Immunofluorescent localization of collagen types I and III, and of fibronectin during feather morphogenesis in the chick embryo

DEVELOPMENTAL

BIOLOGY

94, 93-105 (1982)

Immunofluorescent Localization of Collagen Types I and Ill, and of Fibronectin (during Feather Morphogenesis in the Chick Embryo ANNICK MAU#GER,*MICHEL DEMARCHEZ,* DANIEL HERBAGE,?JEAN-ALEXIS GRIMAUD,~ MICHEL DRUGUET,$ DANIEL HARTMANN,~ AND PHILIPPE SENGEL*,' *Equipe de recherche associee au CNRS 621 “Morphogenese expkrimentale,” Laboratoire de Zoologie et Biologic animate, Uniuersite scientifique et mhdicale de Grenoble, Boite Postale 53 X, 38041 Grenoble, France; tCentre de recherche appliquee de Dermobiochimie, Lyon, France; $Equipe de recherche associke au CNRS 819 “Pathologie cellulaire du foie, ” Institut Pasteur, Lyon, France; §Centre de Radioanalyse, Znstitut Pasteur, Lyon, France Received December 1, 1980; accepted in revised form June 14, 1982 Collagen types I and III were purified from the skin of 3- or 7-week chickens and fibronectin from human serum. Antibodies were railsed in rabbits and used in indirect immunofluorescence on frozen sections of 5- to 16.day chick embryo feather-forming skin. Prior to the formation of dense feather-forming dermis, anticollagen fluorescence was confined to a thin underlining of the dermal-epidermal junction (DEJ), while antifibronectin label was retained on loosely dispersed material in the predermal mesenchyme. Dense feather-forming dermis was characterized by loosening of the anti-collagen label along the DEJ, by its spreading throughout the thickness of dermis, and by an overall densification of antifibronectin label. Feather formation coincided with a decrease of anti-collagen and an increase of antifibronectin label density in the dermal feather condensations and in the core of outgrowing feather buds. By contrast, density of anti-collagen-labeled material was highest and anti-fibronectin-labeled material was lowest in interplumar and glabrous skin. In slanting feather buds and feather filaments, distribution of anti-collagen-labeled material exhibited a type-specific cranial-caudal asymmetry. The microheterogeneous distribution of extracellular matrix components might constitute part of the morphogenetic message that the dermis is known to transmit to the epidermis during the formation of cutaneous appendages. INTRlODUCTION

(Stuart and Moscona, 1967). That this network might be of significance for the formation of feathers is suggested by the fact that it is lacking in the scaleless mutant, the plumage of which is deficient (Goetinck, 1970; Goetinck and Sekellick, 1972; Overton and Collins, 1976) and that treatment with lathyrogens (Goetinck and Sekellick, 1970) or with collagenase (Stuart et al., 1972) inhibits the formation of dermal feather condensations. In this study we report the localization of three major extracellular matrix components, interstitial collagen types I and III and fibronectin, in embryonic skin. Our data reveal that collagen and fibronectin are distributed in a heterogeneous manner which is related to skin morphogenesis.

Collagens and fibronectin (Linder et al., 1975; Stenman and Vaheri, 1978) are major components of extracellular matrix, and have been implicated in morphogenetic interactions (Grobstein, 1967; Slavkin and Greulich, 1975; Lash and Vasan, 1977). In embryonic amniote skin, it is well established that the dermis plays a determinant role in the histogenesis, maintenance, proliferation, and differentiation of the epidermis, and in the organogenesis of cutaneous appendages (Sengel, 1976; Sengel et al., 1980). While it is clear that many precisely timed and precisely located morphogenetic messages are transmittedL from dermis to epidermis, the mode of their transmission remains unknown. Several studies have shown that extracellular matrix components might be important. An acellular exudate from chick embryo limb mesenchyme (McLaughlin, 1961) or a collagenous substrate (Dodson, 1967) is able to maintain an ordered stratification of in vitro cultured epidermis. During the development of cutaneous appendages, a regular network of birefringent collagenase-sensitive fibers appears in the chick skin shortly before or concomitantly with the outgrowth of feather buds ’ To whom all correspondence

MATERIAL

AND METHODS

Samples

Feather-forming skin from the back (spinal tract), wing (scapular and alar tracts), thigh (femoral tract), and ventrum (pectoral tract), as well as skin from adjacent glabrous regions, were examined in White Leghorn or F, offspring from Wyandotte X Rhode Island Red Chick embryos, from 5 to 16 days of incubation

should be addressed.

