the basal lamina are more intensely stained with anti-N-CAM than those deeper into the tissue. At this stage, anti-L-CAM staining is brightest around peridermal cells and the newly generated intermediate cells. The polar staining of basal cells is present but less intense than the staining of intermediate cells. This basal surface staining of the germinative cells is not as distinct around the hinge as it is on cells of the outer surface of the scale. Staining of the superficial dermal cells with anti-L-CAM is not detectable except on cells of lamellar corpuscles. Beyond the definitive scale ridge stage the scutate scale develops by elongation of the ridge so that the apex overlaps the base of the scale distal t o it. Along with these morphogenetic events, the histogenesis of the newly formed inner and outer epidermal surfaces proceeds as increasing numbers of cell layers are generated from the germinative layer of basal cells. Figures 4 and 5 show the pattern of anti-N-CAM staining on the maturing scutate scale from 14 to 19 days of development. Reactivity of dermal cells with antiN-CAM decreases as development progresses. The deepest cells of the superficial dermis retain anti-N-CAM reactivity longer than those nearer the outer surface. As the apex of the scale elongates, the concentration of N-CAM staining on dermal cells near the basal lamina becomes focused around the hinge region of the scale (Figs. 4A,B; 5A,B). The epidermal staining with anti-NCAM continues to be restricted to the outermost embryonic layers. In Figure 5C,D, the large cells of subperiderm are clearly visible, and distinctly stained with anti-N-CAM. As differentiation of the epidermis progresses, the embryonic layers are sloughed and staining with anti-N-CAM is lost, as seen in Figure 4D. Figures 6 and 7 show the pattern of anti-lCAM staining on maturing scutate scales from 16 t o 19 days of development. L-CAM is expressed mainly by the epidermis, except for the lamellar

PRE-PLACODE

N -CAM

L-CAM

Figures 1 and 2

L-CAM AND N-CAM IN AVIAN SCALE DEVELOPMENT

SCALE N-CAM

Fig. 3. Immunofluorescent staining of scutate scales at the definitive scale ridge stage with anti-N-CAM (A,C) and anti-L-CAM (B,D). Anti-N-CAM staining is observed predominantly over the dermal cells. The highest intensity of NCAM staining is concentrated over dermal cells closest t o the basal lamina. Anti-L-CAM staining is observed over the epid-

Fig. 1. Immunofluorescent staining of preplacode stage anterior metatarsal skin with anti-N-CAM (A and C) and anti-L-CAM (B and D). Both anti-N-CAM and anti-L-CAM stain the peridermal cells (pd) and the basal surface of the germinative cells (bc) of the epidermis. Anti-N-CAM stains mesodermal cells with similar intensity t o epidermal staining, while staining of mesodermal cells (m) with anti-L-CAM is barely detectable. Scale bar, 25 pm. Fig. 2. Immunofluorescent staining of anterior metatarsal scutate scale placodes with anti-N-CAM (A,C) and anti-LCAM (B,D). The intensity of staining by both antibodies is greater at this stage than the earlier preplacode stage. Epidermal staining is detected over all cells but the greatest intensity is still observed over peridermal cells and the basal surface of germinative cells. Mesodermal staining by both antibodies is observed. However, anti-N-CAM stains the mesodermal cells with equal intensity t o epidermal staining, while anti-L-CAM staining of mesodermal cells is weaker. This pattern of staining is uniform over placode (pl) and interplacode (ipl) regions. Scale bar, 25 pm.

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RIDGE L-CAM

ermal cells and the lamellar corpuscles (lc) in the dermis. The highest intensity of anti-L-CAM staining is found over peridermal cells, newly generated stratum intermedium cells, and the basal surface of the columnar germinative cells. oss, outer scale surface; h, hinge. Scale bar, 25 pm.

corpuscles in the dermis. As histogenesis and differentiation proceed, the basal cells of the inner scale surface and hinge region become more brightly stained with anti-L-CAM than the more columnar basal cells of the outer scale surface (Fig. 6). All the suprabasal cells eventually express L-CAM on their surfaces. The staining pattern is very distinct and bright around those cells that are in the process of differentiation but, as they become fully keratinized, the cell boundaries are lost, and staining becomes diffuse. This pattern is first observed in the embryonic layers. The large subperiderm cells are clearly outlined by anti-L-CAM, as seen in Figure 7A,B. As cells are added to the stratum intermedium and differentiation of the p-stratum occurs, the subperiderm becomes diffusely stained and is eventually sloughed (Fig. 7C,D).

