GAG - Development - The Company of Biologists

J. Embryol. exp. Morph. 82, 25-40 (1984) Printed in Great Britain © The Company of Biologists Limited 1984

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Exogenous glycosaminoglycans (GAG) are able to modulate avian skin differentiation (epithelial keratinization and feather formation ByE. BECCHETTI, G. STABELLINI, A. CARUSO AND P. CARINCI Institute of Histology and General Embryology, University of Ferrara, Via Fossato di Mortara 64, 44100 Ferrara, Italy

SUMMARY

Several reports have suggested that mesenchymal glycosaminoglycans (GAG) may be involved in the regulatory role of epithelial differentiation. Some researchers have pointed out that exogenous GAG affects extracellular GAG accumulation. We have therefore examined the effect of added GAG on two typical processes of avian skin differentiation: keratinization and feather formation. Glycosaminoglycans, either obtained from fibroblasts cultures (conditioned media) or purified commercially available GAG were administered to 5/6-day chick embryo back skin explants. Control cultures were supported with 199 synthetic medium, chick embryo extract or calf serum. Explants have been examined by histological and histochemical procedures. Skin explants maintained in vitro for 7 days exhibited an epithelial differentiation and a dermal histochemical reactivity which were related to the composition of the culture medium. In conditioned media from dermal fibroblasts, but not from heart or lung fibroblasts, explants always exhibited keratinization. In purified-GAG-containing media, keratinization was observed with condroitinsulphates and not with hyaluronic acid. Keratinization was always related to prevalent accumulation of hyaluronic acid in the underlying mesenchyme whereas feather formation was in relation to deposits of condroitinsulphates in dermis pulp. The above findings demonstrate that exogenous GAG is able to modulate avian skin differentiation and that this regulation is linked to an influence on the mesenchymal GAG pattern.

INTRODUCTION

It is now a well-established fact that epithelial differentiation in several rudiments is developmentally regulated by associated mesenchyme (see Fleischmajer & Billingham, 1968, for a review). However, the nature of the regulatory signals is not yet known. There has been much circumstantial evidence to suggest that glycosaminoglycans (GAG) may be involved in the regulatory role of the mesenchyme, i.e. specific GAG composition in different mesenchymes, their change during development, correlation between onset of normal or altered epithelial differentiation and normal or modified mesenchymal GAG pattern (see review by Carinci, 1981).

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E. BECCHETTI AND OTHERS

Most of these studies have involved the culture of explants in vitro, and naturally the results obtained have emphasized the importance of the culture medium on epithelial development. For example, in chick embryo skin, a classical material used in this type of experiment, epithelial keratinization takes place in vitro in the presence of serum faster than and in similar way to that in vivo (Dodson, 1967; Jensen & Mottet, 1970; Carinci etal. 1976). In contrast, in chick embryo extract the epithelium remains tristratified (Carinci et al. 1976). In addition, in the differentiating explants dermal ground substance changes its histochemical and biochemical pattern correlated with keratinization (accumulation of hyaluronic acid) (Carinci et al. 1975). Feather and scale show normal development in explants supported with synthetic medium (Cohen, 1965); whereas both are inhibited when epithelial growth factor is added (Cohen, 1965). When the initial row of rudiments is damaged at explantation it is reorganized when cultured in a synthetic medium (199), but not in chick embryo extract (Novel, 1973). From these observations one concludes that environmental factors control the differentiation of epithelia, most probably through the mediation of the mesenchyme. Unfortunately, no data are available concerning mesenchymal composition during feather formation. Several researchers have pointed out that exogenous GAG can intervene in extracellular GAG accumulation (Solursh etal. 1974; Weever etal. 1980; Vasan, 1983). It therefore seemed of interest to study whether exogenous GAG was able to interfere with epithelial differentiation, bearing in mind the possible role of GAG as a differentiative signal. Consequently, we examined the effect of added GAG on the two typical processes of skin differentiation (keratinization and feather formation). For this purpose we set up a suitable model consisting of explants of back skin of chick embryos cultured in a synthetic medium (199). To these explants G AGs were administered both as products released by fibroblasts in vitro (conditioned media, in more physiological conditions) and as purified commercially available products, in mixture or isolated. Our results demonstrate that exogenous GAG is able to modulate differentiative processes of avian skin and that this regulation is linked to an influence on the mesenchymal extracellular GAG pattern (prevalence of hyaluronic acid in the dermis underlying keratinized epithelium, prevalence of sulphated GAGs in the dermal plume axis). MATERIALS AND METHODS

