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Adhesion Molecules in Skin Development: Morphogenesis of Feather and Hair. CHENG-MING CHUONG,b HAI-MING CHEN, TING-XING JIANG, AND JENNIFER CHIA Department of Pathology School of Medicine University of Southern California Los Angeles, California 90033
INTRODUCTION One of the key issues in the development and maintenance of skin is the formation of skin appendages such as hair, nail, horn, and feather. The process is formed by complex interactions between the epithelium and the mesenchyme, resulting in the conversion of a flat piece of ectoderm into cutaneous appendages with unique structure and keratin subtypes (reviewed in Ref. 1).The failure of this process leads to various disorders, ranging from congenital malformation to tumors to alopecia? In order to correct these disorders, the mechanism underlying this process must be understood. Cell interaction plays a central role in the formation of skin appendages, and it is essential to identify cell surface molecules involved in this interaction. Recent work has led to the identification of several new adhesion molecule families that mediate cell-cell and cell-substrate adhesion, and they are likely candidates for this process. There are neural cell adhesion molecules (N-CAM), which belong to the immunoglobulin gene superfamily (reviewed in Ref. 3); cadherins, which mediate adhesion in the presence of calcium (reviewed in Ref. 4); tenascin, which is a unique matrix molecule composed of domains homologous to epidermal cell growth factor (EGF); fibronectin type I11 repeat and fibrinogen (reviewed in Ref. 5); and integrins, which serve as cellular receptors for fibronectin, collagen, and other extracellular matrix molecules (reviewed in Ref. 6). These molecules have been shown to be essential during embryonic development and play important physiological roles in the mature adult as well as in regeneration (reviewed in Ref. 7). We have previously shown that N-CAM is expressed during feather induction8v9and that antibodies applied to liver CAM (L-CAM) can alter feather pattern formation.'O In this communication, we demonstrate our continuing study of the expression and function of adhesion molecules in ski development. Because of the distinct pattern and accessibility to experimentation, the feather is a classical model for This work is supported by NIH HD 24301 and the Council for Tobacco Research. C.-M. Cis a recipient of American Cancer Society Junior Faculty Research Award. J. C. is supported by the Edmonson Fellowship, Department of Pathology, University of Southern California. Address for correspondence: Dr. Cheng-Ming Chuong, HMR 204, Department of Pathology, School of Medicine, University of Southern California, 201 1 Zonal Avenue, Los Angeles, California 90033. 263
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studying the induction and morphogenesis of cutaneous appendages. Therefore, we have been studying feather. We have also checked the roles of adhesion molecules in hair for comparison. In this study, we examined the expression of N-CAM, tenascin, integrin, peanut agglutinin (PNA), and platelet-derived growth factor (PDGF) receptor during the morphogenesis of hair and feather. We used feather explant culture and dermal papilla cell cultures to further analyze the function of these adhesion molecules.
MATERIALS AND METHODS Chicken embryos were obtained from Red Wing Farm (Los Angeles, CA) and staged according to Hamburger and Hamilton.” Antibody to PDGF receptor beta chain was prepared against synthesized peptide and provided by Jung-Sun Huang.12 PDGF BB was from R and D Systems (Minneapolis, MN). Antibody to tenascin was from the Developmental Studies Hybridoma Bank. Antibody to integrin beta subunit (CSAT) was a kind gift from Dr. Clayton A. Buck (Wistar Institute, PA). Immunostaining was prepared according to previous publication.7.8 Skin explant cultures were prepared according to Gallin et aL1OWe used a Biorad con-focal microscope. Feather dermal papilla cultures were prepared according to Messenger et ~ 1 . ~ 3
RESULTS Expression of Adhesion Molecules in the Development of the Feather During the morphogenesis of the feather bud and follicle, remarkable molecular heterogeneity defined by the expression pattern of adhesion molecules is observed. In FIGURE lA, the mesodermal cells inside the feather bud appear similar to one another, but different regions in the bud express different types of molecules. These cellular domains probably have special functions, although their actual purpose has yet to be determined. At stage 34, there is an anterior (defined as the side of the bud that forms an obtuse angle to the body surface)-posterior gradient of N-CAM in the feather bud (FIG.1B). In contrast, there is a posterior-anterior gradient of fibronectin (FIG.1C and Ref. 14). Tenascin is present on both the anterior and posterior bud around the bending region where the surface ectoderm evaginates to form the feather bud (FIG.1D). Chondroitin sulfate was shown to be enriched in the anterior bud.I4 By the time a chicken has hatched, its feather follicles have already formed (FIG. 2). The dermal papilla is enriched with N-CAM and tenascin (FIG.2A and B), but is negative for fibronectin (FIG.2C). It is also negative for neuro-glia (Ng)-CAM and L-CAM.8 Above the dermal papilla is a zone of epithelial cells called the “collar,”
FIGURE 1. Differential expression of adhesion molecules in developing feather buds. Immunofluorescent staining of stage 34 chicken dorsal skin. A, phase contrast; B, N-CAM; C, fibronectin; D, tenascin. Note that N-CAM is enriched with the anterior bud (solidarrows), and fibronectin is enriched in the posterior bud (open arrows), whereas tenascin is present in both anterior and posterior buds. Therefore, there are remarkable molecular heterogeneities in the apparently similar mesenchymal cells within the feather bud. Antifibronectin also stains blood vessels. Magnification: 50x. Bar: 100 wm.
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FIGURE 2. Expression of adhesion molecules in feather follicles. Sections from newly hatched chicken skin. A, N-CAM; B, tenascin; C, fibronectin; D, PDGF beta receptor. Dermal papilla: both N-CAM and tenascin are enriched in the dermal papilla, but the other two molecules are not. Collar epithelium: PDGF beta receptors are highly expressed; fibronectin is also present; a low level of N-CAM can be detected. Feather filament: N-CAM is present on the marginal plate epithelia; tenascin is present on the basement membrane and the pulp; fibronectin is all over the pulp. A-C, immunofluorescencepictures. D, alkaline phosphatase secondary antibodies were used. Abbreviations: cl, collar; dp, dermal papilla; ff, feather filament; fs, feather sheath. Magnification: 50x. Bar: 100 pm.
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which surrounds and comes in close contact with the dermal papilla. The feather collar is equivalent to the matrix region of the hair follicle and represents the epithelial cells undergoing active cell proliferation. We found the PDGF receptor beta subunit to be enriched in this collar region (FIG.2D). We examined the distribution of PDGF receptor because it was shown to be a member of the immunoglobulin superfamily and homologous to N-CAM.ISThe complete analysis of PDGF in feather is to be presented elsewhere (Chuong and Huang, unpublished data). The collar stained positive for fibronectin and L-CAM, weakly positive for N-CAM, and negative for tenascin (FIG.2). The feather filament is generated from the collar, and its core contains pulp that is composed of nerves and blood vessels. The pulp is enriched with fibronectin (FIG.2C). Tenascin is also present in the pulp but is limited to the basal lamina underneath the feather filament epithelia (FIG.2B). In the feather filament, N-CAM is expressed on the longitudinal rows of epithelial cells forming the marginal plate. Later, these cells die away, creating a space between the branches of feather barbs that arise from the barb plate. This zebra-stripe staining pattern of N-CAM can be seen clearly in the longitudinal section (FIG.3A) and cross section (FIG.3B) of feather filaments. L-CAM is present on all the epithelia at this stage? Interestingly, the PDGF receptor beta chain is present on the barb plate epithelia that survive and differentiate, thus forming a complementary pattern with N-CAM that stains only the marginal plate epithelia (FIG.3C and D).
