JOURNAL OF MORPHOLOGY 231:161–174 (1997)
Evidence for Participation of the Epidermis in the Deposition
of Superficial Layer of Scales in Zebrafish (Danio rerio):
A SEM and TEM Study
JEAN-YVES SIRE,* ALEXANDRA QUILHAC, JACQUELINE
BOURGUIGNON, AND FRANCOISE ALLIZARD
Université Paris 7, 75251 Paris cedex 05, France
ABSTRACT Comparative studies on scale structure and development in
bony fish have led to the hypothesis that elasmoid scales in teleosts could be
dental in origin. The present work was undertaken to determine whether the
scales in zebrafish (Danio rerio), a species widely used in genetics and
developmental biology, would be an appropriate focus for further studies
devoted to the immunodetection of dental components or to the detection of
the expression of genes coding for various dental proteins in fish scales. The
superficial region of mature and experimentally regenerated scales and its
relationships to the epidermal cover were studied in adult zebrafish using
scanning (SEM) and transmission (TEM) electron microscopy. The elasmoid
scales are relatively large, thin, and are located in the upper region of the
dermis, close to the epidermis. In adults, the surface of the posterior region
appears smooth at the SEM level and is entirely covered by the epidermis.
During regeneration, the relationship of the epidermal cover to the scale
surface is established within 4 days. This interface is easier to study in
regenerating than in mature scales because the former are poorly mineralized. TEM revealed that: (1) the epidermis is in direct contact with the scale
surface, from which it is separated only by a basement membrane-like
structure, (2) there are no dermal elements at the scale surface except at the
level of grooves issuing from the focus and crossing the scale surface radially,
(3) the mineral crystals located in this superficial region are perpendicular to
the scale surface, whereas those located deeper within the collagenous scale
matrix are randomly disposed, and (4) when decalcified, the matrix of the
superficial region of the scale appears devoid of collagen fibrils but contains
thin electron-dense granules, some of which are arranged into layers. The
continuous epidermal covering, the absence of dermal elements, as well as the
fine structure of the matrix and its type of mineralization, strongly suggest
that epidermal products, possibly enamel-like proteins, are deposited at the
scale surface and contribute to the thickening of the upper layer in zebrafish
scales. J. Morphol. 231:161–174, 1997. r 1997 Wiley-Liss, Inc.
In most bony fish the body is protected by
a dermal skeleton of scales that may correspond to the ‘‘exoskeleton’’ that covered the
early vertebrates (e.g., Heterostracans, Osteostracans) (Schultze, ’66, ’77; Reif, ’82;
Smith and Hall, ’90). A clear tendency toward a reduction of the postcranial dermal
skeleton is observed in the course of evolution, and only a few tetrapod species (mainly
reptiles, some amphibians and mammals)
possess dermal sclerifications. In contrast,
r 1997 WILEY-LISS, INC.
bony fish (i.e., osteichthyans) show a considerable diversity of their dermal skeleton
(various types of scales and bony plates) and
of the constituent tissues (various types of
bone, of dentine, enamel, enameloid, and
highly derived tissues) (Francillon-Vieillot
et al., ’90; Zylberberg et al., ’92). For over a
*Correspondence to: Dr. Jean-Yves Sire, Equipe de recherche
‘‘Formations Squelettiques,’’ URA CNRS 1137, Université Paris
7, Laboratoire d’Anatomie comparée, Case 7077, 2, Place Jussieu, 75251 Paris cedex 05, France.
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J.-Y. SIRE ET AL.
decade, one of our goals has been to elucidate the evolutionary routes that have led
from the plesiomorphic structures of the ancestral dermal skeleton to the current diversity of these tissues. A series of comparative
studies was devoted to the structure and
development of the scaled dermal skeleton
in living osteichthyan fish: ganoid scales of
polypterids (Sire et al., ’87; Sire, ’89a) and of
lepisosteids (Sire, ’94), scutes of armored
catfish (Sire and Meunier, ’93; Sire, ’93),
elasmoid scales of cichlids (Sire and Géraudie, ’83), and, recently, bony plates of
syngnathids (Sire, personal observations).