Organ cultures Hubbard fertilized eggs provided by the Selice Incubator Company (Bubano, Imola, Italy), were incubated at 38°C and at 60 % of relative humidity. Fragments of back skin were removed under sterile conditions from embryos of 5-6 days, staged according to the Hamburger-Hamilton table (Hamilton, 1952). They were then placed in culture dishes on membrana testacea following

Exogenous glycosaminoglycans and skin differentiation

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the technique of Bodo and Carinci (1972), in the presence of a suitable culture medium, and incubated for 7 days at 37 °C. In one set of experiments the explants were removed every 2 days together with the membrana testacea, carefully washed in Tyrode's solution and transferred to another culture dish containing the appropriate culture medium (Carinci et al. 1978). Culture media Standard media According to the method of Wolff & Haffen (1962), semisolid natural media were used: an embryo extract medium (hereafter named E) composed of 6 parts of 1 % Agar in Gey's saline, 3 parts of Tyrode's solution, 3 parts of chick embryo extract (DIFCO) and 400 i.u./ml of penicillin and streptomicin; a serum medium (S), in which the chick embryo extract of E was replaced by an equal amount of calf serum (GIBCO); and E+S medium, in which the Tyrode's solution of E was replaced by an equal amount of calf serum. The synthetic medium used was GIBCO medium 199. The 199 was currently used for preparing conditioned and commercially available GAG nutrients. Conditioned media Fragments of back skin, lung or heart, were removed from 7- and 14-day-old chick embryos, cut into small pieces, then washed in Tyrode's solution. They were then dissociated in 0-25 % trypsin (DIFCO 1:300) in PBS (phosphate-buffered saline) without Ca + + and Mg ++ at room temperature for 20-35 min. The cells were recovered by centrifugation and suspended in medium 199 + 20 % serum. Twenty ml of cellular suspension (106 cell/ml) were put in Falconflasks(75 cm2) in an atmosphere of CO2 5 % at 37 °C for 48 h, in order to obtain subconfluent cultures. The culture media were subsequently removed, the cells repeatedly washed with Earle's solution and supplemented by 199 for a further 48 h. Pooled media (three cultures for each preparation) were dialysed, lyophilized and dissolved to obtain a hexosamine concentration of about 400 /ig/ml, on the basis of its determinations, according to Cessi & Piliego (1960). The GAG composition of these nutrients was previously determined (Evangelisti et al. 1979, 1982). Culture medium consisted of 6 parts of 1 % Agar in Gey's saline, 6 parts of conditioned nutrient, plus 400 i.u./ml of penicillin and streptomycin. According to the organs of origin (dermis, lung, heart) and the incubation period (7,14 days) nutrients were named in the following way: D7, D14; L7, L14; H7,Hi4. Culture media containing commercially available GAG The GAG were purchased from the SIGMA Company. Chondroitin 4,6 sulphate (CS), hyaluronic acid (HA), dermatan sulphate (DS) were dissolved in 199