93

0012.1606/82/110093-13$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

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(stages 26 to 42 of Hamburger and Hamilton (1951)). For early stages (5 to 9 days), whole embryos were used, and for later stages (10 to 16 days), pieces of trunk dissected between wing and leg levels were used for the preparation of histological sections. In all, 460 sections derived from three to six embryos of each of the following ages were examined: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 16 days of incubation. Antigens Collagens were prepared from dorsal feather-forming skin of 3- and 7-week old chickens. Acid-soluble type I collagen was extracted by 0.1 M acetic acid and purified according to Piez et al. (1963). Type III collagen was extracted by limited pepsin digestion and purified by fractional precipitation with sodium chloride, according to Rhodes and Miller (1978), as applied to chick skin by Demarchez (1980). For further purification, the collagens were chromatographed on DEAE-cellulose under nondenaturing conditions as described by Byers et al. (1974). Collagen fractions and their cyanogen bromide digests were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, with and without reduction, and by amino acid analysis. Fibronectin was prepared from human serum according to Engvall and Ruoslahti (1977), and further purified by chromatography on Sephadex G-200 to get rid of a slight IgG contaminant. Antibodies New Zealand White rabbits received, every 2 weeks, a subcutaneous injuction of 1 mg of chick native type I or type III collagen, or of human fibronectin. The first injection consisted of the antigen emulsified with complete Freund’s adjuvant, while the subsequent ones (three or four) contained the antigen emulsified with incomplete adjuvant. Non-type-specific collagen antibodies cross-reacting with determinants common to the other collagen types were eliminated by absorbing them on the other type of collagen antigen bound to CNBr-activated Sepharose. Type-specific antibodies were isolated by affinity chromatography on the corresponding collagen-type antigen bound to CNBr-activated Sepharose beads. Monospecificity of the antifibronectin antiserum was tested by immunodiffusion and immunoelectrophoresis against normal human serum and fibronectin-depleted human serum. With either technique, a single precipitation line was obtained with normal human serum; no line was seen when the antibody solution was tested against fibronectin-depleted serum. The specificity and cross-reactivity of the anticolla-

VOL~~ME94,1982

gen antibodies were determined by radioimmunoassays, and further characterized by several controls. Type I and type III collagen were labeled with “‘1 by an enzymatic method (glucose oxidase-lactoperoxidase (Taurog et al., 1979)). In a first step, the immunoreactivity of the antibodies was tested and their optimal dilution were determined: immunoabsorbed antiserum to type I and type III collagen was serially diluted in l/ 100 normal rabbit serum in 0.05 M phosphate-buffered saline (PBS) at pH 7.4. To 100 ~1 of diluted antiserum, 100 ~1 of PBS containing 0.1 ng of 1251-labeled type I or type III collagen, respectively (approximately 10,000 cpm), was added. Tubes were shaken, then incubated at 4°C for 24 hr, after which 100 ~1 of an antiserum to rabbit y-globulins was added. Following incubation overnight in the cold, the precipitate was collected by centrifugation at 5000g for 30 min and the radioactivity of the precipitate was measured in a gamma scintillation counter. Nonspecific binding (NSB) was measured by replacing the anticollagen antiserum by normal rabbit serum. Antigen binding, expressed in percentage, was then calculated as Percentage binding = (cpm in precipitate - cpm of NSB) X loo/total cpm added. In a second step, inhibition experiments were carried out and cross-reactivities were determined for anti-type I and anti-type III antibodies: 100 ~1 of optimally diluted anti-type I or anti-type III collagen antibody (which gave between 30 and 60% binding) was incubated for 24 hr at 4°C with 100 ~1 of cold type I and type III, at concentrations from 0.037 to 74 mg/liter in PBS. Then 100 ~1 of PBS containing 0.1 ng of 1251-collagen of the type corresponding to the antibody to be assayed was added and the radioimmunoassay was carried on as described in the first step. All experiments were performed in triplicate (Fig. 1). Anti-type I collagen antibody exhibited less than 0.1% cross-reaction with type III collagen; anti-type III collagen antibody exhibited 4.1% cross-reaction with type I collagen. Further tests warranted the use of the three types of antibody in type-specific immunolabeling. Indeed antitype I, anti-type III, or antifibronectin fluorescence was extinguished by prior incubation of the antibody solution with the corresponding antigen, and was not extinguished by prior incubation with either of the two other antigens. Furthermore, the absence of disturbing cross-reactivity of anti-type III collagen antibody with type I collagen in the sections was attested by the fact that late embryonic bone or tendon, the collagenous content of which is known to be of type I only, did not show any positive reaction when treated with anti-type III collagen antibody, but fluoresced when treated with antitype I collagen antibody.

MAUGERETAL.