Expression of Adhesion Molecules in the Development of the Hair We also examined the expression of adhesion molecules during different stages of hair development.16 At the hair placode/peg stage, N-CAM is positive on the mesenchyme immediately surrounding the growing hair placode (FIG.4A). N-CAM was also positive on the epithelial placode in a membrane-staining pattern (arrow), which is more obvious in the enlarged panel (FIG.4D). Tenascin is present on the mesenchyme surrounding the placode but appeared to be traced farther outward than N-CAM. Tenascin is also present in the basal lamina underneath the epithelium (FIG. 4B). Because the tenascin-associated proteoglycan contains the PNA binding site and PNA binding cell surface molecules have recently been shown to induce collapse of the growth cone, we also carried out PNA staining. PNA is present on the mesenchyme surrounding the hair placode, but the expression is restricted to the superficial dermis only. This mesenchymal expression is dynamic and disappears soon. PNA is also present on the epithelia and is enriched in the apical surface of placode epithelia (FIG.4C). In the forming hair follicles, N-CAM is present on the dermal papilla, the hair sheath as well as the connective tissue (e.g., muscle) that surround the hair follicles (FIG.5A). In the epithelia, PNA is present in the hair matrix, the inner hair root sheath, the basal lamina of the outer root sheath, and the non-hair forming ectoderm. In the mesenchyme, most of the early PNA expression has disappeared FIG.^^), but it is highly enriched in the region where the surface epithelia invaginate to form the follicle (FIG.5C, arrow). Tenascin is present on the dense connective tissue surrounding the hair follicle sheath. At this stage, tenascin is also enriched in the developing dermis where muscle, tendons, and myotendinous junctions are forming (FIG.5D, E). In the adult, N-CAM remains enriched in the dermal papilla and is also present on the hair sheath. N-CAM is absent in the dermis (FIG.5B). Tenascin is present mainly in the dense connective tissue surrounding the hair follicle (not shown). PNA remains on the hair matrix and root sheath (not shown).
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Function of Adhesion Molecules in the Development of Feather Buds The specific spatial and temporal expression pattern of adhesion molecules during skin appendage development prompted us to look into their function. Due to our establishment of a reproducible culture system, feather explant cultures were used as a model enabling the slightest perturbation to be detectable. We started from stage 32 of the embryonic chicken dorsal skin. At day 0, the condensations were barely visible (FIG.6A), but in 4 days they developed into conical-shaped feather buds (FIG. 6B). This change was particularly impressive when viewed with a confocal microscope. In an optic section, explants cultured for 12 hours showed dermal condensations (FIG.6C). After 4 days in culture, the condensation was transformed into feather buds protruding from the skin surface (FIG.6D). We have tested the perturbation effect of specific antibodies to various adhesion molecules. Interestingly, the reaction of antibodies with several adhesion molecules inhibit feather growth, and the aborted feather patterns show marked differences. The detailed results will be published elsewhere. Here we show the inhibition of N-CAM, tenascin, and the integrin beta subunit by a combination of antibodies. Unlike the control, the buds did not grow but remained as small bumps. The buds also became heterogeneous in size (compare FIG.7A and C). Because of the expression of PDGF receptor in the collar and the barb plate epithelia in feather filament (FIGS.2D and 3C), we hypothesize that PDGF plays a role in feather development. To test this, PDGF BB was added to the culture media. It showed a remarkable effect in enhancing the growth of feather buds. With control, feather germs form cone-shaped buds after 4 days in culture (FIG.7A) and elongate to form slender feather filaments after 8 days in culture (FIG.7B). With PDGF,in 4 days the long, slender feather filaments had already appeared (FIG.7D).
C u b r e of Dermul Papilla Cells Dermal papilla cells have the unusual ability of inducing epithelial cell growth. The molecular basis of this property is analyzed through the culture of dermal papilla cells. We have adapted methods for culturing human hair dermal papilla13to culture feather follicle dermal papilla cells. The cells grew out from the dermal papilla slowly and required 2-3 weeks to form a sheet of cells. Clusters of small, tightly packed cells formed that were surrounded by larger, fibroblast-shaped cells (FIG.8 A, C). N-CAM was positive on all the cells inside the cluster but not on cells outside the cluster (FIG.8 A, B). Enhanced staining of N-CAM was frequently observed at the cellular interface. Within the cluster, tenascin showed an extracellular fibrillar staining pattern. Outside the cluster, tenascin was mostly negative. Although further
FIGURE 3. Expression of adhesion molecules in feather filaments. A, B, C, N-CAM; D, PDGF beta receptor. A, B, N-CAM from newly hatched chicken is visualized by immunofluorescence; C, D, adjacent sections from stage 38 embryo are visualized by alkaline phosphatase. A is a longitudinal section of the feather filament showing that the marginal plate cells (mp) are positive for N-CAM. B is a cross section of a filament showing that the barb plate cells (bp) are negative for N-CAM, while the mp cells and axial plate cells (ap) are positive for N-CAM and appear in regular periodicity. C shows that mp cells stained with anti-N-CAM are positive for the alkaline phosphatase reaction. D shows the opposite staining pattern obtained by staining the PDGF receptor beta subunit with antiserum. Magnifications: A, B, 240x; C, D, 60x. Bars: 100 pm.
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FIGURE 4. Expression of adhesion molecules in developing hair placode. Immunofluorescent staining on E 13 mouse whisker pad. A, N-CAM; B, tenascin; C, P N A D, N-CAM. N-CAM, tenascin, and PNA all are expressed in mesenchyme surrounding the developing hair placode but are distributed differently. N-CAM can be seen on the lateral surface of placode epithelium (arrows in A and D), whereas PNA is more restricted to the apical surface of the placode epithelia. N-CAM can also be seen in nerves (n), which contain larger amounts of N-CAM. Magnifications: A-C, 50x; D, 125x. Bar: 100 pm.
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characterization of these cells is required, the in vitro expression pattern of adhesion molecules is consistent with the in vivo situation in which dermal papilla is positive for both N-CAM and tenascin (FIG.2 A, B).
DISCUSSION Comparative Morphogenesis of Hair and Feather The formation of hair and feather parallel each other in that they both involve induction between epithelium and mesenchyme, cell proliferation, epithelial folding, and mesenchymal condensation. Both follicles contain dermal papilla in the base. New epithelial cells are added to the proximal end and become more differentiated towards the distal portion of the appendages. The end results are skin appendages anchored in follicles and made of specialized keratin. In terms of morphogenesis, hair and feather differ in two major ways. The first is that feather germs form by growing upwards and form buds protruding above the body surface, whereas hair germs begin by forming “epithelial pegs” that grow inside the dermal region. The second difference is that hair ends up as a keratinized cylinder structure, whereas further morphogenetic events take place in feather to generate branched structures. The expression of adhesion molecules in the morphogenesis of hair and feather, however, are fundamentally similar. (1) Both feather and placode epithelia are positive for N-CAM8 (FIG.4D).This transient expression of N-CAM is fundamental and has also been observed in other placodes, including lens placode, otic placode, and the apical ectodermal ridge of limb.17J8 (2) Mesenchymal condensation in feather germs and those surrounding the hair pegs are both positive for N-CAM. Again the presence of N-CAM in mesenchymal condensation is fundamental and has also been observed in precartilaginous condensation and kidney tubule condensation.17J9 (3) As the mesenchymal component expands, it shows heterogeneity in the expression of N-CAM, tenascin, fibronectin, etc., each in different restricted regions. The functional significance has not been determined, but some interesting speculation can be contemplated. For example, both N-CAM and homeoproteins XIHox 1 form an anterior-posterior distribution gradient in the feather bud and may be involved in setting up the anterior-posterior axis of the feather.20In the feather, tenascin is seen in the flanking region of feather buds (FIG.1D); in hair, tenascin and PNA are seen in the “ring” surrounding the hair follicle (FIG.4C and D). These are the regions underneath the bending of epithelia, although one is evaginated and the other is invaginated. Adhesion molecules might exert some mechanical force with their adhesive property during the topological transformation of the epithelial sheet. (4) Both dermal papilla are enriched with N-CAM, even in the adult. (5) Both hair sheath and feather sheath are positive for N-CAM and tenascin, even in the adult.