Figures 4 and 5

L-CAM AND N-CAM IN AVIAN SCALE DEVELOPMENT

Expression of L-CAM and N-CAM during reticulate scale development The ventral footpad of a chicken is covered with round nonoverlapping reticulate scales. Unlike scutate scales, reticulate scales arise as symmetrical elevations without formation of epidermal placodes. Following morphogenesis of the reticulate scale, the suprabasal cells become stratified and differentiate t o form a single, continuous alpha keratinized stratum corneum, which is distinct, especially on the dome, from the inner and outer surfaces of the scutate scale. Because of the differences in morphogenesis and histogenesis of these two scale types, it was significant t o compare the pattern of anti-L-CAM and anti-N-CAM staining of developing reticulate scales with developing scutate scales. Figure 8 shows the pattern of anti-N-CAM staining on early reticulate scales prior to formation of the stratum corneum (Fig. 8A) and on mature scales (Fig. 8B). Aside from the obvious shape difference (cf. ~ i3A~and. 8A) the staining pattern with anti-N-CAM is similar over the two scale types. There is a very low levelof staining on the epidermal cells and a bright stain Over the dermis. As in the early scutate scale, there is a concentration of anti-N-CAM staining Over dermal cells closest to the basal the reticulamina in early reticulate scales. late scale matures 2nd cornification occurs, the level of anti-N-CAM staining decreases until it is not detectable, as is the case with scutate scales (cf. Fig. 4D and 8B). Figure 9 shows developing reticulate scales after staining with anti-L-CAM. The distribution of stain on the early reticulate scale ( ~ i~ ~ A , .B is ) similar to that found on early scutate scale (see Fig. 3B). The embryonic layers and the basal surface of the germinative layer of cells stain with anti-L-CAM. As the scale form develops, the intensity of the staining on the basal surface decreases. As the reticulate scale matures, L-CAM is distributed in the epidermis in the Same pat-

Figs. 4 and 5. Low and high magnification of anti-N-CAM staining on maturing scutate scale. 4A, 4B and 5A are sections of 14-day tissue; 5B, C, and D are sections of 16-day tissue; 4C is 17-day tissue; and 4D is 19-day tissue. Dermal (d) staining gradually decreases as the scale develops. Staining near the basal lamina is focused around the hinge (h) region of the scale. By the time of hatching anti-N-CAM staining is not detectable. N-CAM staining of the epidermis (ep) is concentrated on the outermost embryonic layers of periderm (pd) and subperiderm (spd). OSS, outer scale surface; iss, inner scale surface. Scale bar, 25 pm.

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tern as the inner surface and hinge region of scutate scales. Bright anti-L-CAM staining is observed on all basal and suprabasal cells but, as the cells become cornified the stain appears more diffuse. By contast, on the outer surface of the scutate scale, there is a very low level of anti-lCAM staining on the elongated germinative basal cells. The difference in staining intensity between these different basal cell populations is shown in Figure 10. The more cuboidal basal cells of the reticulate scale (Fig. 10A, open arrow) and inner scutate scale surface and hinge region (Fig. 10B, open arrow) are more intensely stained than the columnar basal cells of the outer scutate scale surface (Fig. 10B, closed arrow).