Immunofluorescent

Collagen and Fibronectin

in Embryonic

95

Feather Development

Staining

Specimens were frozen by immersion in either liquid nitrogen or liquid nitrogen-cooled liquid dichlorofluoromethane (CC&F,). Six-micrometer-thick cryostat sections were cut at -20°C air-dried, and immunolabeled at room temperature by the indirect method. Sections were immersed for 30 min in the antibody solution (1:20 to 1:4 dilution in PBS at pH 7.4 for anti-type I and antitype III antibody, 1:lOO to 1:2000 dilution for antifibronectin antibody), rinsed in PBS, and immersed for another 30 min in fluoroisothiocyanate (FITC)-labeled goat anti-rabbit IgG globulin (Institut Pasteur, Paris) solution (1:67 dilution) containing 70 pg/ml of Evans blue as a background counterstain, which emits a red fluorescence under uv illumination. Sections were mounted in buffered glycerin and observed with a Leitz Ortholux II fluorescence microscope equipped with epi-illumination and I2 filter combination (excitation band 450-490 nm, stop 510 nm). When, at specific stages of feather development (outgrowing and caudally reclining feather buds), the distribution of fluorescence was markedly heterogeneous, pairs of neighboring sections were used to test the accessibility of antigens in the tissues. One section of a given pair was treated by a 100 pg/ml or 1 mg/ml solution of hyaluronidase (Sigma Chemical Co., St. Louis, MO., Type I) in PBS at pH 7.4 for 30 min at room temperature before immunolabeling; the other one served as control and was pretreated with PBS alone. Photographs were taken on either Kodak Ektachrome 50 (push-processed to 200 ASA) or Kodak Ektachrome 200 professional color film. Exposure ranged from 20 set to 3 min. No attempts were made to gain quantitative data on the fluorescence , except when hyaluronidase-treated sections were compared with nontreated control sections; in the latter case, both sections were photographed with an equal exposure time. Black and white prints were obtained from the color transparencies via an internegative on Kodak professional negative orthochromatic film, where most red color was eliminated, leaving the FITC fluorescence against an almost uniformly black background. When necessary, the outer face of the epidermis was indicated on the internegatives by a dotted line of india ink, by following the contours of the red-stained epidermis, which were faintly but clearly visible in most internegatives, but did not show on the paper prints. In some internegatives the redstained epidermal contour was invisible; the dotted line was then drawn by projecting the transparency at the appropriate magnification onto the negative. RESULTS

Observations were made on feather-forming regions, such as the spinal, pectoral, and femoral tracts from 5

%,

b % 100.

t 0.01

0.1

1

10

100 mg/,

FIG. 1. Radioimmunoassay and cross-reactivity of anti-type I (a) and anti-type III collagen antibody (b). Abscissa: concentration of cold type I, or type III collagen. Ordinate: proportion (B/B,) of bound “:Ilabeled (a) type I collagen, and (b) type III collagen, as percentage of initial binding (B,,) to the antibody, used at a dilution of 1:40 (a) and 1:80 (b), respectively.

to 16 days of incubation. Featherless areas, or poorly feathered ones, outside the pterylae, were also observed for comparison. At all stages and with all tested concentrations of antibody the epidermis was constantly negative. In the dermis, fluorescent fibrous, lumpy, or granular structures were seemingly all extracellular, as far as could be judged from ordinary or phase-contrast-transmitted light microscopy. As a rule, labeled structures were

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coarser with anticollagen antibodies than with antifibronectin. Also, they were more closely pericellular with antifibronectin then with anticollagen antibody. Control sections treated with preimmune rabbit serum or with antibody solution previously absorbed with the corresponding antigen did not show any fluorescence after treatment with goat anti-rabbit FITC-labeled IgG. A large part of the features of anti-type-specific labeling is reported in the figure legends and illustrated by Figs. 2-40. Observations are summarized below in chronological order, with reference to the main steps of feather development, namely, the successive formation of feather tracts, feather rudiments, feather buds, and finally feather follicles feather filaments, (Sengel, 1976). Formation

of Feather

Tracts

Prospective feather tracts form, between 5 and 6 days of incubation, as zones where the dermal mesenchyme, by its increased cell density, becomes distinguishable from the looser mesenchyme of the underlying subcutaneous and adjacent featherless zones (Sengel, 1969). Before the densification of the predermal mesenthyme in the prospective spinal tract (at 5 days), antitype I collagen antibody labeled the subepidermal mesenchyme exclusively in its upper portion, over a thickness of approximately one or two cells, along the dermal-epidermal junction (DEJ) (Fig. 2). With anti-

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97

type III collagen antibody, labeling in the prospective spinal pteryla was not detected or very faint. With antifibronectin, sparse label was evenly distributed over the whole depth of predermal mesenchyme. Within differentiated feather tracts (at 5 days in the breast and thigh tracts, at 6 days in the spinal tract), anti-type I collagen antibody labeled the dense featherforming mesenchyme throughout its depth (Figs. 4 and 5). In the still nondensified lateral parts of feather tracts (Fig. 3) and in the prospective featherless areas lateral to the feather tracts (Figs. 4 and 8), only the uppermost part of the still loose predermal mesenchyme was labeled. With anti-type III collagen antibody, label was retained on loose fibrous material, predominantly located along the DEJ in the upper part of the dense dermis (Fig. 7). With antifibronectin, label was distributed over the whole depth of the dense dermis and revealed irregularly shaped fibrous or granular pericellular material (Fig. 6). Formation

of Feather

Rudiments

Feather rudiments appear first, at 7 days, in the middorsal lumbar region of the spinal tract and continue to appear in successive rows during subsequent development until they have all formed by 10 days of incubation. They consist of an epidermal placode and a lensshaped dermal condensation. In regions where rudiments start to form, the three

