Development 100, 501-512 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

501

The structure and distribution of proteochondroitin sulphate during the

formation of chick embryo feather germs

KUNIO KITAMURA

Department of Developmental Biology, Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo, Japan

Summary

The dorsal skin of the chick embryo, in which feathergerm forms, was found to synthesize two proteochon-droitin sulphates, PCS-I and PCS-II and a proteo-heparan sulphate, PHS. A monoclonal antibody(I3B9) was prepared against PCS-I, a higher molecu-lar weight proteochondroitin sulphate. Distributionof PCS-I was immunohistochemically studied usingI3B9. PCS-I was found in the epidermis, basementmembrane and superficial dermis prior to formationof feather rudiments. As the feather rudimentsformed, PCS-I was noted in a condensed area ofdermal cells and in the basement membrane, whilePCS-I decreased remarkably in the epidermal pla-code. The formation of feather buds resulted in adecrease in PCS-I in the region of dermal conden-sation and the basement membrane situated above

this region. PCS-I was asymmetrically distributed inthe feather filaments. The turnover of proteochondro-itin sulphate was studied using autoradiography of[35S]sulphate. Proteochondroitin sulphate in the base-ment membrane and condensed dermis of the featherrudiments showed very slow turnover. On the otherhand, the outgrowth of feather buds caused rapidturnover of proteochondroitin sulphate in the regionof dermal condensation and basement membranesituated above this region. The mechanism for theuneven distribution of PCS-I during feather germformation is discussed.

Key words: proteochondroitin sulphate, feather germ,epidermal placode, dermal condensation, basementmembrane, chick embryo.

Introduction

The feather germ in chick embryo skin is formedthrough epidermal-dermal interactions (Sengel,1976), which cause elongation of epidermal cells(epidermal placode) and condensation of dermal cells(dermal condensation). The distribution patterns ofvarious components of the extracellular matrix duringfeather germ morphogenesis have been immunohis-tochemically elucidated by our group and that ofSengel (Kitamura, 1981; Mauger et al. 19826). Col-lagen types I and III decreased in the region ofcondensation and increased in the surrounding regionof condensation. Fibronectin, however, was found inthe former region. Furthermore, basement mem-brane components, such as laminin and collagen typeIV, showed uniform distribution during feather germformation (Mauger et al. 1982a).

Proteoglycan, a molecule classified according tothe type and distribution of glycosaminoglycans at-tached to the protein core, is also a major extracellu-

lar matrix component (Hascall & Hascall, 1981;Heinegard & Paulsson, 1984). The type, amount anddistribution of glycosaminoglycan or proteoglycanare known to change during tissue and organ develop-ment. Proteochondroitin sulphate changes in theserespects with the formation of cartilage (Kitamura& Yamagata, 1976; Okayama, Pacifici & Holtzer,1976; Shiomura et al. 1984; Matsui, Oohira, Shoji &Nogami, 1986; Kimata et al. 1986). Regional changesin glycosaminoglycan in the basal lamina during thedevelopment of mouse embryonic salivary and sub-mandibular glands have been reported (Bernfield &Banerjee, 1972, 1982). Furthermore, the distributionprofile of proteoheparan sulphate in the basementmembrane changes during mouse kidney tubule andtooth development (Ekblom, 1981; Thesleff et al.1981).

The present study was conducted so as to gain someunderstanding of the structure and distribution ofproteoglycan during feather germ formation. In thisresearch, the chick embryo dorsal skin was found to

502 K. Kitamura

synthesize three types of proteoglycans and, withadvancement of morphogenesis, significant changesin the distribution pattern of the major type ofproteoglycans take place.

Materials and methods

AnimalsFertilized eggs of White Leghorn were obtained commer-cially and incubated at 37°C until use.

Labelling and extractionThe dorsal skin of an 8-day-old embryo, placed on aMillipore filter (HABP04700, Millipore), was labelled for8h with [35S]sulphate (carrier free, New England Nuclear)at 50^Ciml~' in Eagle's MEM containing 10% horseserum and 5 % chick embryonic extracts. The dorsal skinwas detached from the filter and proteoglycan extractedovernight at 4°C with 5 vol. of 20 mM-Tris-HCl buffer,pH7-0 (containing 4M-guanidium chloride, O-lM-6-amino-hexanoic acid, 10mM-EDTA, 5mM-benzaminidine, 10mM-/V-ethylmaleimide and 1 mM-PMSF) by slow rotation. Thesuspension was centrifuged at 20 000 revs min"1 for 30minat 4°C and the residue reextracted with 2 vol. of the samebuffer. The suspension was centrifuged as above. Thesupernatants were combined and macromolecules precipi-tated by the addition of 3 vol. of 95% ethanol containing1-3 % potassium acetate. The precipitates were suspendedin 2 vol. of water and precipitated with ethanol to removeguanidium chloride.