It is compelling to speculate on the evolutional significance of these findings.2l The scale, the skin appendage of the reptile, is a flat plaque on the skin surface and appears similar to an overgrowth epithelial placode. During evolution,
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one more morphogenetic process was evolved in development to form the hair in mammals. To achieve the longer appendage, the placode cells continued to proliferate and grew into the dermis to form the hair peg. In contrast, when the prototype feather evolved in the avian, the newly added morphogenetic process was for the placode epithelia to proliferate and protrude to form the feather bud. Later, the epithelium flanking the feather bud invaginated into the dermis to form a follicle. The follicle forms in hair morphogenesis, too, and is probably the result of convergent evolution. The follicular structure has many advantages: it is a well-protected sac where epithelial-mesenchymal interactions can take place, new epithelial cells can be added to its base, and it provides good anchorage for the longer cutaneous appendage. Our results suggest that adhesion molecules are used repeatedly in similar key morphogenetic steps underlying different developmental processes. In terms of phylogeny, N-CAM appears early and can be detected in the shark. The binding function is highly conserved; frog N-CAM can bind mouse N-CAM. Indeed, N-CAM is also expressed in the scale and is distributed in a more diffuse pattern (unpublished observation). These results are consistent with the hypothesis that during evolution adhesion molecules such as N-CAM were used in different scenarios when developmental mechanisms were evolved to generate novel structures.
Identijicatton of Molecules Involved in the Formation of Skin Appendages To demonstrate that a molecule is involved in a morphogenetic process, we have to show that the molecule and its receptor is expressed during that process, that overexpression or underexpression of that molecule perturbs the end results, and that we can reconstitute the molecular sequence in terms of its upstream regulation and downstream events. We have been using the feather explant culture system as a model for these analyses. In this explant culture, small dermal condensations develop into feather buds in 4 days and become feather filaments in 8 days. This provides an excellent model by which many different cellular processes can be analyzed. Having shown that several adhesion molecules were indeed expressed in feather morphogenesis, we tried to perturb feather development with the addition of antibodies to adhesion molecules. We added antibodies to N-CAM, tenascin, and integrin beta subunit. Notably, feather development was inhibited in each instance. Antibody to integrin had more overall inhibition, but antibody to N-CAM led to feather buds of different sizes, with the distortion of the hexagonal pattern. These results suggest that the adhesion molecules are involved in different parts of the morphogenetic process. The most profound inhibition, however, occurred when a combination of all three antibodies were used.
FIGURE 5. Expression of adhesion molecules in hair follicles. A, B, N-CAM; C, PNA, D, tenascin; E, phase contrast. A, C, D, E, from E 17 mice; B, from adult mouse skin. Note that N-CAM is always present in the dermal papilla (dp) and part of the hair root sheath (hs); mx, hair matrix. N-CAM is also highly expressed in other connective tissues during development but has disappeared in the adult (compare A and B). The keratinized hair in the upper left comer of panel B shows hair shaft autofluorescence. The mesenchymal region flanking the invagination point, described as the region in which the surface epithelium folds in to form the follicle, appears to be a special region enriched for PNA and tenascin (C and D, solid arrows). This region is actually sleeve shaped when seen in three dimensions, as seen by the section tangential to this “sleeve” (C, open arrow). Magnification: 50x. Ear: 100 pm.
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FIGURE 6. Formation of feather buds on embryonic chicken skin explant cultures. A, C, stage 32, beginning of cultures; B, D,4 days after culture. A and B are views using a stereo dissection microscope; C and D are views using a confocal microscope. Note that in the beginning of cultures there are flat small dermal condensations (dc), which developed into feather buds protruding out of the surface of the skin. Magnifications: A, B, 25x; C, D, 175x. Bar: 100 pm.
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FIGURE 7. Perturbation of feather bud formation. Stage 32 embryonic chicken dorsal skin (as shown in FIG.3A) were cultured for 4 days (A) or 8 days (B). C, 4-day culture in the presence of antibodies to integrin beta chain, N-CAM, and tenascin. D, 4-day culture in the presence of PDGF BB chain. Note the inhibition of feather development when the activity of adhesion molecules was reduced. Also note the acceleration of feather growth in the presence of PDGF. Magnification: 25x. Bar: 100 pm.
In our previous work, antibodies to L-CAMled to the disruption of the hexagonal pattern and to the fusion of dermal condensations into horizontal stripes.I0 Goetinck showed that proteoglycan is also involved in this process by using xyloside to obtain a similar perturbed pattern in the feather explant.= In mouse whisker pad cultures, use of antibodies to P cadherin and E cadherin separately inhibits whisker growth, while the two combined have the most notable inhibition.23These studies suggest that multiple adhesion molecules and extracellular matrix components are involved in the moxphogenesis of feather and hair. The sequence of events in which they are involved will have to be determined in the future. Similar multiple involvement of adhesion molecules in cell migration has been reported in cerebellum granule cell migration, neurite fasciculation, and neural crest cell migration (reviewed in Ref. 24).
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Growth factors such as EGF have been shown to increase hair growth. Although previous work has shown mainly the effect of PDGF on mesenchymal ~ e l l s , 2 ~ ~ ~ ~ recent work has shown that PDGF also regulate the proliferation and differentiation of neural ti~sues.2~ Here our observation of enhancement of feather growth by PDGF and the presence of PDGF receptor in feather collar represent other examples of PDGF acting on ectodermal cells. The factors exchanged between dermal papilla and epidermal collar are most interesting; multiple factors may be involved. To further explore this area, dermal papilla cultures are required for molecular analyses.
Adhesion Molecules in Dermal Papilla Cells Dermal papilla cells are unique because they can induce epithelia to grow out a new feather or hair continuously in the adult. The molecular characterization of this ability is of central importance to embryonic induction as well as to growth control of epithelial cell proliferation. We therefore sought to culture these cells. The behavior of dermal papilla cells from feather appears to be similar to that of human hair dermal papillaI3 and mouse whisker.28They grow slowly and form cellular clusters. The cells within clusters were small, round, tightly packed, and positive for both N-CAM and tenascin. The cells outside the clusters were of different shapes and were mostly negative for N-CAM and tenascin. Further molecular and cellular characterization with different markers is obviously required. Jahoda and Oliver have cultured dermal papilla from the whisker of rats and found that cells tend to form clusters. The loss of the ability to form clusters after several generations in culture correlates with the loss of these cells to induce new hair growth.28 This is consistent with our observation that the adhesion molecule N-CAM is involved in the formation of these clusters. It will be interesting to find out whether N-CAM is indeed essential for the induction of new hair.
SUMMARY FIGURE 9 summarizes the morphogenetic process of feather and hair. Hair of feathers are formed from a layer of homogeneously distributed mesenchymal cells. The mesenchymal cells start to condense to form foci in response to some unidentified induction signal (FIG.9B). Several adhesion molecules, including LCAM, N-CAM, integrin, tenascin, as well as proteoglycan, are involved. These adhesion molecules appear to have different roles in this process, because perturbation with specific antibodies leads to different aborted patterns. Hair or feather follicles then form following cell proliferation and epithelial invagination (FIG.9C).
FIGURE 8. Dermal papilla cultures. A, B, immunofluorescence stained for N-CAM; C, phase contrast; D, stained for the presence of tenascin. A, low-power, showing cells forming clusters (c) that were N-CAM positive. Magnification of part of one cluster double stained for N-CAM (B, fluorescein) and tenascin (D, Texas Red). Note that the cells inside the cluster appeared small, round, tightly packed, while the cells surrounding the clusters were flat and dispersed. N-CAM staining was positive on the cells inside the cluster and appeared to be on the cell membrane, but was negative on the outside fibroblastic cells. Tenascin was also enriched inside the cluster with an extracellular matrix staining pattern. Magnification: A, 20x; B-D,100x. Bur: 100 pm.