FIGS. 2-13. Indirect immunolluorescent labeling of types I and III collagen, and of fibronectin (FN) in the integument of 5- to Y-day embryos. Transverse sections. The outer surface of the entirely negative epidermis is indicated by a broken line. FIG. 2. Five-day prospective middorsal spinal pteryla. Antitype I collagen label is restricted to the uppermost part of the predermal mesenchyme (D), along the DEJ. Above the epidermis, the amnion (A), which was left in place to avoid damage to the tissues at freezing, is also labeled. x430. FIG. 3. Six-day

prospective glabrous region lateral to the thoracic spinal pteryla. Anti-type I collagen label is confined to the upper part of the dermis (D), underneath the DEJ. x430. FIG. 4. Six-day femoral skin: on the left, part of the prospective glabrous region (GR) lateral to the femoral pteryla (FP), about half of which is shown, on the right. In the prospective featherless region, anti-type I collagen marks the uppermost part of the dermis only, whereas in the prospective feather tract label extends over the whole depth of the dermis, and is scarce underneath the DEJ. x430. FIG. 5. Still unfeathered lateral region of the 7.day spinal pteryla. Density of anti-type I label, which extends over the whole depth of the dermis, is higher than that within feather rudiments (compare with Fig. 10). X430. FIG. 6. Six-day spinal pteryla. Dermis (D) and subcutaneous mesenchyme (SM) are labeled with antifibronectin antibody, mostly in discrete specks and granules. X430. FIG. 7. Six-day spinal pteryla. Anti-type III collagen label is sparse and restricted to the upper part of the dermis (D), underneath the DEJ. X430. FIG. 8. Seven-day glabrous region lateral to the spinal pteryla. Anti-type I collagen label remains restricted to the uppermost part of the dermis (D), underneath the DEJ (compare with previous stage, Fig. 3). X430. FIG. 9. Dorsal interplumar skin. Note high density of fluorescent anti-type I collagen-labeled material throughout thickness of dermis (compare with feather rudiment, Fig. 10). X430. FIGS. 10-12. Feather rudiments in the spinal pteryla, at 7 (Figs. 10 and 11) and 9 days (Fig. 12). FIGS. 10, 11. With anti-type I collagen (Fig. lo), the dermal condensation (DC), underneath the epidermal feather placode (EP), is labeled throughout its thickness, more densely close to the DEJ than the deeper dermis, whereas with anti-type III collagen (Fig. 11) fluorescent material in the dermal condensation (DC) in sparse. Note, in Fig. 10, a few fibers oriented at right angle to the DEJ in the middle of the feather condensation. X335. FIG. 12. With antifibronectin, label is predominant in the dermal feather condensations (DC) and around blood vessels (BV), whereas it is sparse in interplumar dermis (ID). Note almost continuous label of the DEJ. X135. FIG. 13. Higher magnification of the distribution of antifibronectin label in interplumar skin of the A-day spinal pteryla. Note granular appearance of label and higher density underneath the DEJ. X335

MAUGER ET AL.

Collagen and Fibronectin

kinds of antibody labeled the whole dermis. With antitype I and antitype III collagen antibody, label became less dense within the dermal feather condensations (Figs. 10 and 11) than in th.e adjacent interplumar dermis (Fig. 9). With antifibronectin, the dermal condensations were more heavily labeled than interplumar skin (Figs. 12 and 13). Formation

of Feather

Buds

When rudiments begin to bulge out as convex domes they give rise to feather buds. With anti-type I and antitype III collagen antibody, the center and apex of the bud mesenchyme contained less fluorescent material than its periphery and its base (Figs. 14,15, and 17-19), while the dermis in interplumar skin was heavily stained throughout its thickness. Subcutaneous mesenchyme was almost devoid of positive material. With antifibronectin antibody, label was almost exclusively restricted to the dermal feather condensation, with a maximum of density along the DEJ, while interplumar dermis was almost completely devoid of label (Figs. 16, 20, and 21). In non-feather-forming regions (not shown), with anti-type I and anti-type III antibody, only the upper dermis was fluorescent over a depth of some two to three cells. With antifibronectin antibody, label was scarce. During subsequent development, feather buds begin to slant, assuming their typical cranial-caudal (or, on limbs, proximal-distal) asymmetry. In longitudinal sections of buds, the three kinds of antibody labeling revealed a corresponding cranial-caudal asymmetry in the dermal component of the bud. Characteristically, with anticollagen antibodies th.e label was restricted to the base of the reclining bud, while with antifibronectin antibody, label extended from base to apex of the dermal core in an increasing basal-apical gradient (Figs. 23-25). With the two anticollagen antibodies interplumar skin was densely fluorescent, particularly along the DEJ (Fig. 26 for anti-type I label; not shown for anti-type III label); by contrast, with antifibronectin antibody,

in Embryonic

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Feather Development

label in interplumar skin was almost nonexistent, except a thin fluorescent line underneath the DEJ and around blood vessels (Fig. 27). In order to ascertain whether or not the heterogeneous distribution of label in outgrowing and caudally reclining feather buds was an artifact possibly due to masking of part of the collagen by glycosaminoglycans, IO-day dorsal skin sections were treated by hyaluronidase prior to immunolabeling. Hyaluronidase treatment did not reveal any additional labeling when compared with adjacent nontreated control sections, nor did it change the typical heterogeneous pattern (Figs. 22a,b and 24). In most sections labeled with anticollagen antibodies, hyaluronidase treatment, however, enhanced the intensity of fluorescence. Formation