Column chromatographyThe precipitates from the embryonic skin extract weredissolved with 20 mM-Tris-HCl buffer, pH7-3 (containing7M-urea, 0-2% (w/v) Triton X-100, 0-1 M-aminohexanoicacid, IOITIM-EDTA, 5mM-benzaminidine, 10mM-A'-ethyl-maleimide and 1 mM-PMSF) and applied onto a DEAE-Sephacel column (l-4xl0-0cm, Pharmacia) equilibratedwith the same buffer. The column was eluted by a lineargradient of NaCl (0-05-0-80 M). The [35S]sulphate-contain-ing fractions were separately pooled and precipitated withethanol. The precipitates were dissolved with 50mM-Tris-HCl buffer, pH 70 (containing 4 M-guanidium chloride and0-2 % (w/v) Triton X-100) and applied onto a SepharoseCL-2B or 6B column (l-0x94-0cm, Pharmacia) equilib-rated with the same buffer. The [35S]sulphate-containingfractions were pooled and precipitated with ethanol.

Alkali treatmentThe precipitates of the fractions obtained from the columnchromatography on Sepharose CL-2B and -6B were treatedwith 0-2M-sodium hydroxide for 20 h at 4°C. The solutionwas cooled in an ice bath and neutralized with 1 M-aceticacid. Following the addition of chondroitin-4-sulphate andchondroitin-6-sulphate as carriers, the sample solution wasprecipitated with ethanol and applied onto a SepharoseCL-6B column.

Chondroitinase A BC digestionThe precipitates of the fractions obtained from alkalitreatment followed by gel chromatography were dissolvedin 50 mM-Tris-HCl buffer, pH8-0, and digested with chon-droitinase ABC (final concentration 0-2i.u. ml"1, Seika-gaku Kogyo) for 20 h at 37°C. The digest was applied onto aSepharose CL-6B column.

Nitrous acid degradationFractions not digested by chondroitinase ABC were pooledand precipitated with ethanol. The precipitate was dis-solved in a solution made of equal volumes of 3-6 M-aceticacid and 0-48M-sodium nitrite and allowed to react for100 min at room temperature (Stow, Glasgow, Handley &Hascall, 1982). 0-5M-ammonium sulphamate was added tothe reaction mixture and the sample was then precipitatedwith ethanol and applied onto a Sepharose CL-6B column.

Estimation of chondroitin-4-sulphate and chondroitin-6-sulphateThe relative amounts of 4- and 6-sulphated disaccharides inthe chondroitinase ABC digest of the released glycos-aminoglycans were estimated by the procedure of Saito,Yamagata & Suzuki, 1968.

Isolation and characterization of proteoglycan fromepidermis and dermisThe dorsal skin of an 8-day-old embryo was treated with0-25% EDTA in phosphate-buffered saline (PBS) for10 min at 0°C and the epidermis was dissociated from thedermis with a needle. Epidermis and dermis were labelledseparately with [•"SJsulphate as above. Proteoglycan wasextracted from the labelled epidermis and dermis. DEAE-Sephacel column chromatography, gel chromatography onSepharose CL-2B and -6B and chondroitinase ABC-diges-tion were carried out as above.

Production of monoclonal antibodiesMonoclonal antibodies were prepared according to Galfre& Milstein, 1981. Female Balb/c mice were immunizedwith PCS-I (see Results) purified from the dorsal skin. Thefirst injection (150^g of PCS-I in complete Freund's adju-vant) were administered subcutaneously at several sitesalong the flank. Three booster shots (100^g of PCS-I inincomplete Freund's adjuvant) were administered intraper-itoneally at one month intervals. 4 days prior to its beingsubjected to hybridoma fusion, one mouse was adminis-tered an intravenous injection of 100ng of PCS-I without anadjuvant. This mouse was then sacrificed under a CO2atmosphere and the spleen cells (4X108 cells) were fusedwith mouse myeloma SP2/0 cells (8xlO7 cells) by theaddition of lml of 50% polyethylene glycol 4000 (Merk).The fused cells were suspended in the DME-mediumsupplemented with 15 % (v/v) precolostrum newborn calfserum, 100^M-hypoxanthine, 16f/M-thymidine, 50i.u. ml"1

penicillin and 100 fig ml"1 streptomycin, and plated on six96-well Coaster plates. On the following day, the mediumwas changed to HAT-medium prepared by the adding of0-4ftM-aminopterin to the medium described above. After10 days, screening was conducted for PCS-I by an enzyme-linked immunosorbent assay (ELISA). For the assay,