DISCUSSION In the present study, the expression Of the adhesion L-CAM and N-CAM, has been followed during development Of the avian scutate and reticulate scales. The scutate scales are rectangular, overlapping plates that consist of an Outer surface that a tough pkeratinized stratum corneum and a more pliable hinge and inner surface that elaborates an akeratinized stratum COrneum (Sawyer et a1-7 '86). The reticulate scales are round elevations that elaborate a thick a-keratinized stratum corneum (Sawyer et al., '86). Morphogenesis, histogenesis and differentiation of these structures depends upon complex interactions between the epithelial and underlying mesenchymal tissues from which the scales are formed (Sawyer, '83)-Since L-CAM and N-CAM are thought t o be involved in the adhesion of cell collectives and the induction of moqhogenesis (Hoffman et al., '82; Gallin et al., '83; Edelman, '84; Grumet et al., '84; Chuong et al., '85a,b; Crossin et al., '85; Edelman, '85a,b), a study of the pattern of their expression during avian scale development would provide information concerning the role of these adhesion molecules in epithelial-mesenchymal tissue interactions. Before morphogenesis of the scutate and reticulate scales, the epidermis consists of a layer of cuboidal-shaped germinative cells underlying a transient embryonic layer Of peridermal cells. The dermis consists of a loose collection of mesodermal cells. During this early stage, N-CAM was detected with equal intensity in both epidermis and dermis. After morphogenes~sa n t i - ~ - C ~ ~ staining became predominantly dermal. However, staining with anti-L-CAM was predominantly epidermal at the earliest stages examined

Figures 6 and 7

L-CAM AND N-CAM IN AVIAN SCALE DEVELOPMENT

and remained so throughout development. LCAM and N-CAM were distributed in an identical polar pattern in the early epidermis. Both molecues were expressed on peridermal cells and at the basal surface of germinative cells. This polar distribution persisted during the initial stages of both scutate and reticulate scale morphogenesis. The presence of both adhesion molecules on the peridermal cells suggests that this group of cells had already differentiated into a distinct population separate from the underlying basal cells. The focused expression of the two CAMS only along the epidermal basal surface reflects the relatively loose association of the lateral surfaces of the germinative cells which has been verified by fine structural studies (Sengel, '86). In a recent study of CAM expression during feather development (Chuong and Edelman, '85a,b), NCAM was detected exclusively in the dermis except for transient epidermal expression on the early feather bud. L-CAM was detected only in epidermis and the pattern was uniform over the epidermal cells except for a transient polarity modification in the elevated epidermal placode. These patterns of L-CAM and N-CAM expression further support the thesis that there are early differences between developing feathers and both scutate and reticulate scales (Sawyer and Haake, '86). A critical event in the development of scutate scales is the appearance of epidermal placodes and their interplacode regions. The anterior row begins to form just proximal to the tarsometatarsal joint of the third toe. Discrete populations of cuboidal basal cells become columnar and highly compacted into symmmetric oval-shaped placodes. Interplacode regions develop as loosely associated groups of cuboidal basal cells. The placode cells enter a 36-h nonproliferative phase while the interplacode cells enter a highly proFigs. 6 and 7. Low and high magnification of anti-L-CAM staining of the maturing scutate scale. 6A and 7A are sections of 16-day tissue; 6B and 7B are 17-day tissue; 7C is 18 day tissue; and 6C, 6D, and 7D are 19-day tissue. A region comparable to the boxed region in Fig. 6D is shown in higher magnification in Fig. 10B. All the sections in Fig. 7 show outer scale surface ( 0 s ) . Staining is restricted t o epidermis except for lamellar corpuscles (lc) in the dermis. Basal cells (bc) of the inner scale surface (iss) and hinge (h) region are more brightly stained than the basal cells of the outer scale surface. Suprabasal (sb) cells are all brightly stained, but as cells become keratinized, staining becomes diffuse and intensity weakens. spd, subperiderm; pd, periderm. scale bar, 25 Pm.