From a structural point of view, these studies showed that the elasmoid scales of teleosts could be derived from the ‘‘odontodal’’
tissues (i.e., odontocomplexes; see Ørvig, ’67,
’77) that covered the dermal skeleton in ancestral osteichthyan fish (Sire, ’89a, ’90). This
hypothesis was supported by the existence
of epidermal products in the upper layer of
the scales in cichlids (Sire, ’88) and is consistent with the immunolocalization of mammalian enamel-like antigenic determinants in
the skin covering the scales of carp (Krejsa
et al., ’84) and of mammalian amelogenins
in the ganoine, the ‘‘enamel’’ layer covering
the scales of a polypterid (Zylberberg et al.,
submitted). From a developmental point of
view, the comparison of the events (cytodifferentiation and morphogenesis) leading to
the initiation and formation of an elasmoid
scale (Sire and Géraudie, ’83; Sire et al., ’90)
with those occurring during tooth and dermal bone formation (Huysseune and Sire,
’92; Sire and Huysseune, ’93) support the
hypothesis that scales in teleosts are closer
to teeth than to dermal bone.
In spite of these results, which strongly
suggest the existence of ectodermally derived products in the upper layer of the
elasmoid teleost scale, there is still no information on the type of proteins (enamelins,
amelogenins), and their presence has not
been clearly demonstrated. Before undertaking long-term studies combining immunohistochemistry and molecular biology to detect
epidermal products (i.e., enamel-like proteins) and genes coding for them, we first
needed to find a fish species with scales that
provide favorable material for such investigations. Until now our knowledge on elasmoid scale biology largely has been based on
the cichlid fish, Hemichromis bimaculatus
(Sire, ’87). However, because of increasing
interest in zebrafish (Danio rerio), a cyprinid, and because a large quantity of molecular tools is now available for this species, we
decided to determine whether the zebrafish
scale would be suitable to test our hypothesis on the ‘‘odontodal’’ origin of the elasmoid
scales.
Using scanning and transmission electron
microscopy, the present study describes in
detail the fine structure of the adult zebrafish scale, with particular attention to its
upper region and its relationships to the
epidermal cover. The main features of the
scale structure in zebrafish were previously
reported by Waterman (’70), but he did not
describe the epidermis/scale interface. We
also used experimentally regenerated scales
because scale regeneration, which largely
repeats ontogeny, has proven to be a useful
tool for investigating similar questions in
primitive osteichthyans (Sire et al., ’87; Sire,
’94). Our data strongly support a participation of the epidermis in the deposition of the
upper layer of the scale in zebrafish.
MATERIALS AND METHODS
Animals
Ten adult zebrafish, Danio rerio (30 to 40
mm SL), were bred in 40-liter tanks, in controlled conditions of light (12h/12h) and temperature 25°C, and fed daily on Tetramin
powder and chironomid larvae. Two specimens were killed by an overdose of MS 222
and used for alizarin red staining. Three fish
were anaesthesized in 0.05% MS 222 solution and several scales were removed from
the pectoral region of the left flank (Fig. 1) to
be prepared for scanning electron microscopical (SEM) observations. These fish were allowed to regenerate their scales for 4 or 7
days. They were then over-anaesthesized and
blocks of skin containing regenerated scales
were dissected and fixed for transmission
electron microscopical (TEM) study. Five
other fish were killed and blocks of skin
containing scales were dissected and fixed
for light and TEM studies.
Alizarin red staining
The fish were immersed in 10% formaldehyde for a night, then stored in 70% ethanol
for 2 days. They were cleared in 1% KOH for
1 day and depigmented in 0.02% H2O2 for 2
h, then placed in a solution of 1% KOH
containing 0.5% Alizarin red S (Fluka) for 2
h. After staining, the fish were cleared in a
mixture of 1% KOH and glycerol (v/v) for 3
FINE STRUCTURE OF ZEBRAFISH SCALE
163
Fig. 1. Squamation in zebrafish, Danio rerio (drawing from a 32 mm SL alizarin red stained
and cleared specimen).
days, then stored in pure glycerol and observed with a binocular microscope.