Proteochondroitin sulphate in chick embryo feather germs 503

0-2 jug of PCS-I was coated on each well of a 96-wellmicrotitre plate (Dynatech, Immulon II). After blockingthe plate with DME-medium (containing 15 % precolos-trum new-born calf serum and 15% rabbit serum), theculture supernatant was introduced into each well andincubated at 37°C for 2h, followed by washing in fivechanges of 10 mM-phosphate buffer, pH7-4 (containing0-15M-NaCl, 0-05% Tween 20 and 0-1% BSA). Peroxi-dase-conjugated anti-mouse IgG (Dako) was then added toeach well and followed by incubation at 37 °C for 1 h. Eachwell was washed as above. The peroxidase complex wasreacted with ABTS (Zymed). Well absorbance at 420 nmwas determined with an ELISA plate reader (Corona Co.).The cell line 'I3B9' (see Results) has been cloned by thelimiting dilution method. The subclass of the monoclonalantibody 'I3B9' was determined using a mouse monoclonaltyping kit (Serotec). The specificity of I3B9 was examinedby inhibition ELISA under nonequilibrium conditions(Rennard et al. 1980). The proteoglycan, collagen andfibronectin to be examined were dissolved in 0-02M-sodiumphosphate, 0-15M-NaCl, pH7-2 (200/igml"1), and serialdilutions of these solutions were added to the culturesupernatant obtained from the cloned I3B9 cells (diluted1:5 with PBS) and reacted overnight at 4°C. Portions (50fil)of the mixture were added to the PCS-I-coated wells andincubated for 2h at 37 °C to allow the remaining freeantibody to bind. Succeeding steps in the assay wereperformed as described above. Proteoglycan 'PGH' waspurified from the epiphyseal cartilage of a 13-day-oldembryo (Kimata et al. 1971). Proteoglycan 'PGM' waspurified from the limb buds of a 4-day-old embryo (Kita-mura & Yamagata, 1976). Type I collagen was purified fromthe cranial bones of a 17-day-old embryo (von der Mark,von der Mark & Gay, 1976). Type IV collagen was purifiedfrom chicken gizzard (Mayne & Zettergren, 1980).

Immunohisto chemistryThe dorsal skin of a chick embryo was fixed with 3-5 %formaldehyde in PBS for 30min at 0°C, impregnated with30 % sucrose, embedded in Tissue Tek II compound (MilesScientific), frozen and then cut into 10fim thick sectionswith a cryostat. The frozen sections were placed on apolylysine-coated glass slide and stored at —20°C. Theywere then washed with PBS at room temperature to removethe compound. The epidermal sheet dissociated from thebasement membrane and dermis was fixed with 96 %ethanol and placed on a gelatin-coated glass slide. Both thesections and epidermal sheets were treated with the culturesupernatant of I3B9 for 1 h at 37 °C, followed by washing infive changes of PBS. The secondary antibody consisting ofFTTC-conjugated Ig fractions of goat anti-mouse IgG(diluted 1:100 in PBS containing 1% BSA, Cappel) wasmade to react with each section and sheet for 30min in thedark. The slides were rinsed as above and then mountedusing buffered glycerol. The slides were viewed with a Zeissstandard microscope equipped with a IV FI epifluorescencecondenser (Carl Zeiss). Photographs were taken usingKodak Tri-X film of ASA 400. Some sections were treatedwith 0-1 % (w/v) testicular hyaluronidase (Type IV, Sigma)in PBS for lh at 37°C prior to reaction with I3B9. The

treated sections were washed three times with PBS andsubjected to immunofluorescent staining as above.

RadioautographyThe dorsal skin of various stages was labelled for 1 h with[35S]sulphate at SO^Ciml"1 by the procedure describedabove. For pulse labelling, the labelled skin was washedand immediately fixed for 30 min in Bouin's solution. In thepulse-chase labelling experiments, the labelled skin waswashed and its organ culture continued in [35S]sulphate-freemedium. At a specified time, it was washed again and fixedas above. The fixed skin was dehydrated and embedded inParaplast (Sherwood Medical) and sectioned at 5/j.m. Theslides carrying the sections were dipped into an autoradio-graphic emulsion (Sakura NR-M2, Konishiroku) followedby exposure for 2 weeks in the dark at 4°C. Using atemperature-controlled water bath at 20°C, the slides weredeveloped for 4 min with Konidol X (Konishiroku) andfixed for 8 min with Konifix X (Konishiroku). After washingwith tap water, they were dehydrated and mounted withPermount (Fisher Scientific). Before dipping the slides inthe above emulsion, some of the sections were digestedwith chondroitinase ABC for 1 h at 37 °C.