of Feather

Filaments

and Feather

Follicles

As feather buds elongate, they transform into barb ridge-forming feather filaments, the base of which grows inward into the skin in a cylindrical fashion. At this sbage, the asymmetrical cranial-caudal distribution of fluorescent material was no longer visible. The label of the dermal core of feather filaments decreased in density with both anticollagen antibodies from base to apex, while it increased with antifibronectin antibody (Figs. 28-30 and 32). With anticollagen antibodies, the dermis of the follicle wall was relatively poorly labeled in comparison with the densely fluorescent interfollicular dermis (Figs. 30-32). By contrast, with antifibronectin antibody, label in the follicular dermis was heavier than that in the interfollicular dermis (Figs. 29 and 33). In the portion of the feather filaments rising above the skin, the DEJ around barb ridges in formation was thinly underlined with anticollagen as well as with antifibronectin antibody; in more distal sections, however, label around barb ridges was very faint or absent (Figs. 28 and 29). Blood vessel walls were strongly labeled with antifibronectin (Figs. 29 and 33), more lightly with anticollagen antibody (Figs. 28 and 32). In the subcutaneous mesenchyme, label was rare, if not entirely ab-

FIGS. 14-22. Indirect immunofluorescent labeling of types 1 and III collagen, and of fibronectin (FN), in feather rudiments and feather buds of the 8. to lo-day spinal pteryla. Transverse (Figs. 14-21) and longitudinal (cephalic is to the left) sections (Fig. 22). The outer surface of the entirely negative epidermis is indicated by a dotted line. X230. FIGS. 14-17. In feather rudiments, density of anti-type I (Fig. 14) and anti-type III collagen label (Figs. 15 and 17) is lower in the central and apical parts of the dermal condensations (DC) than that at their periphery or in the adjacent interplumar dermis (ID). On the contrary, with antifibronectin (Fig. 16), label is almost entirely restricted to the dermal condensation (DC). Sections in Figs, 14 and 15 are nondiametrical and pass through the cranial or the caudal half of feather rudiment. Sections in Figs. 16 and 17 are close to diametrical. FIGS. 18-22. In feather buds, with anti-type I collagen antibody (Figs. 18 and 19), density of label is maximal at the base and periphery, and minimal in the center and apex of the dermal core; with antifibronectin antibody (Figs. 20 and 21), density of label is maximal in the dermal condensation and particularly strong along the apical DEJ and blood vessels (BV), whereas it is minimal or inexistent in surrounding interplumar dermis (ID). Figures 18 and 21 show sections passing through apex of buds; Figures 19 and 20 show sections through subapical parts of buds. FIG. 22. Neighboring sections of the same lo-day feather bud. Section a was treated with 1 mg/ml hyaluronidase solution prior to immunolabeling with anti-type I collagen antibody. Section b is the untreated control. Both photographs were equally exposed (113 set). The overall distribution of fluorescent material is unchanged, although fluorescence appears enhanced, in the hyaluronidase-treated section.

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sent. In the underlying dorsal skeletal muscle masses, epimysial and perimysial envelopes contained discontinuously organized fluorescent material (Fig. 28). Between 13 and 16 days in the spinal pteryla, the intracutaneous feather f’ollicle almost terminally differentiates and becomes associated with a complex array of feather (smooth) muscle bundles, which occupy part of the subcutaneous space comprised between the dermis and the dorsal skeletal muscles. Interfollicular dermis was heavily labeled with the two types of anticollagen antibody, while the dermal core of feather filaments and the dermal papilla at their base were less densely labeled, except for the blood vessel walls (Figs. 36, 37, and 40); the dermis of the follicle wall exhibited a density of fluorescent material which was intermediate between the heavy interfollicular and light intrafilament label. With antifibronectin antibody, the follicle wall and dermal papilla of feather filaments were more strongly labeled than the almost completely negative interfollicular d.ermis (Figs. 34, 35, 38, and 39). In the feather muscle bundles, the epimysial envelope was brightly labeled with anti-type I (not shown) and anti-type III collagen antibody (Fig. 40). In some seemingly more advanced bundles, the perimysium was also labeled (not shown). DISCUSSION

The present immuno--histological interstitial collagen and fibronectin

study reveals that are not uniformly

in Embryonic

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101

accumulated in the dermal extracellular matrix during skin development. The heterogeneous distribution is not due to partial and localized masking of collagen molecules by proteoglycans, since treatment of the sections by hyaluronidase did not change the distribution pattern nor reveal any new binding sites of the antibodies. In the differentiating feather tracts the distribution of type I collagen changes from an exclusive thin underlining of the dermal-epidermal junction to an extension over the whole depth of the dermis. Significantly, outside the prospective feather-forming regions collagen remains restricted to the underside of the DEJ. It is therefore likely that the loosening of the distribution of collagen and its removal from the DEJ are related to the acquisition of feather-forming capacity by the dermis. On the contrary, the maintenance of the continuous DEJ collagen deposits might characterize stabilized non-feather-forming skin regions or those that will form feathers at later stages only. Type III collagen appears about 24-48 hr later than type I and is also at first restricted to the uppermost part of the dermis. Fibronectin becomes deposited around dermal cells and along the DEJ in increasing amounts as the dermis matures. In t,he dermal condensation of feather rudiments, as well as in the dermal core of outgrowing feather buds, types I and III collagen become sparse and virtually disappear along the DEJ, while at their basal periphery and in interplumar skin density of collagen