SEM
To remove the soft tissues at their surfaces, isolated scales were immersed in 8%
sodium hypochlorite for several minutes
while monitored with a binocular microscope. The scales were then rinsed, dehydrated, dried, glued on an aluminum support, and covered with a thin layer of gold/
palladium prior to observation with a JEOL
35 SEM.
TEM
Blocks of skin or isolated scales were immersed in a fixative solution containing 1.5%
glutaraldehyde and 1.5% paraformaldehyde
in 0.1 M cacodylate buffer (pH 7.4) for 2 h at
room temperature. After a short rinse in the
buffer containing 10% sucrose, the samples
were postfixed for 2 h in 1% OsO4 in 0.1 M
cacodylate buffer to which 8% sucrose was
added. The samples were subsequently
rinsed in the buffer, dehydrated in a graded
series of ethanol, and embedded in Epon
812. Some samples were decalcified for at
least 7 days in the fixative solution to which
0.1 M EDTA was added (changed every 2
days), then processed as described above.
Histology was studied from 1-µm-thick,
toluidine blue-stained sections. For ultrastructural descriptions, thin sections were
contrasted with uranyl acetate and lead citrate and observed in a 201 Philips EM
operating at 80 kV.
RESULTS
In zebrafish, scales are thin collagenous
plates (,100 µm thick) that are large compared to the size of the fish. They are imbricated, i.e., their anterior region is covered by
the preceding scale from the same longitudinal row and the lateral regions are covered
by two scales of the neighboring rows (Fig.
1). The squamation forms a regular pattern
on the body. On both sides of the caudal
peduncle, four scale rows are present to
which two rows are added, dorsally and ventrally, forming a roof and a keel (Fig. 1).
Seven scale rows cover both sides of the
anterior region to the level of the pelvic fins.
Two other rows cover the belly, from the
anterior region of the anal fin to the region
below the pectoral fin where several other
rows of smaller scales occur. The fins are not
covered by scales except for the basal part of
the caudal fin. Details on the development of
the squamation pattern in zebrafish are
given elsewhere (Sire et al., in press).
Scale surface ornamentation (SEM)
In the pectoral region of a 37 mm SL adult
zebrafish, the scales are roughly rectangular
(1.7 mm wide, 2.1 mm high in Fig. 2) with a
convex posterior region. The scale surface is
separated into two regions: a small anterior
region, which is overlapped by the preceding
and neighboring scales, and a large posterior region, which overlaps the scale behind
and is covered by the epidermis. These regions are characterized by different ornamentations: thin ridges in the anterior region, and grooves and undulations (here
called ripples) in the posterior region (Fig.
2). The ridges, also called circuli, are not
numerous (,20 circuli on the scale in Fig. 2).
They are thin elevations (1.5 µm) that are
disposed around the center of the scale, the
focus, and are parallel to one another and to
the scale margin (Fig. 3). They are tightly
but regularly spaced on the most anterior
region of the scale and more widely spaced
in the lateral parts. The crests of the circuli
are ornamented by numerous, small serrations, ,0.5 µm wide (Fig. 4). In the scales of
adult specimens described here, the focus is
located closer to the anterior rim than to the
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posterior one. This is not the case in young
specimens (personal observations). The focus has an irregular surface from which
issue radial grooves (Figs. 2, 3), also called
radii, which either radiate directly from the
focus or, in the lateral regions, at some distance from it. They cross the posterior region
of the scale to reach its margin perpendicularly. Short ‘‘extra’’ radii are also present
close to the posterior margin, dividing the
space between two radii (Fig. 2). The radii
are thin (10 µm wide), roughly straight
grooves (Figs. 2, 3, 5) in which the superficial layer of the scale is lacking. Each groove
is bordered on both sides by a narrow (5 µm
wide), convex crest with an irregular, rough
surface (Fig. 6). The undulations, or ripples,
are only visible in the posterior region of the
scale (Fig. 2). They are small, regularly disposed, parallel one another and to the scale
margin. Their surface seems smooth at low
magnification (Fig. 7), but at a higher magnification appears covered by numerous small
granules of variable diameter (100–500 nm)
(Fig. 8).