Results

Isolation and characterization of proteoglycanThe dorsal skin of an 8-day-old embryo was meta-bolically labelled in a medium containing [35S]sul-phate. Approximately 95% of the incorporatedradioactivity was solubilized by extracting two times.[35S]Sulphate-labelled macromolecules were chroma-togTaphed on a DEAE-Sephacel column with a NaClgradient (Fig. 1). The macromolecules were eluted astwo peaks at NaCl concentrations of 0-40 M (pooled

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Fig. 1. DEAE-Sephacel chromatography of an extractfrom embryonic chick skin. The column (1-4x10-0cm)was eluted with the indicated NaCl gradient in 20 min-Tris-HCl buffer (see Materials and methods) at10mlh~\ and 2-0ml fractions were collected. The solidhorizontal bars indicate fractions I and II, respectively,which were pooled for further analysis. , NaClconcentration (M).

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Fig. 2. (A) Gel chromatography on Sepharose CL-6B offraction I and (B) gel chromatography on SepharoseCL-2B of fraction II. The Sepharose CL-6B and -2Bcolumns (10x94 cm) were eluted with 50mM-Tris-HClbuffer (see Materials and methods), and 0-6 ml fractionswere collected. The solid horizontal bars indicate thefractions pooled for further analysis. Vo, void volume;V,, column volume.

fractions 105-128) and 0-55 M (pooled fractions 135-155) and designated as fractions I and II, respectively,which were then purified by gel chromatography.Fraction I was chromatographed on a SepharoseCL-6B column and eluted as a single peak with a Kav

of 0-38 (Fig. 2A). Fraction II was chromatographedon Sepharose CL-2B and eluted as a single broadpeak with a Kav of 0-19 (Fig. 2B).

These fractions, following purification, were sub-jected to alkali treatment, chondroitinase ABC diges-tion and nitrous acid degradation to examine theglycosaminoglycans. After each treatment, glycos-aminoglycans were chromatographed on a SepharoseCL-6B column. The glycosaminoglycans of fraction I,

liberated by alkali treatment, eluted as a singlepeak with a Kav of 0-55 (Fig. 3A). About 83 % ofthe released glycosaminoglycans of fraction I weredigested by chondroitinase ABC (Fig. 3B) and therelative amounts of isomeric chondroitin sulphatesobtained were as follows: chondroitin-4-sulphate,62-0%; chondroitin-6-sulphate, 38-0%. The chon-droitinase-resistant fraction (pooled fractions 65-97in Fig. 3B, 17%) were degraded by nitrous acid(Fig. 3C) and the chondroitinase ABC-resistant frac-tion has been shown to be proteoheparan sulphate.The glycosaminoglycans of fraction II, liberated byalkali treatment, were chromatographed on a Sephar-ose CL-6B column and eluted as a single peak with aKav of 0-25 (Fig. 4A). The released glycosaminogly-cans were digested for the most part by chondroiti-nase ABC (Fig. 4B) and the relative amounts ofisomeric chondroitin sulphates in the glycosaminogly-cans of fraction II were as follows: chondroitin-4-sulphate, 23-7%; chondroitin-6-sulphate, 76-3%.

The types and yields (of the solubilized radioac-tivity) of proteoglycans metabolically labelled in thedorsal skin were as follows: high molecular weightproteochondroitin sulphate (designated as PCS-I),54-8%; low molecular weight proteochondroitin sul-phate (designated as PCS-I I), 37-5% and proteo-heparan sulphate (designated as PHS), 7-7%.

The labelled epidermis and dermis were separatelyextracted and chromatographed on a DEAE-Sepha-cel column and both were found to contain fraction Iand II (Fig. 5). Gel chromatography on SepharoseCL-2B and digestion with chondroitinase ABC indi-cated fraction II from the epidermis and dermis to bethe same as PCS-I (Fig. 6A,B). No further analysis offraction I in epidermis and dermis was made.

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Fig. 3. Gel chromatography on Sepharose CL-6B of (A) fraction I treated with 0-2M-NaOH, (B) glycosaminoglycansdigested with chondroitinase ABC and (C) chondroitinase ABC-resistant glycosaminoglycans treated with nitrous acid.The conditions of gel chromatography were the same as those in Fig. 2. The solid horizontal bars indicate the fractionspooled. Vo, void volume; V,, column volume.