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in the ratio 5:3:1 (corresponding to that determined in D7) (Evangelisti et al. 1982) at the following final concentration: 520/^g/ml, 260]Ug/ml, 26/ig/ml. In some experiments, we used as a dissolving system E instead of 199. CS (at the 142-8/ig/ml final concentration), HA (at the 85-7/ig/ml, 142-8//g/ml final concentrations), DS (at the 31-4/ig/ml, 57-1/ig/ml, 142-8 /ig/ml final concentrations) were separately dissolved in 199. Culture medium was composed of 6 parts of 1 % Agar in Gey's saline balanced, 6 parts of GAG (mixed or individual) and 400 i. u ./ml of penicillin and streptomycin. Treatment with testicular hyaluronidase Treatment with testicular hyaluronidase (Merck) was used in order to examine both the role of the GAG present in the culture media in the regulation of the morphogenetic events (by means of a pretreatment) and the importance of extracellular GAG pattern (by means of an addition to culture media). For the former the culture media were incubated at 40 °C for 48 h with 770//g/ml of enzyme. For the latter the enzyme was added to the media at final concentrations of 280 ^g/ml for the whole in vitro period. Histological and histochemical investigations Cultures were fixed in buffered formalin at 4°C for 3-4 h, and routine histological procedures were followed. Serial sections were cut at 4-7 fim and stained for morphological examination with haematoxylin-eosin. The following terminology will be used for embryonic feathers (Sengel, 1971). Feather rudiments comprise a dermal condensation and an overlying epidermal placode; they are flat or just starting to bulge out. Feather buds bulge out above the surface of the skin; they consist of epidermal sheath and dermal pulp. Feather filaments are elongated buds characterized by the differentiation of barb ridges. Where no precise stage is meant, the word feather will be used indifferently for any one of the three mentioned structures. For the histochemical analysis, we used a set of procedures in order to detect glycoproteins (GP) and glycosoaminoglycans (GAG) and to distinguish between different GAG: sequential staining with Alcian blue 8 GX (Fluka) 1 % in 0-3 Mor 0-025M-MgCh solution and PAS, after diastase (Hoechst 0-1% in 0-2Mphosphate buffer, pH6, 37 °C, l h ) ; sequential staining with Alcian blue 1 % in 0-3M-MgCl2 solution and Alcian yellow GXS (Chroma), 1 % in 0-025M-MgCl2 solution (thereafter referred to as Ab-Ay). The GAG were classified into different classes by Alcian staining, owing to the different critical electrolyte concentration at which the anionic polymers change from binding dye to binding Mg ++ (Scott & Dorling, 1965). Sections were also incubated with testicular hyaluronidase (Merck, lmg/ml 0-lM-phosphate buffer, pH7-6, 6h at 37 °C). Control sections were incubated in buffer alone. Histochemical data are fully reported in Table 5. PAS positivity not abolished by diastase digestion was indicative of GP


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presence; staining with Alcian blue in 0-3M-MgCl2 which was not removed by testicular hyaluronidase was indicative of chondroitin sulphate B (dermatan sulphate, DS); staining with Alcian blue in 0-3M-MgCl2, which was hyaluronidase sensitive was indicative of chondroitin sulphates A/C (CS); staining with Alcian yellow in 0-025 M-MgCk was indicative of hyaluronic acid (HA), the reactivity of more acidic groups being blocked by previous 0-3M-MgCl2 Alcian blue staining (Zaniboni & Carinci, 1968). In preliminary experiments no reactivity was detected with Alcian blue in the presence of 0-5-0-8 M-MgCk. Fragments from the back skin of 5/6- and 13-day embryos were examined by means of the above procedures. RESULTS

Our results are based on 248 cultures and will be described with regard to: a) assessment of the model; b) effect of conditioned media; c) effect of commercially available GAG; d) effect of treatment with testicular hyaluronidase; e) effect of sequential GAG supplementation. For reference data, the first to be described will be the observations on skin from 5/6- and 13-day embryos, i.e. at the explantation time and after a chronological period corresponding to that of in vitro maintenance. Shin development in ovo 5/6- day back skin presented a bistratified epithelial lamina (external layer composed of flat cells and basal layer of cuboid cells). There were no visible signs of feather development. Dermis was loose with a ground substance that was faintly PAS positive and well stained in Ab-Ay at 0-3 M-MgCk Alcian blue but the staining was completely abolished after testicular hyaluronidase digestion (indicating that CS is the prominent GAG) (see Table 5). At 13 days feather filaments curved in a caudal direction were present. The feather epithelium was thicker owing to an increase in the intermediate and superficial layers. The dermis pulp was rich in ground substance strongly reacting with 0-3 M-MgCk Alcian blue in Ab-Ay; this reaction was partially abolished by the treatment with testicular hyaluronidase (more prominence of CS and DS). The epithelium of the interplumar skin was three-layered (basal, intermediate and superficial) and the dermis was PAS positive and reacted to 0-3 M-MgCk Alcian blue in Ab-Ay. Skin development in vitro a) In standard culture media In this set of experiments we studied the effect of E, S, E+S on the two morphogenetic events under consideration (keratinization and feather formation) (Table 1).

EMB82

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Table 1. Epithelial histogenesis of 5/6- day back skin explants maintained 7 days in vitro Medium

Cultures number

Three layered

Squamous layers

Keratinization

Feather

13 13 14 10

13 7 — —

6 — 1

— 14 9

11 12 — 7

18 11 12 13

— — — 4 7

1 4 2 2 1 —

17 7 10 7 — —

15 10 10 11 8 7

A) Standard* 199 E S

E+S B) Conditioned* D7

H7 L7 D14 H14 L14

8 8

8

* see Material and Methods.