Scale location in the skin
The zebrafish scales are located in the
outer part of the dermis, and roughly parallel the skin surface (Figs. 9–11). They are
inserted into spaces in the dermis (scale
pockets) completely bounded by organized
mesenchymal tissues except posteriorly, on
the outermost surface, where the basal layer
of the epidermis contacts the scale directly.
Hence, a great percentage of the upper scale
surface is covered by the epidermis, which
moreover forms a fold around the free posterior margin of the scale (Fig. 9). On the
upper surface, the epidermis is several cell
layers thick with numerous specialized cells,
mainly mucous and club cells. In contrast,
the epidermal fold located at the deep scale
surface is only two cell layers thick and
devoid of specialized cells (Figs. 9, 10). The
dense dermis (5stratum compactum) forms
a relatively thin layer (50–100 µm thick) in
the deep region of the skin facing the lateral
musculature and below the scales. Sheets of
loose dermis (5stratum laxum) emanate
from the dense dermis and separate the
scale pockets from one another. Posteriorly,
these dermal sheets are connected to the
epidermal folds. A thin layer of loose connective tissue is also present between the deep
surface of the scale and the epidermal fold in
the posterior region (Fig. 10). The stratum
laxum is composed of a loose network of
collagen fibrils in which elongated fibroblasts, pigment cells, and capillary blood
vessels are located. Only the very anterior
region of the scale is surrounded by dense
dermis. The bottom of each scale pocket is
covered by a thin cellular sheet, called the
scale pocket lining (Whitear et al., ’80), which
separates the pocket from the dermis below
(Fig. 10). This ‘‘epithelium’’ is composed of a
single layer of flat fibroblast-like cells linked
by desmosomes, and it is covered on both
sides by a thin basement membrane, as previously described in other teleost fish (Whitear et al., ’80; Sire, ’89b). The scale pocket
lining is lacking in the posterior region facing the epidermal fold (Fig. 10). The deep
surface of the scale is covered by flat scaleforming cells, which form a thin ‘‘epithelium’’ (5hyposquama of Waterman, ’70). Pigment cells are frequently seen in the space
between the hyposquama and the scale
pocket lining cells (Fig. 10). In the anterior
region, isolated scale-forming cells (5episquama of Waterman, ’70) cover the scale
surface where it is at a distance from the
epidermis or covered by the neighboring
scales. The episquamal cells are not seen at
Figs. 2–8. Scanning electron micrographs of Danio
rerio scale surface after sodium hypochlorite treatment.
Scales from the pectoral region of a 37 mm SL specimen,
in the region indicated by an asterisk in Figure 1.
Anterior margin is to the left.
Fig. 2. The surface of the zebrafish scale is separated
into two regions by the anterior limit of the epidermal
cover (dotted line). The anterior region is characterized
by circular ridges, the circuli (c). The posterior region is
ornamentated with radial grooves, the radii (r) and
semi-circular undulations, or ripples (s). f, focus.
Fig. 3. Anterior region showing the circuli (c) and
the focus (f), from which some radii originate (r).
Fig. 4. The surface of a circulus (c) is irregular and
shows small serrations (arrows).
Fig. 5. In the posterior region, several radii (r) interrupt the semi-circular ripple(s).
Fig. 6. A radius is composed of a central groove
surrounded by two convex crests showing an irregular
surface.
Fig. 7. The ripples are slight undulations of the scale
surface without any prominent ornamentation.
Fig. 8. High magnification of the scale surface in the
posterior region showing numerous small, rounded granules. Bars 5 200 µm (345; Fig. 1), 100 µm (3120, Fig. 5),
50 µm (3250, Fig. 3), 10 µm (31,100, Fig. 7), 2 µm
(35,000, Fig. 4, 34,000, Fig. 6), 1 µm (38,500, Fig. 8).
FINE STRUCTURE OF ZEBRAFISH SCALE
Figures 2–8
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J.-Y. SIRE ET AL.
