J. Embryol. exp. Morph. 82, 25-40 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
25
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).
26
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
27
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
28
E. BECCHETTI AND OTHERS
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
Exogenous glycosaminoglycans and skin differentiation
29
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
30
E. BECCHETTI AND OTHERS
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).
Exogenous glycosaminoglycans and skin differentiation
V
31
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
32
E. BECCHETTI AND OTHERS
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
—
—
—
4
<_fi
142-8 /ig/ml
57-1 ^g/ml
31-4/ig/ml
4
9
4
8
DS
1—» 1—»
isolated GAG*
3
7
1
1
—
1
1
2
3
5
2
8
4
7
8
8
Exogenous glycosaminoglycans and skin differentiation
Figs 7-12. Histological (haematoxylin-eosin; Figs 9, 10) and histochemical
(sequential staining Alcian blue in 0-3 M-MgCb and Alcian yellow in 0-025 M-MgCb;
photographed with a Leitz CB 16 5 blue filter; Figs 7, 8, 11, 12) pictures of chick
embryonic skin explants cultured for 7 days as follows.
Fig. 7. In D7: dermis pulp strongly reactive with Alcian blue. X250.
Fig. 8. In D7: mesenchyme underlying keratinized epithelium weakly reactive
with Alcian blue. x250.
Fig. 9. In H14: feather filaments and interplumar tristratified epithelium. x400.
Fig. 10. In GAG containing 199: epidermal keratinization and feather filaments.
X250.
Fig. 11. In GAG containing 199: dermis pulp strongly reactive with Alcian blue
X250.
Fig. 12. In GAG containing 199: mesenchyme underlying keratinized epithelium
weakly reactive with Alcian blue X400.
33
34
E. BECCHETTI AND OTHERS
17
Figs 13-18. Histological (haematoxylin-eosin) pictures of chick embryonic skin
explants cultured for 7 days as follows.
Fig. 13. In 142-8 jug/ml DS containing 199: epidermal keratinization. xlOO.
Fig. 14. In 142-8 jug/ml HA containing 199: feather buds and filaments, interplumar tristratified epithelium. x250.
Fig. 15. In 199+testicular hyaluronidase: lack of feathers. xlOO.
Fig. 16. In S+testicular hyaluronidase: unkeratinized epithelium. X250.
Fig. 17. 142-8 ^g/ml CS containing 199/199/199: feather filaments and interplumar tristratified epithelium. xlOO.
Fig. 18. 199/142-8/ig/ml CS containing 199/199: epidermal keratinization.
X250.
Both at 260/ig/ml and 26/ig/ml cultures exhibited the same behaviour.
Epithelial keratinization occurred in about 80 % of cases. The underlying dermis
was strongly reactive to 0-025 M-MgCb Alcian yellow in Ab-Ay (prevalent
presence of HA) and was PAS positive. There were numerous feather buds and
feather filaments; in the latter, the stratified squamous epithelium was thicker on
the posterior edge of the feather which curved to one side as in vivo (Fig. 10).
The dermis pulp was loose and rich in material reacting to 0-3M-MgCl2 Alcian
Exogenous glycosaminoglycans and skin differentiation
35
blue in Ab-Ay and partially digested after treatment with testicular
hyaluronidase (index of CS and DS) (Figs 11,12). A similar pattern was observed
when GAG mixtures were added to E.
The results concerning the individual classes of GAG are noted in Table 2.
Skin maintained in CS at the concentration of 142-8 jUg/ml keratinized.
Feathers were present in moderate numbers in the form of rudiments, buds and
filaments.
Skin maintained in DS reached a different degree of differentiation according
to the DS concentration. At a dose of 31-4/ig/ml numerous feather buds and
feather filaments were present, whereas interplumar epithelium remained at
three to four layers, or further stratified but did not keratinize. In contrast at
higher doses the stratified squamous epithelium showed extended keratinization and a few abnormal feather rudiments and feather buds were present (Fig.
13). Skin explants maintained in HA (at 85-7/ig/ml and 142-8 jug/ml) showed
three to four layered interplumar epithelium which never keratinized, an
abundance of feather buds, and elongated filaments, which were bent and
covered by a very high epithelium (as many as ten or more layers) above all on
the posterior edge (Fig. 14). Barb ridges were at times observed. The dermal
pulp was loose.