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MESENCHYMAL CONDENSATION
CELL PROLIFERATION
EPITHELIAL INVAGI NATlON
DIFFERENTIATION
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The dermal papilla is enriched with N-CAM and tenascin, whereas the feather collar (equivalent of hair matrix) is enriched with L-CAM and PDGF receptor. Epithelial cells in the feather collar receive a signal from the dermal papilla and are able to continue to divide. Several growth factors, such as PDGF and EGF, may be involved. As epithelial cells are pushed upwards, they differentiate and keratinize in a cylindrical structure into hair. In feather, another morphogenetic event takes place to form the branched structure. The epithelial cylinder of the feather shaft invaginates to form rows of cells that die to become space and create the secondary branch or barbs (FIG. 9D). N-CAM is enriched in the cells destined to die and appears to form the border of cell groups within which the “death signal” is transmitted. In some, but not all, feathers the same process is repeated, in a way analogous to fractal formation, to form the tertiary branches or the barbules (FIG. 9E). Thus, in each step of the morphogenesis of feather and hair, different adhesion molecules are expressed and are involved in different functions: induction, mesenchymal condensation, epithelial folding, and cell death, depending on different scenarios. We have just begun to elucidate these molecular events.
REFERENCES 1. SENGEL, P. 1976. Morphogenesis of Skin. Cambridge University Press. New York, NY. & S. L. ROBBINS. 1989. Robbins Pathologic Basis of Disease. 2. COTRAN, R. S., V. KUMAR 4th edit. W. B. Saunders Co. Philadelphia, PA. G. M. 1988. Topobiology: An introduction to molecular embryology. Basic 3. EDELMAN. Books. New York, NY. M. 1988. The cadherins: Cell-cell adhesion molecules controlling animal mor4. TAKEICHI. phogenesis. Development 102: 639-655. 5. ERICKSON, H. P. & M. A. BOURDON. 1989. Tenascin: An extracellular matrix protein prominent in specialized embryonic tissues and tumors. Annu. Rev. Cell. Biol. 5: 7 1-92. 0. R. 1987. Integrins: A family of cell surface receptors. Cell 48: 549-554. 6. HYNES. 7. CHUONG, C.-M. 1990. Adhesion molecules N-CAM and tenascin in embryonic development and tissue regeneration. J. Craniofacial Gene. Dev. Biol. 10: 147-161. 8. CHUONG. C.-M. & G. M. EDELMAN. 1985. Expression of cell adhesion molecules in embryonic induction. I. Morphogenesis of nestling feathers. J. Cell Biol. 101: 10091026. 1985. Expression of cell adhesion molecules in 9. CHUONG. C.-M. & G. M. EDELMAN. embryonic induction. 11. Morphogenesisof adult feathers. J. Cell Biol. 101: 1027-1043.
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FIGURE 9. Schematic drawing showing adhesion molecules in the development of skin appendages using N-CAM as an example. A, a piece of ectoderm with mesenchyme underneath. B, the circles represent dermal condensation. N, N-CAM expressing dermal condensation. C, following cell proliferation and epithelial invagination, the major shaft of the skin appendages, hair, or feather rachis is formed. Large N in panels C-E represents N-CAM expressing dermal papilla. D, occurring in feather only, the epithelial cylinder forms alternating rows of cells that either die or are keratinized to generate branches (barbs) inserted on the rachis. E,this process repeats in a way similar to the formation of a fractal. The result is the tertiary branch (barbules) inserted on the secondary branch. In panels D and E, the smaller and smallest Ns represent epithelial cells that express N-CAM (marginal and axial plate, respectively) and die, to become spaces between barbs and barbules, respectively. The barb plate and barbule plate epithelia cells differentiate to express special kinds of keratin and become feather proper.
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10. GALLIN. W. I., C.-M. CHUONG, L. H. FINKEL & G. M. EDELMAN. 1986. Antibodies to L-CAM perturb inductive interactions and alter feather pattern and structure. Proc. Natl. Acad. Sci. USA 83: 8235-8239. 11. HAMBURGER,^. & H. HAMILTON. 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88: 49-92. 12. HUANG. S. S. & J. S. HUANG. 1989. Rapid turnover of the platelet-derived growth factor receptor in sis-transformed cells and reversal by suramin. J. Biol. Chem. 263: 1260812619. 13. MESSENGER, A. G., H. J. SENIOR & S. S. BLEEHEN. 1986. The in vitro properties of dermal papilla cell lines established from human hair follicles. Br. J. Dermatol. 1 1 4 425-430. 14. MAUGER,A., M. DEMARCHEZ, D. HERBAGE, J.-A. GRIMAUD. M. DRUGUET, D. HARTMA”& P. SENGEL. 1982. Irnmunofluorescent localization of collagen types I and 111, and of fibronectin during feather morphogenesis in the chicken embryo. Dev. Biol. 9 4 93105. 15. WILLIAMS, L. T. 1989. Signal transduction by the platelet derived growth factor receptor. Science 243: 1564-1570. 1952. The development of mouse vibrissae in vivo and in 16. DAVIDSON, P. & M. H. HARDY. vitro. J. Anat. 86: 342-356. 17. CROSSIN. K. L.,C.-M. CHUONG & G. M. EDELMAN. 1985. Expression sequences of cell adhesion molecules. Proc. Natl. Acad. Sci. USA 82: 6942-6946. 18. RICHARDSON. G., K. L. CROSSIN, C.-M. CHUONG & G. M. EDELMAN. 1987. Expression Of cell adhesion molecules in embryonic induction. 111. Development of the otic placode. Dev. Biol. 119: 217-230. & H. M. CHEN. 1990. Differential roles of N-CAM, tenascin 19. CHUONG. C.-M., X.T. JIANG and fibronectin in the adhesive property of limb bud cells: Formation of precartilage mesenchymal condensation. J. Cell Biol. 111 (suppl.): Abstr. No. 1500. 20. BEREITER-HAHN, J., A. G. MATOLTSY & K. S. RICHARDS. 1984. Biology of the Integument, Vol. 2. Vertebrates. Springer-Verlag. New York, NY. 21. CHUONG. C.-M., G. OLIVER. S. TING. B. JEGALIAN, H. M. CHEN& E. M. DE ROBERTIS. 1990.Gradient of homeoproteins in developing feather buds. Development 1 1 0 10211030. 22. GOETINCK, F. PAUL& D. L. CARLONE. 1988. Altered proteoglycan synthesis disrupts feather pattern formation in chick embryonic skin. Dev. Biol. 127: 179-186. 23. HIRAI, Y.,A. NOSE,S. KOBAYASHI & M. TAKEICHI. 1989. Expression and role of E- and P-cadherin adhesion molecules in embryonic histogenesis. 11. Skin morphogenesis. Dev. Biol. 105: 271-277. 24. CHUONG, C.-M. 1990. Differential roles of multiple adhesion molecules in cell migration: Granule cell migration in cerebellum. Experientia 46: 892-899. 25. RUSSEL, R., E. W. RAINES & D. F. BOWEN-POPE. 1986. The biology of platelet-derived growth factor. Cell 46: 155-169. 26. HELDIN. C.-H. & B. WESTERMARK. 1990. Platelet-derived growth factor: Mechanism of action and possible in vivo function. Cell Regul. 1: 555-566. 27. SASAHARA, M., W. U. FRIES,E. RAINES, A. M. GOWN, L. E. WESTRUM, M. P. FORSCH. D. T.BONTHRON, R. Ross & T. COLLINS. 1991. PDGF B-chain in neurons of the central nervous system, posterior pituitary, and in a transgenic model. Cell 64: 217-227. 1984. Vibrissa dermal papilla cell aggregative behavior 28. JAHODA, C. A. B. & R. F. OLIVER. in vivo and in vitro. J. Embryol. Exp. Morphol. 79: 211-224.