Fig. 9. Schematical drawing from 1-µm-thick, serial, longitudinal sections of the skin in
adult Danio rerio (female, 37 mm SL) showing the relationships of the scales with the
surrounding tissues. d, dermis; e, epidermis; l, lipid; m, muscle; sp, scale pocket.
the scale surface where it is covered by the
epidermis (Fig. 10).
Scale structure
Descriptions of the scale structure proper
can be found in Waterman (’70), but the
general organization is briefly recalled here
for better understanding.
Both in transverse and longitudinal sections, the zebrafish scale appears to be
clearly constituted of two regions: a superficial, thin, well-mineralized, woven-fibered
layer, called the external layer, and a deep,
thick, partially mineralized, lamellar layer,
called isopedine (Figs. 10, 11). In the latter
the collagen fibrils are regularly arranged
into layers forming a plywood-like structure
in which the direction of the fibrils changes
from one layer to another by ,90°. The mineralization progresses only a short distance
from the external layer downward and a
large part of the isopedine is unmineralized,
even in adults. Cells are never observed in
the scale tissues. The scale organization is
interrupted at regular intervals by radial
grooves. Here, the well-mineralized external
layer is replaced by an unmineralized, loose,
collagenous tissue, whereas the isopedine
below is entirely unmineralized and its collagenous matrix devoid of background substance (Figs. 10, 11). The surface of the radii
is covered by a roughly convex dermal space
containing fibroblast-like cells and blood vessels embedded within a loose matrix. These
grooves provide the only places where dermal components are located at the scale
surface in areas where it is covered by the
epidermis. Indeed, elsewhere on its whole
posterior region, the scale surface is only
separated from the epidermal basal layer
cells by a 100-nm-thick layer that looks like
a basement membrane (Figs. 12, 13), which
is fairly ‘‘typical’’ on Figure 17.
When decalcified, the woven-fibered external layer of a mature scale is seen to be
covered by a thin layer (1 µm thick in average) that lacks collagen fibrils but is rich in
an electron-dense, fine, granular material
(Fig. 13). This layer is similar to the limiting
layer previously described in cichlid scales
(Schönbörner et al., ’79; Sire, ’85), and we
have chosen to conserve this name. Some
granules are aligned within this limiting
layer, and they form dense lines parallel to
the scale surface (Fig. 13). The surface of
this layer is in direct contact with the basement membrane-like structure immediately
below the epidermal basal layer cells. The
mineral crystals located in the limiting layer
of the scale are oriented perpendicularly to
its surface, and this contrasts with the random disposition of the mineral crystals
within the woven-fibered external layer below (Fig. 12; see also Fig. 17). The crystals
appear to be attached directly to the deep
surface of the basement membrane.
In 4-day regenerated scales the epidermis
is already in contact with the upper surface
in several regions (Fig. 14). Mineralization
has started in this region of the scale but
only a few crystals are deposited (Fig. 15).
This loose organization of the crystals facilitates sectioning and allows better observation of the features at the scale surface than
FINE STRUCTURE OF ZEBRAFISH SCALE
167
Fig. 10. Detail of the framed region in Figure 9, schematically drawn from thin sections.
The rectangle is detailed on Figures 12 and 13. e, epidermis; ef, epidermal fold; ld, loose dermis;
pc, pigment cell; r, radius; sc, scale; sfc, scale-forming cell; sp, scale pocket; spl, scale pocket
lining.
in mature, well-mineralized scales. Patches
of thin granular matrix, organized into small
spherules or ovoid structures, are observed
at the scale surface, in the region immediately below the epidermal basal layer (Fig.
15). These patches contain radially oriented
mineral crystals forming ‘‘urchin-like’’ structures that differ from the randomly disposed
crystals in the collagen matrix of the external layer of the scale (Fig. 16). This repre-
sents the anlage of the limiting layer. Facing
the scale surface, the epidermal basal layer
cells are cuboidal and juxtaposed. Their cytoplasm is rich in organelles such as RER
cisternae, mitochondria, Golgi saccules, and
small vesicles, some of them fusing with the
cell membrane, which has irregular contours. There is no lamina densa of the basement membrane interposed between the epidermal basal cells and the scale surface. In
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Fig. 11. One-µm-thick section of the skin from a 35
mm SL Danio rerio (undecalcified sample). Scales are
parallel to the skin surface and their well-mineralized
superficial layer is closely apposed to the epidermal (e)
basal layer cells (arrows). Radial grooves are visible
(arrowheads). Bar 5 50 µm (3300).