Histochemical findings on isolated GAG maintained explants were consistent
with the above described patterns.
Table 3. Effect of hyaluronidase addition
Medium
Cultures
Three
Squamous
number
layered
layers
Keratinization
Feather
E*
S*
199*
7
4
4
6
1
1
3
3
—
—
—
1
2
S**
D7**
4
7
2
1
4
4
1
3
1
enzyme added to skin explants maintained in different nutrients
'* enzymatic pretreatment of the media
Table 4. Effect of sequential CS supplementation
Medium*
CS/199/199
199/CS/199
Cultures
number
Three
layered
6
6
5
1
Squamous
layers
1
Keratinization
4
Feather
1 — 6
5
*CS indicates the 142-8 |ug/ml condroitin 4,6 sulphate containing nutrient; sequence indicates the type of nutrient supplemented to explants every 48 hours.
— I—
a
6 days
b
a
b
13 days
a
b
199
a
b
E
a
b
S
in vitro
a
* 5/6-day explants maintained in vitro for 7 days.
a, mesenchyme underlying interplumar epithelium; b, feather dermis; Ab, Alcian blue in 0-3 M MgCh; Ay, Alcian yellow in 0-025 M
Symbols: — none: +— very weak; + weak; + + increase in reactivity; + + + strong.
Ab/PAS after
diastase
Ab/Ay after
hyaluronidase
Ab/Ay
Sequential
staining
in vivo
Table 5. Dermal histochemical reactivity of back skin
b
D7
a
b
199 + GAG
C/3
W
H
o
H
H
w
m
w
w
n
n
Exogenous glycosaminoglycans and skin differentiation
37
d) Effect of treatment with testicular hyaluronidase
Significant effects were observed in the set of experiments in which testicular
hyaluronidase was added during explantation in vitro (see Table 3). In 199,
explants showed a drastic reduction of feathers in number so that the skin
appeared smooth with tristratified epithelium (Fig. 15).
In S, the epithelium stratified but did not achieve keratinization as it should.
The feathers were either not present or remained at rudiment stage (Fig. 16).
After addition of serum pretreated with hyaluronidase the explants showed
keratinization in 100 % of the cultures, feathers were scarcely represented and
only at the rudiment stage.
After pretreatment of D7 the skin keratinized in 60 % of explants, whereas
feather buds and feather filaments were present.
e) Time sequence
Skin explants presented a different degree of differentiation according to
whether the CS containing 199 was added in the first or second 48 h (see Table 4).
In the first case, the cultures presented feather filaments in which, at times,
barb ridges were present. Interplumar epithelium remained at three to four
layers (Fig. 17).
In the second case, the feathers were less numerous and at the stage of rudiments or buds, whereas the interplumar epithelium was stratified, squamous and
keratinized (Fig. 18).
DISCUSSION
In vivo 5/6-day back skin presents a bistratified epithelium without any signs
of feather differentiation. At 13 days feather filaments have differentiated and
the interplumar epithelium is tristratified. The 5/6-day mesenchyme shows a
uniform morphological as well as GAG pattern. The 13-day dermis presents
marked differences between the region underlying interplumar epithelium and
that found in the plume axis. These data are in agreement with previous
biochemical observations (Pane et al. 1974).
When 5/6-day skin explants are maintained in vitro for 7 days, the course of
development observed is related to the composition of the culture medium. In
199, as in E, feather formation takes place in a similar morphological and
chronological way as in vivo, keratinization is lacking. In S there is an earlier
keratinization than in ovo and no feather formation, even if signs of a few
rudiments do exist. In E+S both keratin and a slower feather formation than in
E alone can be seen.
From this group of experiments it is evident that keratinization and feather
formation are differentiative processes which are regulated independently of one
another and that an earlier keratinization slows down the feather morphogenesis.