FIGS. 23-31. Indirect immunofluorescent labeling of types I and III collagen, and of fibronectin (FN) in asymmetrical feather buds (Figs. 23325), feather filaments (Figs. 26-30), and feather follicles (Figs. 28-31). Transverse (Fig. 28) and longitudinal (cephalic is to the left) sections (Figs. 23-27, 29-31). The outer surface of the entirely negative epidermis is indicated by a broken line. FIGS. 23-25. Asymmetrical distribution of labeled material in the dermal core (D) of slanting buds in the lo-day spinal (Figs. 23-24) and 12-day crural (Fig. 25) pterylats. X210. FIGS. 23, 24. With anti-type I (Fig. 23) and anti-type III collagen antibody (Fig. 24), labeled material is predominant at the base of the bud, and distributed according to a decreasing (type I) or increasing (type III) craniallcaudal density gradient; the apex of the buds is almost entirely devoid of label, Note: section in Fig. 24 was treated with hyaluronidase (1 mg/ml) prior to immunolabeling. FIG. 25. With antifibronectin, density of labeled material is maximal at the apex and around caudal base of bud, whereas it is sparse under its cranial slope. Blood vessels (BV) are conspicuously marked. FIGS. 26, 27. Lower magnification (X85) of reclining early feather filaments from the lo-day spinal (Fig. 26) and 12-day crural (Fig. 27) pterylae, illustrating the difference in density of labeled material with anti-type I collagen (Fig. 26) and antifibronectin antibody (Fig. 27) between the cranial and the caudal base, and between the dermal core (D) of the filament and the interplumar dermis (ID). BV, blood vessels. FIG. 28. Overview of part of the 12.day spinal pteryla together with subcutaneous tissues, labeled with anti-type I collagen antibody. The interplumar dermis (ID) is brightly fluorescent, while labeled material is sparse at the base of the feather filaments (filaments 1 and 3) and in their dermal core (D). The DEJ inside the feather filaments is but faintly marked around barb ridges (BR) in filament 2, cut transversely at about the middle of its length, and also underneath the still undifferentiated epidermis of filament 3; in filament 4, cut near its apex, the DEJ around the differentiating barb ridges (BR) is not labeled. The subcutaneous mesenchyme (SM) is almost devoid of fluorescent material, in contrast to the underlying skeletal musculature (MU). X85. FIG. 29. Overview of part of the 13.day spinal pteryla, labeled with antifibronectin antibody. The interplumar dermis (ID) is but sparsely labeled, except along the DEJ, while the follicular dermis (FD) and dermal core (D) are more densily labeled. Note conspicuous label around barb ridges (BR) and blood vessels (BV). X85. FIG. 30. Fourteen-day interplumar skin, feather follicle and lower part of feather filament in the spinal pteryla, labeled with anti-type I collagen antibody. The interplumar dermis (ID) is heavily packed with fluorescent material, while the follicular dermis (FD) is less densely labeled and over a lesser depth. Fluorescent material is abundant at the base of the filament (future dermal papilla, DP), and becomes sparser as one moves toward its apex, where it is completely absent. The DEJ inside the lower part of the feather filament is clearly marked. In the subcutaneous mesenchyme (SM), density of fluorescent material is low in comparison to that of interplumar dermis. X110. FIG. 31. Section passing tangentially through the edge of a 14.day feather follicle, labeled with anti-type I collagen antibody, to show the transversely oriented circular fibers in the follicle wall. Note also the difference in density of fluorescent material between the interplumar dermis (ID) and the follicular dermis (FD). The “black hole” corresponds to a sliver of tangentially cut epidermis (E) from the follicle wall. X270.

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deposits increases. This :is indicative of a possible stabilizing role of collagen in skin, and is reminiscent of similar processes in other developing organs, like the salivary gland and lung (Grobstein and Cohen, 1965; Bernfield and Wessells, l970; Wessells, 1970; Spooner and Faubion, 1980; Bern.field, 1981). By contrast, distribution of fibronectin in feather rudiments and feather buds is almost complementary to that of interstitial collagen: the dermal condensation becomes enriched in fibronectin, with a maximum along the DEJ, while in interplumar skin fibronectin becomes scarce. During subsequent development, when feather buds acquire a cranial-caudal (or proximodistal) asymmetry, the distribution pattern of the three types of antigen becomes specifically asymmetric. A similar heterogeneity was also described for type I collagen and type III procollagen in the pulp of the mouse tooth germ (Lesot et al., 1978). It seems straightforward to relate the asymmetrical distribution of collagens and fibronectin in the dermal core of feather buds to their orientation with respect to the cephalocaudal axis of the embryo. It is noteworthy that sulfated proteoglycans in outgrowing buds likewise exhibit a decreasing basal-apical and cranial-caudal distribution gradient (Sengel et al., 1962). Thus type I collagen and sulfated proteoglycans might offer a stabilized base on which the cranial epidermis of the bud can grow out at a faster rate than the caudal epidermis. The distribution of type III collagen fibers with a max-