Figs. 12, 13. Danio rerio. Transmission electron micrographs of the region indicated by a rectangle in
Figure 10. The asterisks in these figures indicate the
same locations.
Fig. 12. Undecalcified sample. The mineral crystals
in the limiting layer at the surface of the superficial
region (*) are densely packed. This organization contrasts with the randomly disposed mineral crystals in
the external layer below. e, epidermis.
Fig. 13. EDTA decalcified sample. The limiting layer
of the scale (*) is rich in fine granules that in some areas
are linearly arranged, forming dense layers (arrowheads). This matrix contrasts with that of the external
layer(s) below. Moreover the scale surface is in direct
contact with the epidermal (e) basal layer cells (arrows).
Bars 5 250 nm (340,000, Fig. 12; 360,000, Fig. 13).
Fig. 14. Danio rerio. One-µm-thick section of the
skin containing a 4-day-regenerated scale. It is already
well formed and in some areas, the epidermis (e) is in
contact with their surface (arrows). d, dermis. Bar 5 50
µm (3300).
Figs. 15–17. Danio rerio. Transmission electron micrographs of 4 day- (Figs. 15, 16) and 7-day- (Fig. 17)
regenerated scales in the region covered by the epidermis (e). Undecalcified samples.
Fig. 15. Cuboidal, juxtaposed epidermal basal layer
cells are directly facing (arrows) the superficial layer(s)
of the scale that shows rounded patches of granular
matrix. i, isopedine.
Fig. 16. Detail of the epidermis/scale limit as in
Figure 15. Patches of thin granular matrix (arrows) are
located immediately adjacent to invaginations (arrowheads) of the plasmalemma of the epidermal cells. These
patches are invaded by mineral crystals that are radially arranged. There is no basement membrane between
the epidermal cells and the scale matrix.
Fig. 17. Detail of the upper region of a well-formed
scale during the initial phase of matrix mineralization.
A lamellar basement membrane-like structure (arrowheads) constitutes the only interface between scale and
epidermis. The arrow points to an urchin-like structure
located close to the epidermal basal layer cells. Elsewhere at the scale surface, the crystals are perpendicular to the deep surface of the basement membrane.
Bars 5 1 µm (39,000, Fig. 15); 250 nm (360,000, Figs.
16, 17).
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7-day regenerated scales the limiting layer
is thicker and more mineralized than previously. The urchin-like structures are rare
and the mineral crystals are now perpendicularly oriented towards the scale surface. The
plasmalemma of the epidermal basal layer
cells lining the scale matrix is more regular
than in the previous stage and the anlage of
the basement membrane-like structure is
appearing (Fig. 17).
DISCUSSION
Our SEM study of the scale surface and
the detailed description of the upper layer of
the scale using TEM completes and adds
information to the previous description of
the scale structure in zebrafish (Waterman,
’70). Moreover, the present work brings new
data on the relationships between the scale
surface and the epidermis in teleost fish.
Indeed the observations presented above
strongly suggest that the epidermal basal
layer cells produce substances that are directly incorporated into the superficial region of the scale.
Scale structure and location
In zebrafish, a cyprinid, the scales are
relatively large compared to the size of the
fish, they are superficially located in the
skin, they are easy to manipulate, and they
regenerate as rapidly as do the scales of
cichlid fish (Sire and Géraudie, ’84). Moreover they obviously belong to the elasmoid
type as defined by Bertin (’44), and their
structure is similar to that found in all elasmoid scales described until now in teleosts
(review in Meunier, ’83; Whitear, ’86; Sire,
’87). These findings, and the recent description of developmental stages (Sire et al., in
press), demonstrate that zebrafish scales are
favorable material for further investigations
dealing with scale biology in general, and
scale development in particular.