Adhesion Molecules in Skin Development: Morphogenesis of Feather and Hair. CHENG-MING CHUONG,b HAI-MING CHEN, TING-XING JIANG, AND JENNIFER CHIA Department of Pathology School of Medicine University of Southern California Los Angeles, California 90033
INTRODUCTION One of the key issues in the development and maintenance of skin is the formation of skin appendages such as hair, nail, horn, and feather. The process is formed by complex interactions between the epithelium and the mesenchyme, resulting in the conversion of a flat piece of ectoderm into cutaneous appendages with unique structure and keratin subtypes (reviewed in Ref. 1).The failure of this process leads to various disorders, ranging from congenital malformation to tumors to alopecia? In order to correct these disorders, the mechanism underlying this process must be understood. Cell interaction plays a central role in the formation of skin appendages, and it is essential to identify cell surface molecules involved in this interaction. Recent work has led to the identification of several new adhesion molecule families that mediate cell-cell and cell-substrate adhesion, and they are likely candidates for this process. There are neural cell adhesion molecules (N-CAM), which belong to the immunoglobulin gene superfamily (reviewed in Ref. 3); cadherins, which mediate adhesion in the presence of calcium (reviewed in Ref. 4); tenascin, which is a unique matrix molecule composed of domains homologous to epidermal cell growth factor (EGF); fibronectin type I11 repeat and fibrinogen (reviewed in Ref. 5); and integrins, which serve as cellular receptors for fibronectin, collagen, and other extracellular matrix molecules (reviewed in Ref. 6). These molecules have been shown to be essential during embryonic development and play important physiological roles in the mature adult as well as in regeneration (reviewed in Ref. 7). We have previously shown that N-CAM is expressed during feather induction8v9and that antibodies applied to liver CAM (L-CAM) can alter feather pattern formation.'O In this communication, we demonstrate our continuing study of the expression and function of adhesion molecules in ski development. Because of the distinct pattern and accessibility to experimentation, the feather is a classical model for This work is supported by NIH HD 24301 and the Council for Tobacco Research. C.-M. Cis a recipient of American Cancer Society Junior Faculty Research Award. J. C. is supported by the Edmonson Fellowship, Department of Pathology, University of Southern California. Address for correspondence: Dr. Cheng-Ming Chuong, HMR 204, Department of Pathology, School of Medicine, University of Southern California, 201 1 Zonal Avenue, Los Angeles, California 90033. 263
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studying the induction and morphogenesis of cutaneous appendages. Therefore, we have been studying feather. We have also checked the roles of adhesion molecules in hair for comparison. In this study, we examined the expression of N-CAM, tenascin, integrin, peanut agglutinin (PNA), and platelet-derived growth factor (PDGF) receptor during the morphogenesis of hair and feather. We used feather explant culture and dermal papilla cell cultures to further analyze the function of these adhesion molecules.
MATERIALS AND METHODS Chicken embryos were obtained from Red Wing Farm (Los Angeles, CA) and staged according to Hamburger and Hamilton.” Antibody to PDGF receptor beta chain was prepared against synthesized peptide and provided by Jung-Sun Huang.12 PDGF BB was from R and D Systems (Minneapolis, MN). Antibody to tenascin was from the Developmental Studies Hybridoma Bank. Antibody to integrin beta subunit (CSAT) was a kind gift from Dr. Clayton A. Buck (Wistar Institute, PA). Immunostaining was prepared according to previous publication.7.8 Skin explant cultures were prepared according to Gallin et aL1OWe used a Biorad con-focal microscope. Feather dermal papilla cultures were prepared according to Messenger et ~ 1 . ~ 3
RESULTS Expression of Adhesion Molecules in the Development of the Feather During the morphogenesis of the feather bud and follicle, remarkable molecular heterogeneity defined by the expression pattern of adhesion molecules is observed. In FIGURE lA, the mesodermal cells inside the feather bud appear similar to one another, but different regions in the bud express different types of molecules. These cellular domains probably have special functions, although their actual purpose has yet to be determined. At stage 34, there is an anterior (defined as the side of the bud that forms an obtuse angle to the body surface)-posterior gradient of N-CAM in the feather bud (FIG.1B). In contrast, there is a posterior-anterior gradient of fibronectin (FIG.1C and Ref. 14). Tenascin is present on both the anterior and posterior bud around the bending region where the surface ectoderm evaginates to form the feather bud (FIG.1D). Chondroitin sulfate was shown to be enriched in the anterior bud.I4 By the time a chicken has hatched, its feather follicles have already formed (FIG. 2). The dermal papilla is enriched with N-CAM and tenascin (FIG.2A and B), but is negative for fibronectin (FIG.2C). It is also negative for neuro-glia (Ng)-CAM and L-CAM.8 Above the dermal papilla is a zone of epithelial cells called the “collar,”
FIGURE 1. Differential expression of adhesion molecules in developing feather buds. Immunofluorescent staining of stage 34 chicken dorsal skin. A, phase contrast; B, N-CAM; C, fibronectin; D, tenascin. Note that N-CAM is enriched with the anterior bud (solidarrows), and fibronectin is enriched in the posterior bud (open arrows), whereas tenascin is present in both anterior and posterior buds. Therefore, there are remarkable molecular heterogeneities in the apparently similar mesenchymal cells within the feather bud. Antifibronectin also stains blood vessels. Magnification: 50x. Bar: 100 wm.
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FIGURE 2. Expression of adhesion molecules in feather follicles. Sections from newly hatched chicken skin. A, N-CAM; B, tenascin; C, fibronectin; D, PDGF beta receptor. Dermal papilla: both N-CAM and tenascin are enriched in the dermal papilla, but the other two molecules are not. Collar epithelium: PDGF beta receptors are highly expressed; fibronectin is also present; a low level of N-CAM can be detected. Feather filament: N-CAM is present on the marginal plate epithelia; tenascin is present on the basement membrane and the pulp; fibronectin is all over the pulp. A-C, immunofluorescencepictures. D, alkaline phosphatase secondary antibodies were used. Abbreviations: cl, collar; dp, dermal papilla; ff, feather filament; fs, feather sheath. Magnification: 50x. Bar: 100 pm.
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which surrounds and comes in close contact with the dermal papilla. The feather collar is equivalent to the matrix region of the hair follicle and represents the epithelial cells undergoing active cell proliferation. We found the PDGF receptor beta subunit to be enriched in this collar region (FIG.2D). We examined the distribution of PDGF receptor because it was shown to be a member of the immunoglobulin superfamily and homologous to N-CAM.ISThe complete analysis of PDGF in feather is to be presented elsewhere (Chuong and Huang, unpublished data). The collar stained positive for fibronectin and L-CAM, weakly positive for N-CAM, and negative for tenascin (FIG.2). The feather filament is generated from the collar, and its core contains pulp that is composed of nerves and blood vessels. The pulp is enriched with fibronectin (FIG.2C). Tenascin is also present in the pulp but is limited to the basal lamina underneath the feather filament epithelia (FIG.2B). In the feather filament, N-CAM is expressed on the longitudinal rows of epithelial cells forming the marginal plate. Later, these cells die away, creating a space between the branches of feather barbs that arise from the barb plate. This zebra-stripe staining pattern of N-CAM can be seen clearly in the longitudinal section (FIG.3A) and cross section (FIG.3B) of feather filaments. L-CAM is present on all the epithelia at this stage? Interestingly, the PDGF receptor beta chain is present on the barb plate epithelia that survive and differentiate, thus forming a complementary pattern with N-CAM that stains only the marginal plate epithelia (FIG.3C and D).