38
E. BECCHETTI AND OTHERS
The histochemical dermal reactivity demonstrates that keratinization is always
related to a prevalent accumulation of HA in the underlying mesenchyme,
whereas feather formation is in relation to deposits of CS in the dermis pulp. The
correlation between epithelial differentiation and mesenchymal GAG pattern is
confirmed by the experiments with testicular hyaluronidase. The enzyme added
during maintenance of cultures prevents both feather formation (in E) and
keratinization (in S).
The addition of exogenous GAG modifies the course of the differentiation
processes studied. This effect is not generic but correlated to the type and concentration of the GAG added, which would suggest we are dealing with a specific
effect corresponding to a definite regulatory mechanism. This is why, for example, we observe a different ability to promote keratinization between D14, on the
one hand, and H14 and L14 , on the other, and another difference in the stimulation of feather formation between D7, L7 and H7. The GAG contents in these
media are different: in D7 , and even more so in H7 , CS clearly prevails over HA,
whereas in L7, CS and HA are in equal amount (Evangelisti et al. 1979, 1982).
Furthermore, pretreatment of D7 with testicular hyaluronidase markedly
reduces the ability to promote both keratinization and feather formation; this
supports the possibility that the effect of the culture medium depends on its GAG
content.
Pretreatment of serum, on the other hand, has no effect; this suggests that the
stimulatory keratinization serum factor(s) is (are) not GAG.
The results obtained following addition of mixtures of commercial GAG
(CS+HA+DS) in relative concentrations corresponding to those present in D7
confirm the regulatory role of exogenous GAG. The explants show keratinization and feather formation, and the mesenchyme presents a different composition of extracellular GAG in the tracts underlying keratinized epithelium
(prevalence of HA) or in those forming the feather axis (prevalence of CS).
Among the individual GAG, DS and CS provoke keratinization of the
epithelium, HA a better development of feathers.
It is interesting to note that sulphate GAGs elicit an earlier keratinization and
a slower feather morphogenesis.
To the best of our knowledge, the reported data are the first direct demonstration that exogenous GAGs play a regulatory role in epithelial differentiation. As
far as the mechanism involved is concerned, in agreement with previous observations and hypotheses (Toole & Trelstad, 1971; Bernfield etal. 1973; Hart, 1976;
Carinci et al. 1976), it is tempting to believe that this takes place through a
modification in mesenchymal GAG pattern. In fact, the extracellular GAG
pattern of the mesenchyme both in ovo and in vitro, is always related to the
nature of the evolution of the epithelium: prevalence of HA in the case of
keratinization; prevalence of CS in the case of feather formation.
Furthermore, the sequential addition of CS at the subsequent times, in which
fibroblasts were shown to possess a different ability in kind of released GAG, has
Exogenous glycosaminoglycans and skin differentiation
39
effects on keratinization when the mesenchyme is able to produce HA (second
48 h), but has no effect whatsoever when this ability is lacking (first 48 h) (Carinci
etal. 1978).
The results obtained with individual GAG allow us to suggest a possible means
by which the pattern of extracellular GAG is regulated. It may consist of a feedback mechanism. In fact, when CS or DS are added there is an accumulation of
HA in the mesenchyme (with the correlated differentiation process of the
epithelium, i.e. keratinization), whereas when HA is added, there is an accumulation of sulphated GAG and epithelium remains three-layered, but feathers
develop to a greater extent.
Research is presently being carried out in an attempt to discover this mechanism. Its identification would allow us to demonstrate a level of autoregulation of
differentiation.
This work was in part supported by a grant from the Ministero della Pubblica Instruzione
(Italy).
REFERENCES
BERNFIELD, M. R., COHN, R. H. & BANERJEE, S. D. (1973). Glycosaminoglycans and
epithelial organ formation. Am. Zool. 13, 1067-1083.
BODO, M. A. & CARINCI, P. (1972). Membrana testacea: a support for organ culture. Experientia 28, 1397.
CARINCI, P., PANE, G. L., SIMONELLI, L. & ZANIBONI, G. (1975). A correlation between
mesenchymal composition and epidermal differentiation in chick embryonic skin.
Experientia 31, 586-587.
CARINCI, P., SIMONELLI, L., BUBOLA, G. & PETTAZZONI, G. (1976). An ultrastructural analysis
of chick embryonic skin development in vitro. J. exp. Zool. 197, 389-402.