In zebrafish scales, the surface of the posterior region is relatively smooth except
along the radial grooves. These grooves are
thought to have two important roles, i.e.,
mechanical and trophic. First, the absence
of mineralization at their level allows the
large posterior region of the scale to be more
flexible; second, nutrients can be brought to
the epidermis through the dermal components which are located at their surface.
Elsewhere, the epidermis is in direct contact
with the scale surface, from which it is only
separated by a basement membrane. This
surface lacks the typical ornamentations that
have been described in this region for other
elasmoid scales (e.g., Hughes, ’81; Sire, ’86;
Lippitsch, ’92). The ornamentation of the
scale surface seems to be related to the distance from the epidermis. The surface is
smooth in zebrafish where the epidermis is
in close contact with the scale, and it is
ornamented where the epidermis is at a
short distance as, e.g., in cichlids (Sire, ’86).
Such a relationship is also illustrated by the
scales in some Clupeiformes. They have a
smooth surface in the region covered by the
epidermis, and this corresponds to a tight
covering of the scale by epidermis (Sire, personal observations). In the ornamented
scales of cichlids, tubercles develop around
anchoring fibers that attach the epidermis
to the scale surface (Sire, ’85, ’86). Such
anchoring fibers are not present in zebrafish,
and one can ask how the epidermis maintains its relation to the scale surface. A similar question has been raised in primitive
osteichthyans, the polypterids and the lepisosteids, where the epidermis also is in close
contact with the surface of ganoid scales
(Sire et al., ’87; Sire, ’94). Here, a characteristic basement membrane-like structure
separates the well-mineralized scale surface, ganoine (an enamel), from the epidermal basal layer (Zylberberg et al., ’85). This
unmineralized layer has been called the ganoine membrane (Sire, ’94), and it probably
contains adhesive substances that allow the
epidermis to remain in contact with the scale
surface during swimming activity (Zylberberg et al., ’85; Sire, ’94). The thin basement
membrane-like structure described in the
present study, at the epidermis-scale interface in zebrafish, could have a similar function.
From a comparison of the data available
on the fine structure of the elasmoid scales
in teleosts (e.g., Brown and Wellings, ’69;
Yamada, ’71; Kobayashi et al., ’72; Lanzing
and Wright, ’76; Schönbörner et al., ’79; Zylberberg and Meunier, ’81; Sire and Géraudie, ’83; Zylberberg et al., ’84), it appears
that the extent of the so-called limiting layer
at the external layer surface is directly related to the epidermal covering, i.e., the limiting layer exists only on the scale surface,
that is directly covered by the epidermis or
lies at a small distance from it (see also Sire,
’85). Elsewhere, i.e., in the anterior part of
the scale not covered by the epidermis, the
FINE STRUCTURE OF ZEBRAFISH SCALE
external layer proper is only present. The
degree of development of the epidermal covering also depends on the position of the
scale in the dermis and on the degree of
imbrication of the scales, both being related
to the ecological adaptation of the species,
mainly with regard to a mechanical protection against the substratum (Burdak, ’79).
In this way, thick scales, a large extent of
mineralization and a thick limiting layer,
are interpreted as a reinforcement of the
protection of the skin (Sire, ’85, ’86). It would
appear that zebrafish scales, which are superficially located in the dermis, are thin,
incompletely mineralized, and have a poorly
developed superficial layer, probably constitute a poor mechanical protection.
A general tendency toward reduction of
the dermal skeleton is observed during the
evolution of several fish groups, and it is
largely admitted that such a reduction permitted the exploitation of a wide range of
ecological niches (e.g., Schultze, ’77; Meunier, ’83). Zebrafish scales could represent a
type of reduction of the scale cover that
results in superficialization in a small-size
fish, in contrast to another type of reduction
that results in deep embedding in the dermis (as in Anguilla anguilla, e.g.) (Zylberberg et al., ’84).