Expression of Adhesion Molecules in the Development of the Hair We also examined the expression of adhesion molecules during different stages of hair development.16 At the hair placode/peg stage, N-CAM is positive on the mesenchyme immediately surrounding the growing hair placode (FIG.4A). N-CAM was also positive on the epithelial placode in a membrane-staining pattern (arrow), which is more obvious in the enlarged panel (FIG.4D). Tenascin is present on the mesenchyme surrounding the placode but appeared to be traced farther outward than N-CAM. Tenascin is also present in the basal lamina underneath the epithelium (FIG. 4B). Because the tenascin-associated proteoglycan contains the PNA binding site and PNA binding cell surface molecules have recently been shown to induce collapse of the growth cone, we also carried out PNA staining. PNA is present on the mesenchyme surrounding the hair placode, but the expression is restricted to the superficial dermis only. This mesenchymal expression is dynamic and disappears soon. PNA is also present on the epithelia and is enriched in the apical surface of placode epithelia (FIG.4C). In the forming hair follicles, N-CAM is present on the dermal papilla, the hair sheath as well as the connective tissue (e.g., muscle) that surround the hair follicles (FIG.5A). In the epithelia, PNA is present in the hair matrix, the inner hair root sheath, the basal lamina of the outer root sheath, and the non-hair forming ectoderm. In the mesenchyme, most of the early PNA expression has disappeared FIG.^^), but it is highly enriched in the region where the surface epithelia invaginate to form the follicle (FIG.5C, arrow). Tenascin is present on the dense connective tissue surrounding the hair follicle sheath. At this stage, tenascin is also enriched in the developing dermis where muscle, tendons, and myotendinous junctions are forming (FIG.5D, E). In the adult, N-CAM remains enriched in the dermal papilla and is also present on the hair sheath. N-CAM is absent in the dermis (FIG.5B). Tenascin is present mainly in the dense connective tissue surrounding the hair follicle (not shown). PNA remains on the hair matrix and root sheath (not shown).
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Function of Adhesion Molecules in the Development of Feather Buds The specific spatial and temporal expression pattern of adhesion molecules during skin appendage development prompted us to look into their function. Due to our establishment of a reproducible culture system, feather explant cultures were used as a model enabling the slightest perturbation to be detectable. We started from stage 32 of the embryonic chicken dorsal skin. At day 0, the condensations were barely visible (FIG.6A), but in 4 days they developed into conical-shaped feather buds (FIG. 6B). This change was particularly impressive when viewed with a confocal microscope. In an optic section, explants cultured for 12 hours showed dermal condensations (FIG.6C). After 4 days in culture, the condensation was transformed into feather buds protruding from the skin surface (FIG.6D). We have tested the perturbation effect of specific antibodies to various adhesion molecules. Interestingly, the reaction of antibodies with several adhesion molecules inhibit feather growth, and the aborted feather patterns show marked differences. The detailed results will be published elsewhere. Here we show the inhibition of N-CAM, tenascin, and the integrin beta subunit by a combination of antibodies. Unlike the control, the buds did not grow but remained as small bumps. The buds also became heterogeneous in size (compare FIG.7A and C). Because of the expression of PDGF receptor in the collar and the barb plate epithelia in feather filament (FIGS.2D and 3C), we hypothesize that PDGF plays a role in feather development. To test this, PDGF BB was added to the culture media. It showed a remarkable effect in enhancing the growth of feather buds. With control, feather germs form cone-shaped buds after 4 days in culture (FIG.7A) and elongate to form slender feather filaments after 8 days in culture (FIG.7B). With PDGF,in 4 days the long, slender feather filaments had already appeared (FIG.7D).
C u b r e of Dermul Papilla Cells Dermal papilla cells have the unusual ability of inducing epithelial cell growth. The molecular basis of this property is analyzed through the culture of dermal papilla cells. We have adapted methods for culturing human hair dermal papilla13to culture feather follicle dermal papilla cells. The cells grew out from the dermal papilla slowly and required 2-3 weeks to form a sheet of cells. Clusters of small, tightly packed cells formed that were surrounded by larger, fibroblast-shaped cells (FIG.8 A, C). N-CAM was positive on all the cells inside the cluster but not on cells outside the cluster (FIG.8 A, B). Enhanced staining of N-CAM was frequently observed at the cellular interface. Within the cluster, tenascin showed an extracellular fibrillar staining pattern. Outside the cluster, tenascin was mostly negative. Although further
FIGURE 3. Expression of adhesion molecules in feather filaments. A, B, C, N-CAM; D, PDGF beta receptor. A, B, N-CAM from newly hatched chicken is visualized by immunofluorescence; C, D, adjacent sections from stage 38 embryo are visualized by alkaline phosphatase. A is a longitudinal section of the feather filament showing that the marginal plate cells (mp) are positive for N-CAM. B is a cross section of a filament showing that the barb plate cells (bp) are negative for N-CAM, while the mp cells and axial plate cells (ap) are positive for N-CAM and appear in regular periodicity. C shows that mp cells stained with anti-N-CAM are positive for the alkaline phosphatase reaction. D shows the opposite staining pattern obtained by staining the PDGF receptor beta subunit with antiserum. Magnifications: A, B, 240x; C, D, 60x. Bars: 100 pm.
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FIGURE 4. Expression of adhesion molecules in developing hair placode. Immunofluorescent staining on E 13 mouse whisker pad. A, N-CAM; B, tenascin; C, P N A D, N-CAM. N-CAM, tenascin, and PNA all are expressed in mesenchyme surrounding the developing hair placode but are distributed differently. N-CAM can be seen on the lateral surface of placode epithelium (arrows in A and D), whereas PNA is more restricted to the apical surface of the placode epithelia. N-CAM can also be seen in nerves (n), which contain larger amounts of N-CAM. Magnifications: A-C, 50x; D, 125x. Bar: 100 pm.
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characterization of these cells is required, the in vitro expression pattern of adhesion molecules is consistent with the in vivo situation in which dermal papilla is positive for both N-CAM and tenascin (FIG.2 A, B).
DISCUSSION Comparative Morphogenesis of Hair and Feather The formation of hair and feather parallel each other in that they both involve induction between epithelium and mesenchyme, cell proliferation, epithelial folding, and mesenchymal condensation. Both follicles contain dermal papilla in the base. New epithelial cells are added to the proximal end and become more differentiated towards the distal portion of the appendages. The end results are skin appendages anchored in follicles and made of specialized keratin. In terms of morphogenesis, hair and feather differ in two major ways. The first is that feather germs form by growing upwards and form buds protruding above the body surface, whereas hair germs begin by forming “epithelial pegs” that grow inside the dermal region. The second difference is that hair ends up as a keratinized cylinder structure, whereas further morphogenetic events take place in feather to generate branched structures. The expression of adhesion molecules in the morphogenesis of hair and feather, however, are fundamentally similar. (1) Both feather and placode epithelia are positive for N-CAM8 (FIG.4D).This transient expression of N-CAM is fundamental and has also been observed in other placodes, including lens placode, otic placode, and the apical ectodermal ridge of limb.17J8 (2) Mesenchymal condensation in feather germs and those surrounding the hair pegs are both positive for N-CAM. Again the presence of N-CAM in mesenchymal condensation is fundamental and has also been observed in precartilaginous condensation and kidney tubule condensation.17J9 (3) As the mesenchymal component expands, it shows heterogeneity in the expression of N-CAM, tenascin, fibronectin, etc., each in different restricted regions. The functional significance has not been determined, but some interesting speculation can be contemplated. For example, both N-CAM and homeoproteins XIHox 1 form an anterior-posterior distribution gradient in the feather bud and may be involved in setting up the anterior-posterior axis of the feather.20In the feather, tenascin is seen in the flanking region of feather buds (FIG.1D); in hair, tenascin and PNA are seen in the “ring” surrounding the hair follicle (FIG.4C and D). These are the regions underneath the bending of epithelia, although one is evaginated and the other is invaginated. Adhesion molecules might exert some mechanical force with their adhesive property during the topological transformation of the epithelial sheet. (4) Both dermal papilla are enriched with N-CAM, even in the adult. (5) Both hair sheath and feather sheath are positive for N-CAM and tenascin, even in the adult.