CARINCI, P., STABELLINI, G. & BECCHETTI, E. (1978). Serum-dependent avian skin differentiation in vitro: time sequence of induced events. /. Embryol. exp. Morph. 45, 25-35.
CARINCI, P. (1981). The mesenchyme during development: composition of the ground
substance, regulatory factors, embryological significance. Bas. Appl. Histochem. 25,
267-277.
CESSI, C. & PILIEGO, F. (1960). The determination of aminosugars in the presence of
aminoacids and glucose. Biochem. J. 77, 508-510.
COHEN, S. (1965). The stimulation of epidermal proliferation by a specific protein (EGF).
Devi Biol. 12, 394-407.
DODSON, J. W. (1967). The differentiation of epidermis. I. The interrelationship of epidermis
and dermis in embryonic chicken skin. /. Embryol. exp. Morph. 17, 83-105.
EVANGELISTI, R., PAGLIARINI, A., STABELLINI, G. & CARUSO, A. (1979). Sintesi di
glicosaminoglicani in cultura primaria di fibroblasti embrionali. Bas. Appl. Histochem.
23/suppl, XVIII.
EVANGELISTI, R., STABELLINI, G., VENTUROLI, A. & CARINCI, P. (1982). Glycosaminoglycan
synthesis by embryonic fibroblasts is age-dependent and modulated by environmental factors. Experientia 38, 616-617.
FLEISCHMAJER, R. & BILLINGHAM, R. E. (1968). Epithelial-Mesenchymal Interactions.
Baltimore: Williams & Wilkins.
HAMILTON, H. L. (1952). Lillie's Development on the Chick. An Introduction. New York:
H/Holt.
HART, G. W. (1976). Biosynthesis of glycosaminoglycans during corneal development. /. biol.
Chem. 251, 6513-6521.
40
E. BECCHETTI AND OTHERS
H. M. & MOTTET, N. K. (1970). Ultrastructural features of defective in vitro
keratinization of chick embryonic skin. /. Cell Sci. 6, 485-509.
NOVEL, G. (1973). Feather pattern stability and reorganization in cultured skin. /. Embryol.
exp. Morph. 51, 27-50.
PANE, G. L., BECCHETTI, E. & CARINCI, P. (1974). Biochemical composition of chick embryonic
skin. Int. J. Biochem. 5, 579-583.
SCOTT, J. E. & DORLING, J. (1965). Differential staining of glycosaminoglycans
(mucopolysaccharide) by alcian blue in salt solution. Histochemie 5, 221-233.
SENGEL, P. (1971). The organogenesis and arrangement of cutaneous appendance in birds.
Adv. Morph. 9, 181-230.
SOLURSH, M., VAEREWYCK, S. A. & REITER, R. S. (1974). Depression by hyaluronic acid of
glycosaminoglycan synthesis by cultured chick embryo chondrocytes. Devi Biol. 41,
233-244.
TOOLE, B. P. & TRELSTAD, R. L. (1971). Hyaluronate production and removal during corneal
development in chick. Devi Biol. 26, 28-35.
VASAN, N. (1983). Analysis of perinotochordal materials. 2. Studies on the influence of
proteoglycans in somite chondrogenesis. J. Embryol. exp. Morph. 73, 263-274.
WEVER, J., SCHACHTSCHABEL, D. D., SLUKE, G. & WEVER, G. (1980). Effect of short or long
term treatment with exogenous glycoaminoglycans on growth and glycosaminoglycan
synthesis of human fibroblast in culture. Mech. age. Dev. 14, 89-99.
WOLFF, E. & HAFFEN, K. C. (1952). Sur une m6thode de culture d'organes embryonnaires in
vitro. Texas Rep. Biol. med. 10, 463-472.
ZANIBONI, G. & CARINCI, P. (1968). Caratterizzazione istochimica dei polisaccaridi acidi. IV.
Colorazione in presenza di diverse concentrazioni saline. Boll. Soc. Ital. Biol. Sper. 44,
670-673.
JENSEN,
(Accepted 23 February 1984)