Proteochondroitin sulphate in chick embryo feather germs 505

Characterized of monoclonal antibody 13B9Monoclonal antibodies were prepared against PCS-I,the main proteoglycan in the dorsal skin of an 8-day-old embryo. Eighteen clones were found to bepositive for PCS-I by ELISA. Among these, themonoclonal antibody, I3B9, which showed thehighest activity in ELISA, was characterized andfound to be of the IgG 2b subclass. To determine thespecificity of I3B9, inhibition ELISA was carried outagainst various proteoglycans, such as PCS-I, chon-droitinase ABC-digested PCS-I, PCS-II, PGH puri-fied from embryonic cartilage and PGM purified fromthe limb bud of a stage-24 chick embryo. I3B9 reactedwith PCS-I and chondroitinase ABC-digested PCS-I,but not with PCS-II or PGH (Fig. 7A). PGM weaklycross reacted with I3B9 (Fig. 7A). Neither did it react

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Fig. 6. (A) Gel chromatography on Sepharose CL-2B offraction II from epidermis and dermis.(B) Gel chromatography on Sepharose CL-6B of thechondroitinase ABC-digest of fraction II from epidermisand dermis. The conditions of gel chromatography werethe same as those in Fig. 2. The solid horizontal barindicates the fractions pooled, x x, epidermis;9 # , dermis; Vo, void volume; V,, column volume.

Fig. 4. Gel chromatography on Sepharose CL-6B of(A) fraction II treated with 0-2M-NaOH and(B) glycosaminoglycans digested with chondroitinaseABC. The conditions of the gel chromatography were thesame as those in Fig. 2. The solid horizontal bar indicatesthe fractions pooled. Vo, void volume; V,, columnvolume.

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Fig. 5. DEAE-Sephacel chromatography of extractsfrom epidermis and dermis. The conditions ofchromatography were the same as those of Fig. 1. Thesolid horizontal bar indicates fraction II, pooled forfurther analysis, x x, epidermis; # • , dermis;

, NaCl concentration (M).

001 0-1 1-0 10 100Concentration of inhibitor (jigml"')

Fig. 7. Inhibition ELISA of monoclonal antibody 'I3B9'for (A) various proteoglycans, and (B) collagens andfibronectin. All plates were coated with PCS-I preparedfrom chick embryo dorsal skin. (A) The followinginhibition solutions were used: • , PCS-I; O,chondroitinase ABC-digested PCS-I; • , PCS-II; D,PGH; A, PGM. (B) The inhibition solutions were: • ,PCS-I; • , collagen type I; D, collagen type IV; A,fibronectin.

506 K. Kitamura

with collagen type I or IV, nor fibronectin (Fig. 7B).Thus, I3B9 appears highly specific for PCS-I and maypossibly recognize the core protein of PCS-I as anepitope.

Distribution of PCS-I during feather germ formationin chick embryos

In the dorsal skin of a 6-day-old embryo, fromwhich no feather rudiments had formed, PCS-I was

present in both the epidermis and superficial dermis(Fig. 8A). Basement membrane also stained withI3B9 (Fig. 8A). In a 7-day-old embryo, feather rudi-ments composed of an epidermal placode and der-mal condensation were noted. Epidermal placodesformed at the site of the presumptive feather werecharacterized by vertically elongated epidermal cells.Dermal cells beneath each epidermal placode wereprogressively condensed and stained with I3B9

Fig. 8. Indirect immunofluorescencestaining of PCS-I. (A) 6-day-oldembryo: no epidermal placode anddermal condensation. PCS-I is stainedin the epidermis and superficialdermis. Basement membrane alsoshows the presence of PCS-I.(B) 7-day-old embryo: epidermalplacode and slight dermalcondensation (formation of a featherrudiments). Weak staining with I3B9 isseen in the region of the epidermalplacode, compared with the regionsurrounding the placode. The dermalcondensation area is stained withI3B9. The basement membrane isuniformly stained with I3B9.(C) 8-day-old embryo: protrusion of afeather rudiment (formation of afeather bud). Epidermis in the featherbud is hardly stained with I3B9. Theapex region of dermal condensationand the basement membrane situatedabove the apex region of the dermalcondensation are very weakly stainedwith I3B9. (D) 9-day-old embryo:weak staining with I3B9 has extendedthroughout the entire area of dermalcondensation. However, the basementmembrane and dermis in the basalregion of feather bud are stained withI3B9. (E) 10-5-day-old embryo:elongation of feather bud in aposterior direction (formation of afeather filament). Intense staining withI3B9 is apparent in the epidermis,basement membrane and dermis in theanterior region of the feather filament,while the posterior region has stainedonly very weakly with I3B9. Bar,

27 fim.

Proteochondroitin sulphate in chick embryo feather germs 507

Fig. 9. Indirect immunofluorescence staining of PCS-I(magnified images). (A) Feather rudiment of a 7-5-day-old embryo: the centre of the epidermal placode hashardly stained with I3B9. Dermal condensation andbasement membrane situated above it are stronglystained with I3B9. (B) Feather bud of an 8-5-day-oldembryo: the epidermis in the feather bud shows very littlestaining with I3B9. The apex region of dermalcondensation and the basement membrane situated aboveits apex region are also very weakly stained with I3B9.However, dermis and basement membrane in the basalregion of the feather bud show intense I3B9 staining.Bar, 11 fim.