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liferative state (Sawyer, '72a,b; Tanaka and Kato, '83a,b; Tanaka et al., '87). During this period, the dividing interplacode cells move into the proximal side of the nondividing cell population of the placode, change to a columnar morphology, and cease mitotic activity. It is during this movement and change of interplacode cells into nondividing placode cells that the scutate scale takes on its asymmetric shape and becomes elevated above the surface of the shank. After this period of morphogenesis, all cells return to active cell cycles and stratification of the scale begins. The epidermal placode and interplacode stage is required for normal development of the scutate scale. Tissue recombinant experiments have shown that without placode formation the dermis does not receive the required signals to become a competent inducer of scutate scale development (Sawyer and Abbott, '72; McAleese and Sawyer, '81,'82; Dhouailly and Sawyer, '84). Expression of L-CAM and N-CAM in the epidermis was observed during the placode stage. In spite of the differences between the cell populations of the placode and interplacode regions, there was no obvious difference observed in the distribution of the two adhesion molecules. As in the preplacode stage, both L-CAM and N-CAM were focused on peridermal cells and at the basal surface of all germinative cells. This same pattern was observed during morphogenesis of reticulate scales, which occurs without placode formation. The finding that antiL-CAM and anti-N-CAM staining did not discriminate between the very different cell populations of the placode and interplacode regions indicates that these adhesion molecules are not responsible for defining the borders of these morphogenetically important cell groups. Epidermal placodes also form during feather morphogenesis, but unlike scutate scale placodes, the feather placodes are associated with discrete nonproliferating dermal condensations. This difference is clearly reflected in the pattern of NCAM distribution. During the preplacode stage of feather development, dermal cells were found to be evenly positive for N-CAM (Chuong and Edelman, ,851, as are the dermal cells of scutate scales (Fig. 1).This uniform dermal distribution of NCAM persisted and increased in intensity during scutate scale placode formation (Fig. 2) but, at the sites of feather placodes, dermal condensations appeared and became highly N-CAM positive relative t o the interplacode regions. N-CAM-positive cells were observed transiently in the feather epidermal placode, while in scutate scale develop-

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Fig. 8. Anti-N-CAM staining of 14-day (A) and 19-day (B) reticulate scales. Staining is seen throughout the dermis (d) of the early reticulate scale but is most intense near the basal lamina. There is also a detectable level of staining of

Fig. 9. Anti-L-CAM staining of developing reticulate scales. On 14-day reticulate scales (A,B) epidermal staining is seen over the outermost embryonic layers (el) and a t the basal surface of the germinative basal cells fbc). As stratification occurs (C), at 18 days, anti-L-CAM brightly

embryonic layers of epidermis (ep). In the mature reticulate scale, staining with anti-N-CAM was not detectable in either dermis or epidermis. scale bar, 25 km.

stains all the cells of the epidermis. The cornified layers display a diffuse and weak level of stain (D), at 19 days. A region comparable to the boxed region is shown in higher magnification in Fig, 10A. scale bar, 25 km.

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Fig. 10. Anti-L-CAM staining of 19 day reticulate scale (A) and scutate scale (B). The reticulate scale region (A) is comparable to the boxed region in Fig. 9D, and the scutate scale region (B) is comparable to the boxed region shown in Fig. 6D. The open arrow (A) points to the reticulate scale germinative cells, which are brightly stained with anti-LCAM. The open arrow (B) points to the germinative cells of

the hinge and inner surface of the scutate scale, which are identical in shape and anti-L-CAM staining to the germinative cells of the reticulate scale. The closed arrow in (H) points t o the more columnar basal cells of the scutate scale outer surface, which stain only weakly with anti-L-CAM. scale bar, 25 Fm.

ment N-CAM was expressed continually over the peridermal cells and at the basal surface of the germinative cells in both the placode and interplacode regions. These differences in N-CAM expression further demonstrate that while development of scutate scales and feathers depend on epidermal placode formation, the associated events of morphogenesis are very different. Late in morphogenesis of the scutate scale, a higher concentration of anti-N-CAM staining was detected in the dermal cells closest to the basal lamina around the hinge and inner scale surface. A similar concentration of anti-N-CAM staining was detected in the dermal cells closest to the basal lamina in all areas of the developing reticulate scale. It is possible that this regionally focused N-CAM expression is related to the final histological outcome of the tissues. The germinative cells above these N-CAM concentrations, (hinge/inner scutate scale surface and all the reticulate scale surface) will give rise to akeratinizing suprabasal cells, while the other re-

gions (outer scutate scale surface) will give rise t o p-keratinizing cells. In both scutate and reticulate scale development, N-CAM expression was limited to early embryonic events and morphogenesis. As histogenesis and differentiation of the scales occurred, the dermal N-CAM staining diminished. N-CAM expressed in the embryonic layers of periderm and subperiderm was lost as the layers cornified and were sloughed at hatching. The simultaneous expression of both L-CAM and NCAM in these embryonic layers sets them apart from the tissue of the scale proper. This difference in character of the embryonic layers seems t o be determined very early in development, probably before the placode stage. L-CAM expression appeared to be primarily associated with ongoing events of histogenesis and differentiation. Anti-L-CAM staining was detected in the basal cells of the reticulate scale and scutate scale hinge and inner surface that ultimately gave rise t o an a-stratum but seemed to be