It is compelling to speculate on the evolutional significance of these findings.2l The scale, the skin appendage of the reptile, is a flat plaque on the skin surface and appears similar to an overgrowth epithelial placode. During evolution,
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one more morphogenetic process was evolved in development to form the hair in mammals. To achieve the longer appendage, the placode cells continued to proliferate and grew into the dermis to form the hair peg. In contrast, when the prototype feather evolved in the avian, the newly added morphogenetic process was for the placode epithelia to proliferate and protrude to form the feather bud. Later, the epithelium flanking the feather bud invaginated into the dermis to form a follicle. The follicle forms in hair morphogenesis, too, and is probably the result of convergent evolution. The follicular structure has many advantages: it is a well-protected sac where epithelial-mesenchymal interactions can take place, new epithelial cells can be added to its base, and it provides good anchorage for the longer cutaneous appendage. Our results suggest that adhesion molecules are used repeatedly in similar key morphogenetic steps underlying different developmental processes. In terms of phylogeny, N-CAM appears early and can be detected in the shark. The binding function is highly conserved; frog N-CAM can bind mouse N-CAM. Indeed, N-CAM is also expressed in the scale and is distributed in a more diffuse pattern (unpublished observation). These results are consistent with the hypothesis that during evolution adhesion molecules such as N-CAM were used in different scenarios when developmental mechanisms were evolved to generate novel structures.
Identijicatton of Molecules Involved in the Formation of Skin Appendages To demonstrate that a molecule is involved in a morphogenetic process, we have to show that the molecule and its receptor is expressed during that process, that overexpression or underexpression of that molecule perturbs the end results, and that we can reconstitute the molecular sequence in terms of its upstream regulation and downstream events. We have been using the feather explant culture system as a model for these analyses. In this explant culture, small dermal condensations develop into feather buds in 4 days and become feather filaments in 8 days. This provides an excellent model by which many different cellular processes can be analyzed. Having shown that several adhesion molecules were indeed expressed in feather morphogenesis, we tried to perturb feather development with the addition of antibodies to adhesion molecules. We added antibodies to N-CAM, tenascin, and integrin beta subunit. Notably, feather development was inhibited in each instance. Antibody to integrin had more overall inhibition, but antibody to N-CAM led to feather buds of different sizes, with the distortion of the hexagonal pattern. These results suggest that the adhesion molecules are involved in different parts of the morphogenetic process. The most profound inhibition, however, occurred when a combination of all three antibodies were used.
FIGURE 5. Expression of adhesion molecules in hair follicles. A, B, N-CAM; C, PNA, D, tenascin; E, phase contrast. A, C, D, E, from E 17 mice; B, from adult mouse skin. Note that N-CAM is always present in the dermal papilla (dp) and part of the hair root sheath (hs); mx, hair matrix. N-CAM is also highly expressed in other connective tissues during development but has disappeared in the adult (compare A and B). The keratinized hair in the upper left comer of panel B shows hair shaft autofluorescence. The mesenchymal region flanking the invagination point, described as the region in which the surface epithelium folds in to form the follicle, appears to be a special region enriched for PNA and tenascin (C and D, solid arrows). This region is actually sleeve shaped when seen in three dimensions, as seen by the section tangential to this “sleeve” (C, open arrow). Magnification: 50x. Ear: 100 pm.
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FIGURE 6. Formation of feather buds on embryonic chicken skin explant cultures. A, C, stage 32, beginning of cultures; B, D,4 days after culture. A and B are views using a stereo dissection microscope; C and D are views using a confocal microscope. Note that in the beginning of cultures there are flat small dermal condensations (dc), which developed into feather buds protruding out of the surface of the skin. Magnifications: A, B, 25x; C, D, 175x. Bar: 100 pm.
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FIGURE 7. Perturbation of feather bud formation. Stage 32 embryonic chicken dorsal skin (as shown in FIG.3A) were cultured for 4 days (A) or 8 days (B). C, 4-day culture in the presence of antibodies to integrin beta chain, N-CAM, and tenascin. D, 4-day culture in the presence of PDGF BB chain. Note the inhibition of feather development when the activity of adhesion molecules was reduced. Also note the acceleration of feather growth in the presence of PDGF. Magnification: 25x. Bar: 100 pm.
In our previous work, antibodies to L-CAMled to the disruption of the hexagonal pattern and to the fusion of dermal condensations into horizontal stripes.I0 Goetinck showed that proteoglycan is also involved in this process by using xyloside to obtain a similar perturbed pattern in the feather explant.= In mouse whisker pad cultures, use of antibodies to P cadherin and E cadherin separately inhibits whisker growth, while the two combined have the most notable inhibition.23These studies suggest that multiple adhesion molecules and extracellular matrix components are involved in the moxphogenesis of feather and hair. The sequence of events in which they are involved will have to be determined in the future. Similar multiple involvement of adhesion molecules in cell migration has been reported in cerebellum granule cell migration, neurite fasciculation, and neural crest cell migration (reviewed in Ref. 24).
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Growth factors such as EGF have been shown to increase hair growth. Although previous work has shown mainly the effect of PDGF on mesenchymal ~ e l l s , 2 ~ ~ ~ ~ recent work has shown that PDGF also regulate the proliferation and differentiation of neural ti~sues.2~ Here our observation of enhancement of feather growth by PDGF and the presence of PDGF receptor in feather collar represent other examples of PDGF acting on ectodermal cells. The factors exchanged between dermal papilla and epidermal collar are most interesting; multiple factors may be involved. To further explore this area, dermal papilla cultures are required for molecular analyses.
Adhesion Molecules in Dermal Papilla Cells Dermal papilla cells are unique because they can induce epithelia to grow out a new feather or hair continuously in the adult. The molecular characterization of this ability is of central importance to embryonic induction as well as to growth control of epithelial cell proliferation. We therefore sought to culture these cells. The behavior of dermal papilla cells from feather appears to be similar to that of human hair dermal papillaI3 and mouse whisker.28They grow slowly and form cellular clusters. The cells within clusters were small, round, tightly packed, and positive for both N-CAM and tenascin. The cells outside the clusters were of different shapes and were mostly negative for N-CAM and tenascin. Further molecular and cellular characterization with different markers is obviously required. Jahoda and Oliver have cultured dermal papilla from the whisker of rats and found that cells tend to form clusters. The loss of the ability to form clusters after several generations in culture correlates with the loss of these cells to induce new hair growth.28 This is consistent with our observation that the adhesion molecule N-CAM is involved in the formation of these clusters. It will be interesting to find out whether N-CAM is indeed essential for the induction of new hair.
SUMMARY FIGURE 9 summarizes the morphogenetic process of feather and hair. Hair of feathers are formed from a layer of homogeneously distributed mesenchymal cells. The mesenchymal cells start to condense to form foci in response to some unidentified induction signal (FIG.9B). Several adhesion molecules, including LCAM, N-CAM, integrin, tenascin, as well as proteoglycan, are involved. These adhesion molecules appear to have different roles in this process, because perturbation with specific antibodies leads to different aborted patterns. Hair or feather follicles then form following cell proliferation and epithelial invagination (FIG.9C).
FIGURE 8. Dermal papilla cultures. A, B, immunofluorescence stained for N-CAM; C, phase contrast; D, stained for the presence of tenascin. A, low-power, showing cells forming clusters (c) that were N-CAM positive. Magnification of part of one cluster double stained for N-CAM (B, fluorescein) and tenascin (D, Texas Red). Note that the cells inside the cluster appeared small, round, tightly packed, while the cells surrounding the clusters were flat and dispersed. N-CAM staining was positive on the cells inside the cluster and appeared to be on the cell membrane, but was negative on the outside fibroblastic cells. Tenascin was also enriched inside the cluster with an extracellular matrix staining pattern. Magnification: A, 20x; B-D,100x. Bur: 100 pm.