(Figs8B, 9A). The epidermal placode, however,only weakly stained (Fig. 8B). This was particularlyevident in the centre of an epidermal placode of a7-5-day-old embryo (Fig. 9A). The decrease in PCS-Iin the epidermal placode was also observed in theepidermal sheet of a 7-5-day-old embryo, detachedfrom the basement membrane and dermis by EDTAtreatment (Fig. 10). That is, PCS-I decreased more inthat particular part of the epidermal placode regionwhere epidermal cells were packed together, thanin the region surrounding the epidermal placode(Fig. 10). The basement membrane situated beneaththe epidermal placode stained with I3B9 (Figs8B,9A).

In an 8-day-old embryo, feather rudiments couldbe seen to start bulging out and develop into feather

Fig. 10. Immunofluorescence micrograph of an epidermalsheet of dorsal skin of a 7-day-old embryo, stained byindirect immunofluorescence for PCS-I. The region of theepidermal placode has stained to a lesser degTee than thatof the surrounding epidermal placode. Bar, 27 fim.

buds, in which dermal condensation was quite appar-ent. Feather bud formation brought about changes inthe distribution of PCS-I. The apex region of thedermal condensation and basement membrane situ-ated above the apex region of the dermal conden-sation stained only very weakly with I3B9, as did alsothe epidermis of the feather buds (Figs 8C, 9B). Withthe growth of feather buds in a 9-day-old embryo, thisarea of weak staining expanded so as to encompass allthe dermal condensation (Fig. 8D). However, thebasement membrane and dermis in the basal regionof the feather buds stained with I3B9 (Fig. 8C,D).The epidermis, basement membrane and dermis ofthe interplumar region showed strong immunofluor-escence (Fig. 8B-D).

By day 10-5, the feather buds elongated in aposterior direction and developed into feather fila-ments. Asymmetrical distribution of PCS-I was notedin these feather filaments. PCS-I was abundant in theepidermis, basement membrane and dermis of theanterior region of the feather filaments (Fig. 8E). Theposterior region stained only very weakly with I3B9(Fig. 8E).

Control sections treated with culture medium andthe culture supernatant of I3B9, previously absorbedby PCS-I, showed no fluorescence (not shown).Treatment with testicular hyaluronidase had no sig-nificant effect on these staining patterns (not shown).

Labelling patterns of [35S]sulphateThe turnover of PCS-I in the feather rudiments andbuds was determined by pulse-chase experimentswith [35S]sulphate. Following the incorporation of[35S]sulphate for lh , the basement membrane situ-ated above the dermal condensation in the featherrudiments as well as the area of the dermal conden-sation itself were strongly labelled (Fig. 11A). Most

508 K. Kitamura

11A

B

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Fig. 11. [35S]Sulphate autoradiograms of feather rudiments. (A) The dorsal skin of a 7-day-old embryo was labelled forlh . The basement membrane situated above the dermal condensation is heavily labelled. The area of the dermalcondensation is also strongly labelled. (B) The labelled section was digested by chondroitinase ABC, causing most ofthe label to disappear. (C) 4h chase. The amount of label has decreased somewhat in the basement membrane anddermal condensation of the feather rudiments. Bar, 21fim.

of the label was eliminated by the chondroitinaseABC digestion, thus indicating it to be virtually allproteochondroitin sulphate (Fig. 11B). After chasefor 4h, the label in the basement membrane abovethe dermal condensation and the area of dermalcondensation was found to be very weakly chased(Fig. 11C).

Following the [35S]sulphate incorporation, thefeather buds were found to have the same labellingpatterns as those of the feather rudiments. That is,the area of dermal condensation and basement mem-brane above the dermal condensation were stronglylabelled (Fig. 12A). However, label in the featherbuds was rapidly chased for a period of 2 h (Fig. 12B).After chase for 4h, decrease in the amount of labelwas conspicuous in the apex region of the dermalcondensation and basement membrane above theapex region of the dermal condensation (Fig. 12C).Most of the label disappeared following chase for 6h(Fig. 12D).

Discussion

The extracellular matrix is considered to performimportant functions in morphogenesis. During the

formation of chick embryo feather germs composedof epidermal placode and condensed dermis, changesin the distribution profiles of collagen types I and IIIand fibronectin have been reported (Kitamura, 1981;Mauger et al. 1982b). Sengel, Bescol-Liversac &Guillam (1962) have also demonstrated changes inthe continuous labelling patterns of [35S]sulphateduring feather formation. The present study providesdetailed clarification of the structure and distributionof proteoglycans during feather germ formation andconfirms and extends the scope of the studies ofSengel et al. (1962).