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absent from the basal cells of the scutate scale staggerer mice. Proc. Natl. Acad. Sci. U.S.A., 79:70367040. outer surface which gave rise to a beta stratum. Thus, L-CAM expression distinguished between Edelman, G.M., W.J. Gallin, A. Delouvee, B.A. Cunningham, and J.-P. Thiery (1983) Early epochal maps of two different two basal cell populations undergoing determinacell adhesion molecules. Proc. Natl. Acad. Sci. U.S.A., tion toward different end points. In addition, anti80:4384-4388. L-CAM staining was intense on all suprabasal Gallin, W.J., G.M. Edelman, and B.A. Cunningham (1983) Characterization of L-CAM, a major cell adhesion molecule cells undergoing keratinization in both reticulate from embryonic liver cells. Proc. Natl. Acad. Sci. U.S.A., and scutate scales. As cornification occurred, the 80: 1038- 1042. anti L-CAM stain became diffuse and eventually Gallin, W.J., C.-M. Chuong, L.H. Finkel, and G.M. Edelman disappeared. Thus, L-CAM expression also ap(1986) Antibodies to liver cell adhesion molecule perturb peared t o be associated with the general process of inductive interactions and alter feather pattern and structure. Proc. Natl. Acad. Sci. U.S.A., 83:8235-8239. cell differentiation in these scales.

ACKNOWLEDGMENTS This work was supported by grant HD 18129 from the National Institute of Child Health and Human Development. We wish t o thank Mr. Clint Cook for photographic assistance. LITERATURE CITED Bell, E., and Y.T. Thathachari (1963) Development of feather keratin during embryogenesis of the chick. J. Cell Biol., 16:215-223. Chuong, C.-M., and G.M. Edelman (1984) Alterations in neural cell adhesion molecules during development of different regions of the nervous system. J. Neurosci., 4:23542368. Chuong, C.-M., and G.M. Edelman (1985a) Expression of cell adhesion molecules in embryonic induction. I. Morphogenesis of nestling feathers. J . Cell Biol., 101:10091026. Chuong, C.-M., and G.M. Edelman (1985b) Expression of cell adhesion molecules in embryonic induction. 11. Morphogenesis of adult feathers. J. Cell Biol., 101:1027-1043. Crossin, K.L., C.-M. Chuong, and G.M. Edelman (1985) Expression sequences of cell adhesion molecules. Proc. Natl. Acad. Sci. U.S.A. 82:6942-6946. Dhouailly, D. (1975) Formation of cutaneous appendages in dermo-epidermal recombinations between reptiles, birds and mammals. Wilhelm Roux’s Arch., 177:323-340. Dhouailly, D., and R.H. Sawyer (1984) Avian scale development. XI. Initial appearance of the dermal defect in scaleless skin. Dev. Biol., 105:343-350. Dhouailly, D., G.E. Rogers, and P. Sengel (1978) The specification of feather and scale protein synthesis in epidermal-dermal recombinants. Dev. Biol., 65:58-68. Dwyer, N.K. (1971) Chick scale morphogenesis: Early events in the formation of overall shank and individual scale shape. Masters thesis, University of Massachusetts, Amherst, Massachusetts. Edelman, G.M. (1984) Cell adhesion and morphogenesis: The regulator hypothesis. Proc. Natl. Acad. Sci. U.S.A., 81: 1460-1464. Edelman, G.M. (1985a) Cell adhesion and the molecular process of morphogenesis. Annu. Rev. Biochem., 54:135-169. Edelman, G.M. (1985b) Expression of cell adhesion molecules during embryogenesis and regeneration. Exp. Cell Res., 161:1-16. Edelman, G.M., and C.-M. Chuong (1982) Embryonic to adult conversion of neural cell adhesion molecules in normal and

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Expression of the cell adhesion molecules, L-CAM and N-CAM during avian scale development.

To examine the involvement of cell adhesion molecules in the inductive epithelial-mesenchymal interactions during avian scale development, a study of ...
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