Explants of back skin, at the end of in vitro maintenance in 199, presented a great number of feather rudiments, feather buds and feather filaments, without, however, barb ridge differentiation (Fig. 1). The dermis pulp was strongly positive to 0-3.MgCl2 M Alcian blue in Ab-Ay and sensitive to hyaluronidase (indicating an increase of sulphated GAG) (Fig. 2). The interplumar epithelium had three layers (basal, intermediate, superficial). The underlying dermis was loose with a ground matrix positive to Alcian blue in 0-3 M-MgCk and sensitive to testicular hyaluronidase. An analogous pattern was observed after maintenance in E. At times interplumar epithelium exhibited four to five layers without keratinization. The explants maintained in S presented a pluristratified epithelium with numerous squamous layers and keratin (Fig. 3). A few feather rudiments were observed. The dermal intercellular substance underlying keratinized epithelium was PAS positive and clearly reacted to 0-025 M-MgCk Alcian yellow, sensitive to hyaluronidase (accumulation of hyaluronic acid) (Fig. 4). In the dermis underlying the feather rudiments the ground substance reacted to Alcian blue in 0-3 MMgCk and was partially digested by testicular hyaluronidase (index of CS and DS). Skin maintained in E plus S presented both keratinized epithelium and feather in rudiment and bud stage. The histochemical features of the ground substance were analogous to those already described (alcianophilic to 0-025 MMgCk and PAS positivity in the dermis of keratinized interplumar skin, alcianophilic to 0-3 M-MgCk in the dermis pulp).


V

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S ^ '

Figs 1-6. Histological (haematoxylin-eosin; Figs 1, 3, 5, 6) and histochemical (sequential staining Alcian blue in 0-3 M-MgCl2 and Alcian yellow in 0-025 M-MgCb; photographed with a Leitz CB 16 5 blue filter; Figs 2,4) pictures of chick embryonic skin explants cultured for 7 days as follows. Fig. 1. In 199: feather filaments and interplumar tristratified epithelium. xlOO, Fig. 2. In 199: dermal pulp strongly reactive with Alcian blue. X250. Fig. 3. In S: epidermal keratinization. X400. Fig. 4. In S: dermal ground substance weakly reactive with Alcian blue. X400. Fig. 5. In H7: epidermal keratinization and feather buds. x250. Fig. 6. InD?: both featherfilamentsand interplumar epithelium keratinized. x 100. b) In conditioned culture media In the explants supplied with conditioned culture media from 7-day fibroblasts (D7, L7, H7) the epithelium keratinized, whatever the conditioned medium (see

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Table 1). However, a few differences may be demonstrated: in L7, the explants appeared to attain keratinization on the edges of the feather buds and of filaments, a fact which was also demonstrated in H7 where, however, the layers of keratinization were greater (Fig. 5). In D7, the cultures presented feather at rudiment or bud stage with uniform keratinization on the whole surface of the culture (Fig. 6). The ground substance of the dermal pulp showed a prevalent positivity to 0-3 M-MgCb Alcian blue in Ab-Ay, (Fig. 7) partially sensitive to treatment with testicular hyaluronidase (CS and DS), whereas the dermis underlying keratinized epithelium exhibited alcianophilia at a molar concentration of 0-025 in MgCl2 (HA) (Fig. 8). Different patterns were observed in explants, maintained in conditioned culture media from 14-day fibroblasts (Table 1). In D14 the skin explants reached keratinization, feather rudiments and feather buds were formed; whereas in H14 and L14 a pattern similar to that in 199 was observed (interplumar epithelium was tristratified with evidence of feather rudiments, buds and filaments) (Fig. 9). c) In culture media containing GAG mixtures or single classes The explants maintained in media containing mixtures of GAG at concentrations over 260 /ig/ml degenerated or exhibited signs of damage whether GAG were added to 199 or to E (see Table 2). Table 2. Epithelial histogenesis of 5/6-day back skin explants maintained 7 days

in vitro Medium

Cultures number

Dead culture

Three layered

1

1** 1 2

1 1

3 1

Squamous Keratinization layers

Feather

GAG mixtures* in 199

520/ig/ml 260/ig/ml 26/zg/ml

5 8 8

3 — —

inE

520/ig/ml 260/^g/ml 26/xg/ml

4 11 14

2 — —

1** 6 6

1 4 7

2** 7 12

5 12

2

HA

142-8 |Ug/ml 85-7 ^g/ml

CS

142-8/ig/ml

10

* see Material and Methods ** suffering cultures

— — —

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