Proteoglycans in the dorsal skinThe dorsal skin of an 8-day-old embryo synthesizedthree types of proteoglycans, PCS-I, PCS-II and PHS(Figs 1-4). Lever & Goetink (1976) have observed inthe skin of chick embryos two types of proteoglycansdiffering in molecular weight. In the present study,a proteoglycan fraction with low molecular weight(fraction I) was found to consist of PCS-II and PHS.PCS-I and PGM are proteochondroitin sulphatessynthesized by the noncartilageous mesenchymal tis-sues of chick embryos (Kitamura & Yamagata, 1976;Okayama et al. 1976; Kimata et al. 1986). No analysis

Proteochondroitin sulphate in chick embryo feather germs 509

12A

B

Fig. 12. [35S]Sulphate autoradiograms of feather buds. (A) The dorsal skin of an 8-5-day-old embryo was labelled for1 h. Much label can be seen in the area of the dermal condensation and the basement membrane above the dermalcondensation. (B) 2h chase. The label has decreased in the area of dermal condensation and basement membraneabove the dermal condensation. (C) 4h chase. The amount of label has decreased markedly in the apex region of thedermal condensation and basement membrane above the apex region of the dermal condensation. (D) 6h chase.Further decrease in the label in the dermal condensation and basement membrane of the feather buds is evident. Bar,21 jim.

was made of the protein core of PCS-I in this studyand thus the present author is not in a position to saywhether PCS-I is the same as PGM. However, sincePGM showed weak cross reactivity with I3B9, it maypossibly be a PCS-I similar to proteochondroitinsulphate (Kimata et al. 1986).

PCS-I was biochemically demonstrated to be pres-ent not only in dermis but epidermis as well (Figs 5,6). Since the ectoderm of a chick embryo limb budproduces PGM (Kimata et al. 1986) and the corneaepithelium and epidermis of chick embryos syn-thesize collagen type I (Linsenmayer, Smith & Hay,

1977; Kitamura, unpublished data), it is possible thatthe embryonic epithelium and epidermis may alsosynthesize extracellular matrix components peculiarto mesenchyme at an early stage in chick develop-ment. Although the functions of PCS-I in the epider-mis remain obscure, PCS-I may possibly contribute tostabilization of the dermis.

Proteochondroitin sulphate-I in the epidermisPCS-I was immunohistochemically detected in theepidermis of the skin of a 6-day-old embryo and theinterplumar skin of 7- to 10-5-day-old embryos

510 K. Kitamura

(Fig. 8). The ectoderm of a limb bud has also beenreported to stain with anti-PGM antibody (Kimata etal. 1986). Although PCS-I in the epidermis is appar-ently intracellular at microscopical levels (Fig. 10,Vertel, Barkman & Morrell, 1985), the strict localiz-ation of PCS-I in the epidermis should be examinedimmunohistochemically by electron microscopy.

A marked decrease in PCS-I in the epidermalplacode, exceeding that in the epidermis surroundingit, was noted following formation of the epidermalplacode (Figs8B, 9A, 10). Experimental results ofthe hyaluronidase treatment of sections ruled out thepossibility that this decrease detected by immunoflu-orescence results from the masking of PCS-I withother molecules such as hyaluronate. Similar localdecrease in PCS-I was also noted in the epidermalplacode of scale germs (Kitamura, unpublisheddata). Furthermore, collagen type I also decreased inthe epidermal placode of feather and scale germs(Kitamura, unpublished data). It has recently beenreported that epithelial morphology influences theamount of collagen produced (Sugrue & Hay, 1986).Although the actual mechanism for this decreaseremains to be clarified, morphological changes inepidermal cells during epidermal placode formationmay inhibit the synthesis of PCS-I. The decrease inPCS-I in the epidermis of the presumptive featherregion may be one of the first indications of feathergerm morphogenesis. Furthermore, a conspicuousdecrease in PCS-I continued to persist in the epider-mis of feather buds and filaments (Fig. 8C-E). Thus,a transition of epidermis appears to occur in theregion of feather germ formation from the undiffer-entiated state. However, a transition would notnecessarily indicate overt differentiation of epidermisas represented by /S-keratinization (Haake, Konig &Sawyer, 1984).

Proteochondroitin sulphate-I in the basementmembraneIt is well known that the basement membrane con-tains proteoheparan sulphate synthesized by epi-thelial cells (Kanwar, Hascall & Farquhar, 1981). Thedistribution of PHS during feather germ formationhas not been examined due to lack of an antibodyspecific for PHS from chick embryonic skin. Thebasal lamina of mouse salivary gland has been foundto contain chondroitin sulphate from epithelium(Cohn, Banerjee & Bernfield, 1977). In this study,PCS-I was identified immunohistochemically in thebasement membrane of chick embryo dorsal skin(Fig. 8A,B). PCS-I in the basement membrane of thisskin may derive from the dermis since PCS-I wasfound along the basement membrane under theepidermal placode where PCS-I was noted to de-crease (Figs8B, 9A). Fibronectin in the basement

membrane of developing tooth has also beenreported to be produced exclusively by mesenchymalcells (Hurmerinta, Kuusela & Thesleff, 1986).