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imum around the caudal base of the feather bud may likewise be interpreted as the formation of a firm foundation over which the growing bud leans backward. The high density of fibronectin in the dermal core might promote cell movement during outgrowth. It is important to remember that this orientation of feathers is strictly epidermis dependent, as demonstrated by epidermal-dermal recombination experiments, where the cephalocaudal axis of the epidermis was turned by 180” or 90” with respect to that of the dermis (Sengel, 1958; Novel, 1973). If a causal relationship holds between the heterogeneous distribution of extracellular matrix constituents and the orientation of feathers, it would mean that the deposition of these macromolecules in the matrix is under epidermal control. The terminal differentiation of the feather filament is characterized by the ingrowth of the feather follicle at the base and by the formation of barb ridges in the portion remaining above the surface of the skin. Interestingly, the asymmetrical cranial-caudal distribution of collagens and fibronectin at the base of the filament vanishes as the follicle forms. The decreasing basal-apical gradient of collagens and increasing basal-apical gradient of fibronectin, however, persist throughout the elongation of the feather filament, with maximal density of collagen fibers at the periphery of the follicle base; again, the collagen pattern closely resembles that of sulfated proteoglycans (Sengel et al., 1962). Inside the upper portion of feather filaments, type I and type III

FIGS. 32-40. Indirect immunofluorescent labeling of types I and III collagen, and of fibronectin (FN) in the skin of 13- to 16-day embryos. Transverse (Figs. 35 , 36, 38, and 39) and longitudinal (cephalic is to the left) sections (Figs. 32-34, 37, and 40). The outer surface of the entirely negative epidermis is indicated by a broken line. FIGS. 32, 33. Thirteen-day feather follicles. FIG. 32. With anti-type III collagen antibody, label is restricted to interfollicular dermis (ID), blood vessel walls (BV), and subcutaneous musculature (MU). The follicular dermis (FD) is barely labeled, and the dermis inside the feather filament (D) is almost completely negative. X85. FIG. 33. With antifibronectin antibody, fluorescent material is dense in the dermal core (D) of the feather filament, when compared with the sparse label of interfollicular dermis (ID). In the follicle wall (FD), heavy label is confined to the upper (subepidermal) part of the dermis, along the DEJ. BV, blood vessels. x170. FIG. 34. With antifibronectin antibody, distribution of fluorescent material in this 16.day feather follicle is similar to that at the earlier stage (see Fig. 33), with the difference that now interfollicular dermis (ID) is completely devoid of label. DEJ, dermal-epidermal junction of follicle wall. X170. FIG. 35. Dermal papilla (DP) at the base of a 16.day feather follicle, labeled with antifibronectin antibody. Note the presence of label in the papilla, in the upper (subepidermal) part of the follicular dermis (FD), along the DEJ, and between barb ridges (BR). x210. FIG. 36. Transversely cut 14.day feather filament, labeled with anti-type I collagen antibody (compare with Fig. 39). The DEJ around barb ridges (BR) is thinly underlines& compared with the heavily packed label of underlying interplumar dermis (ID). Besides the central main blood vessel with its fluorescent rim, the poorly labeled dermal core (D) contains several other patchy fluorescent structures associated with smaller blood vessels. ~335. FIG. 37. Sixteen-day feather follicle labeled with anti-type I collagen antibody. Note high density of labeled material in interplumar dermis (ID) and comparatively lower density in follicular dermis (FD) and dermal papilla (DP). FM, feather muscle bundles. X210. FIG. 38. Thirteen-day feather follicle labeled with antifibronectin antibody. Note almost complete absence of labeled material in the interplumar dermis (ID), compared to the distinctly labeled follicular dermis (FD) and dermal papilla (DP). x210. FIG. 39. Transversely cut 13-day feather filament, labeled with antifibronectin antibody (compare with Fig. 36). Interplumar dermis (ID), dermal core of filament (D), ancl DEJ around barb ridges (BR) are faintly fluorescent, compared to the bright rim of the central blood vessel (BV). x210. FIG. 40. Overview of skin and subcutaneous tissues, with base of feather follicle and feather muscle bundles (arrows), labeled with anti-type III collagen antibody. Note the difference of label density between brightly fluorescent interplumar dermis (ID), less densely marked follicular dermis (FD), and faintly fluorescent dermal papilla (DP). E, epidermis of feather follicle. x95.