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MESENCHYMAL CONDENSATION
CELL PROLIFERATION
EPITHELIAL INVAGI NATlON
DIFFERENTIATION
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The dermal papilla is enriched with N-CAM and tenascin, whereas the feather collar (equivalent of hair matrix) is enriched with L-CAM and PDGF receptor. Epithelial cells in the feather collar receive a signal from the dermal papilla and are able to continue to divide. Several growth factors, such as PDGF and EGF, may be involved. As epithelial cells are pushed upwards, they differentiate and keratinize in a cylindrical structure into hair. In feather, another morphogenetic event takes place to form the branched structure. The epithelial cylinder of the feather shaft invaginates to form rows of cells that die to become space and create the secondary branch or barbs (FIG. 9D). N-CAM is enriched in the cells destined to die and appears to form the border of cell groups within which the “death signal” is transmitted. In some, but not all, feathers the same process is repeated, in a way analogous to fractal formation, to form the tertiary branches or the barbules (FIG. 9E). Thus, in each step of the morphogenesis of feather and hair, different adhesion molecules are expressed and are involved in different functions: induction, mesenchymal condensation, epithelial folding, and cell death, depending on different scenarios. We have just begun to elucidate these molecular events.
REFERENCES 1. SENGEL, P. 1976. Morphogenesis of Skin. Cambridge University Press. New York, NY. & S. L. ROBBINS. 1989. Robbins Pathologic Basis of Disease. 2. COTRAN, R. S., V. KUMAR 4th edit. W. B. Saunders Co. Philadelphia, PA. G. M. 1988. Topobiology: An introduction to molecular embryology. Basic 3. EDELMAN. Books. New York, NY. M. 1988. The cadherins: Cell-cell adhesion molecules controlling animal mor4. TAKEICHI. phogenesis. Development 102: 639-655. 5. ERICKSON, H. P. & M. A. BOURDON. 1989. Tenascin: An extracellular matrix protein prominent in specialized embryonic tissues and tumors. Annu. Rev. Cell. Biol. 5: 7 1-92. 0. R. 1987. Integrins: A family of cell surface receptors. Cell 48: 549-554. 6. HYNES. 7. CHUONG, C.-M. 1990. Adhesion molecules N-CAM and tenascin in embryonic development and tissue regeneration. J. Craniofacial Gene. Dev. Biol. 10: 147-161. 8. CHUONG. C.-M. & G. M. EDELMAN. 1985. Expression of cell adhesion molecules in embryonic induction. I. Morphogenesis of nestling feathers. J. Cell Biol. 101: 10091026. 1985. Expression of cell adhesion molecules in 9. CHUONG. C.-M. & G. M. EDELMAN. embryonic induction. 11. Morphogenesisof adult feathers. J. Cell Biol. 101: 1027-1043.
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FIGURE 9. Schematic drawing showing adhesion molecules in the development of skin appendages using N-CAM as an example. A, a piece of ectoderm with mesenchyme underneath. B, the circles represent dermal condensation. N, N-CAM expressing dermal condensation. C, following cell proliferation and epithelial invagination, the major shaft of the skin appendages, hair, or feather rachis is formed. Large N in panels C-E represents N-CAM expressing dermal papilla. D, occurring in feather only, the epithelial cylinder forms alternating rows of cells that either die or are keratinized to generate branches (barbs) inserted on the rachis. E,this process repeats in a way similar to the formation of a fractal. The result is the tertiary branch (barbules) inserted on the secondary branch. In panels D and E, the smaller and smallest Ns represent epithelial cells that express N-CAM (marginal and axial plate, respectively) and die, to become spaces between barbs and barbules, respectively. The barb plate and barbule plate epithelia cells differentiate to express special kinds of keratin and become feather proper.
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10. GALLIN. W. I., C.-M. CHUONG, L. H. FINKEL & G. M. EDELMAN. 1986. Antibodies to L-CAM perturb inductive interactions and alter feather pattern and structure. Proc. Natl. Acad. Sci. USA 83: 8235-8239. 11. HAMBURGER,^. & H. HAMILTON. 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88: 49-92. 12. HUANG. S. S. & J. S. HUANG. 1989. Rapid turnover of the platelet-derived growth factor receptor in sis-transformed cells and reversal by suramin. J. Biol. Chem. 263: 1260812619. 13. MESSENGER, A. G., H. J. SENIOR & S. S. BLEEHEN. 1986. The in vitro properties of dermal papilla cell lines established from human hair follicles. Br. J. Dermatol. 1 1 4 425-430. 14. MAUGER,A., M. DEMARCHEZ, D. HERBAGE, J.-A. GRIMAUD. M. DRUGUET, D. HARTMA”& P. SENGEL. 1982. Irnmunofluorescent localization of collagen types I and 111, and of fibronectin during feather morphogenesis in the chicken embryo. Dev. Biol. 9 4 93105. 15. WILLIAMS, L. T. 1989. Signal transduction by the platelet derived growth factor receptor. Science 243: 1564-1570. 1952. The development of mouse vibrissae in vivo and in 16. DAVIDSON, P. & M. H. HARDY. vitro. J. Anat. 86: 342-356. 17. CROSSIN. K. L.,C.-M. CHUONG & G. M. EDELMAN. 1985. Expression sequences of cell adhesion molecules. Proc. Natl. Acad. Sci. USA 82: 6942-6946. 18. RICHARDSON. G., K. L. CROSSIN, C.-M. CHUONG & G. M. EDELMAN. 1987. Expression Of cell adhesion molecules in embryonic induction. 111. Development of the otic placode. Dev. Biol. 119: 217-230. & H. M. CHEN. 1990. Differential roles of N-CAM, tenascin 19. CHUONG. C.-M., X.T. JIANG and fibronectin in the adhesive property of limb bud cells: Formation of precartilage mesenchymal condensation. J. Cell Biol. 111 (suppl.): Abstr. No. 1500. 20. BEREITER-HAHN, J., A. G. MATOLTSY & K. S. RICHARDS. 1984. Biology of the Integument, Vol. 2. Vertebrates. Springer-Verlag. New York, NY. 21. CHUONG. C.-M., G. OLIVER. S. TING. B. JEGALIAN, H. M. CHEN& E. M. DE ROBERTIS. 1990.Gradient of homeoproteins in developing feather buds. Development 1 1 0 10211030. 22. GOETINCK, F. PAUL& D. L. CARLONE. 1988. Altered proteoglycan synthesis disrupts feather pattern formation in chick embryonic skin. Dev. Biol. 127: 179-186. 23. HIRAI, Y.,A. NOSE,S. KOBAYASHI & M. TAKEICHI. 1989. Expression and role of E- and P-cadherin adhesion molecules in embryonic histogenesis. 11. Skin morphogenesis. Dev. Biol. 105: 271-277. 24. CHUONG, C.-M. 1990. Differential roles of multiple adhesion molecules in cell migration: Granule cell migration in cerebellum. Experientia 46: 892-899. 25. RUSSEL, R., E. W. RAINES & D. F. BOWEN-POPE. 1986. The biology of platelet-derived growth factor. Cell 46: 155-169. 26. HELDIN. C.-H. & B. WESTERMARK. 1990. Platelet-derived growth factor: Mechanism of action and possible in vivo function. Cell Regul. 1: 555-566. 27. SASAHARA, M., W. U. FRIES,E. RAINES, A. M. GOWN, L. E. WESTRUM, M. P. FORSCH. D. T.BONTHRON, R. Ross & T. COLLINS. 1991. PDGF B-chain in neurons of the central nervous system, posterior pituitary, and in a transgenic model. Cell 64: 217-227. 1984. Vibrissa dermal papilla cell aggregative behavior 28. JAHODA, C. A. B. & R. F. OLIVER. in vivo and in vitro. J. Embryol. Exp. Morphol. 79: 211-224.