Although structural changes in the basement mem-brane have been observed during the morphogenesisof various organs (Ekblom et al. 1981; Bernfield &Banerjee, 1978; Thesleff et al. 1980), no temporo-spational changes in the distribution of laminin andcollagen type IV have been detected during feathergerm formation (Mauger et al. 1982a). PCS-I was alsouniformly detected along the basement membraneduring the formation of feather rudiments (Figs 8B,9A). However, its distribution in the basement mem-brane of feather buds differed from that in thebasement membrane of feather rudiments. The de-crease in PCS-I was conspicuous in the basementmembrane at the top of feather buds (Figs 8C,9B).This decrease was examined autoradiographically.Proteochondroitin sulphate in the basement mem-brane of the feather buds was less stable than that inthe feather rudiments (Figs 11A,C, 12A-D). Thislack of stability was similar to that of basal laminarglycosaminoglycans in submandibular morphogenesis(Bernfield & Banerjee, 1982). It should be noted thatPCS-I in both the basement membrane and con-densed dermis decreased at the same time.

Proteochondroitin sulphate-I in the dermisA decrease in collagen type I in the condensed regionof dermal cells has been noted to occur soon afterthe start of dermal condensation (Kitamura, 1981;Mauger et al. 19826). The decrease in PCS-I wasdifferent from that of collagen type I. It was immuno-histochemically detected in the area of dermalcondensation of feather rudiments (Figs8B, 9A).Autoradiography also showed proteochondroitin sul-phate to be synthesized in the area of dermal conden-sation of feather rudiments and to have a very slowturnover (Fig. 11 A,C). Thus, accumulated PCS-Imay possibly be responsible in part for the conden-sation of dermal cells in the presumptive area of thefeather germ. Fibronectin may perform the samefunction (Kitamura, 1981; Mauger et al. 1982).

Following rudiment protrusion, the decrease inPCS-I started from the top region of the dermalcondensation (Figs 8C, 9B) and proceeded to its coreregion (Fig. 8D). In the region of dermal conden-sation, the decrease in PCS-I did not occur as a resultof its being masked by other molecules, such ashyaluronate, from the hyaluronidase treatment.Pulse labelling with [35S]sulphate showed strongincorporation of [35S]sulphate into the area ofdermal condensation and basement membrane situ-ated above it (Fig. 12A). This indicates the highsynthetic activity of proteochondroitin sulphate in thedermal cells of the condensed area. The incorporated

Proteochondroitin sulphate in chick embryo feather germs 511

label, however, subsequently underwent rapid turn-over, which was particularly remarkable in the apexregion of feather buds (Fig. 11B-D). From thesefindings along with the results of immunohistochemi-cal and autoradiographic analysis, PCS-I is thusshown to undergo rapid degradation not only in thebasement membrane but also in the condensed der-mis during feather bud growth.

Mesenchymal cells have been reported to degradeepithelial basal lamina glycosaminoglycans (Smith &Bernfield, 1982). Thus, condensed dermis in featherbuds likely retains a set of enzymes that degrade thecore protein and glycosaminoglycans of PCS-I in thebasement membrane and condensed dermis. Acti-vation of these enzymes may be essential for theprotrusion of feather rudiments. Furthermore, itshould be emphasized that the decrease in PCS-I inthe epidermal placode occurs prior to that in PCS-I inthe basement membrane and condensed dermis. Thisdecrease in the epidermal placode may cause thedermis to lose its stability and its subsequent morpho-genetic activation of dermis in the presumptive regionof feather germs.

PCS-I was found in the basement membrane anddermis in the basal region of the feather buds(Fig. 8D). It was also present in a large amount inthe basement membrane and dermis in the anteriorregion of feather filaments (Fig. 8E). As assumed forcollagen types I and III, PCS-I in feather buds andfilaments may also function to maintain the basestructure necessary for feather germ protrusion anddirectional elongation (Mauger et al. 19826). Thepresent data strongly indicate that PCS-I is quitelikely involved in epidermal-dermal interactions inthe morphogenetically active region of feather germ.

I acknowledge with deep gratitude the support of Drs Y.Kato and T. Higashinakagawa through this study. I wish tothank Dr S. Tanaka for many valuable discussions, Miss M.Sezaki for excellent technical assistance and Mrs Y. Murak-ami for typing my manuscript.

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{Accepted 5 March 1987)