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collagens are rather sparse, except along the DEJ of fully developed barb ridges whose basement membrane is underlined by a thin and continuous deposit mainly of type I collagen. In younger barb ridges, which are still in the process of being formed, collagen is less abundant along the DEJ. Thus again, collagen appears to be laid down preferentially in zones to become stabilized, while it is absent or less dense where morphogenetic movements are still under way. Conversely, fibronectin is most abundant in the dermal core of feather filaments, where barb ridges are being formed. In short, in interplumar dermis, the two types of interstitial collagen are most abundant and fibronectin is rare, while an attenuated density of collagenous fibers and a high content of fibronectin are maintained around the follicle wall and inside the feather filament, throughout the period of their ingrowth and elongation. These observations confirm those of Kitamura (1981) and extend them to type III collagen as well as to stages of feather development beyond the early feather bud. They have been extended recently to scale-forming tarsometatarsal skin, and to abnormal glabrous skin from hydrocortisone-treated or scaleless mutant chick embryos (Mauger et al., 1982). They can be interpreted as follows. At sites where morphogenetic movements are actuated, as in the bulge of the outgrowing bud, a rigid collagenous framework would be a hindrance. On the contrary, in regions where histological stability of the integument is needed, as in the interplumar skin or precisely along the peripheral base of buds, the collagenous fibrous architecture might offer a firm foundation for the centrally located outgrowing tissues. Thus the fibrous collagenous deposits in the dermal extracellular matrix might exert a negative site-specific control on an innate propensity of skin to grow out. Localized production of collagenases and/or other hydrolases might liberate the prospective bud zones from their initial collagen-dependent rigid restraint. The accumulation of fibronectin in the morphogenetically active regions might exert a movement-promoting action on dermal as well as on overlying epidermal cells. This interpretation is in agreement with the view, gained from the observation of other morphogenetic systems, that fibronectin accumulates in regions of high cell motility (Critchley et al., 1979; Newgreen and Thiery, 1980; Mayer et al., 1981) and that collagen is deposited in regions to be immobilized (Bernfield, 1981). It is supported by results of in. vitro cell cultures, which show that fibronectin has a stimulating effect on neural crest cell movement (Greenberg et al., 1981), whereas collagen substrates slow down neural crest cell migration (Davis and Trinkaus, 1981) and stabilize the basal lamina of mammary epithelial cells (David and Bernfield, 1979). In conclusion, the spatial microheterogeneity of the

VOLUME 94. 1982

dermal collagenous network and of extra- and pericellular fibronectin might represent part of the morphogenetic message that the dermis is known to transmit to the epidermis during skin and feather development. Zones of histological stability are characterized by early, increasing deposits of interstitial collagen and by sparse production of fibronectin, whereas zones of morphogenetic activity become enriched in fibronectin and remain relatively deprived of interstitial collagens until the cutaneous appendages have finally completed their morphogenesis. The authors are grateful for the expert assistance of Chantal Buffevant (Centre de Recherche de Dermobiochimie, Lyon), Christian Repiquet, and Mireille Heyden (Institut Pasteur, Lyon), Yolande Bouvat, and Jacqueline Lana (Laboratoire de Zoologie et Biologie animale, USM Grenoble). This work was supported by a research grant from the DGRST (BRD 79-7-1220), by the CNRS (ERA Nos. 621 and 819, and RCP No. 533), and by the Universitk scientifique et medicale de Grenoble. REFERENCES BERNFIELD, M. R. (1981). Organization and remodeling of the extracellular matrix in morphogenesis. In “Morphogenesis and Pattern Formation” (T. G. Connelly, L. L. Brinkley, and B. M. Carlson, eds.), pp. 139-162. Raven Press, New York. BERNFIELD, M. R., and WESSELLS, N. K. (1970). Intra- and extracellular control of epithelial morphogenesis. Deu. Biol. (Suppl.), 4, 195-249. BYERS, P. H., MC KENNEY, K. H., LICHTENSTEIN, V. P., and MARTIN, G. R. (1974). Preparation of type III procollagen and collagen from rat skin. Biochemistry 13, 5243-5247. CRITCHLEY, D. R., ENGLAND, M. A., WAKEI,Y, J., and HYNES, R. 0. (1979). Distribution of fibronectin in the ectoderm of gastrulating chick embryos. Nature (London) 280, 498-500. DAVID, G., and BERNFIELD, M. R. (1979). Collagen reduces glucosaminoglycan degradation by cultured mammary epithelial cells: Possible mechanism for basal lamina formation. Proc. Nat. Acad. Sci. USA 76, 786-790. DAVIS, E. M., and TRINKAUS, J. P. (1981). Significance of cell-to-cell contacts for the directional movement of neural crest cells within a hydrated collagen lattice. J. Embryol. Exp. Morphol. 63, 29-51. DEMARCHEZ, M. (1980). “DBveloppement de la peau et des phaneres chez le poulet: Etude biochimique et immunochimique du collag&ne et analyse ultrastructurale de la jonction dermo-Bpidermique, chez l’animal normal ou traiti! d l’hydrocortisone.” These de doctorat de sp&ialit& de Biologie cellulaire, Universitk scientifique et medicale de Grenoble, France, October 31, 1980. DODSON, J. W. (1967). The differentiation of epidermis. I. The interrelationship of epidermis and dermis in embryonic chicken skin. J. Embryol. Exp. Morphol. 17, 83-105. ENGVALL, E., and RUOSLAHTI, E. (1977). Binding of soluble form of fibroblast surface protein, fibronectin to collagen. Znt. J. Cancer 20, l-5. GOETINCK, P. F. (1970). Epithelial-mesenchymal interactions and collagen synthesis in the establishment of feather patterns in the chick embryo. J. Cell Biol. 47, 72a. GOETINCK, P. F., and SEKELLICK, M. J. (1970). Early morphogenetic events in normal and mutant skin development in the chick embryo and their relationship to alkaline phosphatase activity. Deu. Biol. 21, 349-363.

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