Scale-epidermis relationships
The present ultrastructural study of the
relationships of the epidermis to the scale
surface in zebrafish scales shows that (1) in
adult specimens, the epidermis is in direct
contact with the whole outer surface of the
posterior region of the scale, from which it is
separated only by a thin basement-membrane like structure, (2) there are no dermal
cells in between except opposite the radial
grooves, (3) the limiting layer is devoid of
collagen fibrils, but it is rich in fine granules
arranged into electron-dense lines suggesting a periodical deposit, (4) in this layer, the
mineral crystals are oriented perpendicularly to the scale surface, and (5) in regenerating scales the epidermal basal layer cells
are directly in contact with the scale surface
within 4 days after scale removal, and they
show evidence of protein synthesis in the
vicinity of the scale surface. The latter has
thickened after 7 days of regeneration.
These morphological observations strongly
suggest the existence of epidermal products
that are deposited at the surface of the external layer of the scales in zebrafish, thus
constituting a thin limiting layer.
171
Epidermis/scale surface relationships
In adult zebrafish, the epidermal basal
layer cells are always seen in contact with
the posterior region of the scale surface, but
they do not seem to be synthetically active,
judged by a cytoplasm that is poor in organelles and rich in microfilaments (probably
cytokeratins). An intimate epidermal covering has been reported for this species by
Waterman (’70), but without further description or comment. Experimentally regenerated scales have been found to be a useful
tool, because at a given time they repeat the
events that occur during ontogeny, albeit on
a larger scale (Sire and Géraudie, ’84; Sire et
al., ’87; Sire, ’94). This technique allowed us
to show that the epidermal basal layer cells
rapidly contact the scale surface and that
they are involved in the deposition of some
substance on it. This conclusion is based on
the fact that (1) they have a cytoplasmic
content rich in organelles known to be involved in protein synthesis, (2) they immediately contact the scale surface with an irregular contour of the cell membrane, after 4
days, and (3) the limiting layer is seen to be
thicker after 7 days without any major
change in the epidermal cells. In adult specimens, the ‘‘inactive’’ aspect of the epidermal
cells is undoubtedly related to a slowing
down, or an arrest, in the deposition of epidermal products at the scale surface, because such substances probably are only deposited periodically (see below).
Recent observations of the scale surface in
some Clupeiformes have also revealed a direct covering by the epidermis (Sire, personal observations), but in the other elasmoid scales described until now (see reviews
in Whitear, ’86; Sire, ’87), the outer layer of
the posterior region was always found to be
separated from the epidermis by a narrow
dermal space. However, in the cichlid Hemichromis bimaculatus there are indications
of possible epidermal basal layer cell participation in scale material deposit (Sire, ’88).
Moreover, the fine structure of the scale is
known only for a few teleosts compared to
the 23,000 or more species that exist (Nelson, ’94). Among these works little attention
is paid to the relationship between the epidermis and the scale surface (Sire, ’85, ’88),
because these studies are generally concerned with systematics (e.g., Hughes, ’81;
Lippitsch, ’92, ’93).
Until now, direct epidermal contact has
only been described in the ganoid scales of
172
J.-Y. SIRE ET AL.
polypterids and lepisosteids. Consequently,
this would be a plesiomorphic character for
teleosts. The superficial layer of the ganoid
scales, ganoine, has recently been demonstrated to be entirely formed by the inner
epidermal cells, and consequently to be ‘‘true’’
enamel (Sire et al., ’87; Sire, ’94, ’95) as in
tetrapod teeth. In zebrafish, the events occurring at the scale surface during regeneration
are similar to those described during the
deposit of the ganoine: active, roughly cuboidal cells are in direct contact with the scale
surface, deposition of substances, then formation of a basement membrane-like layer
at the interface, and a rest phase. The only
difference we could find is related to the
amount of material produced in the surfaces
of the two scales: a thin (,1 µm in adult)
limiting layer in zebrafish compared to a
thick (,100 µm) ganoine layer in polypterids.
Formation of the limiting layer
In elasmoid scales, as first reported by
Schmidt (’51) and Lerner (’53) using polarized light, the limiting layer covering the
posterior region of the scale is characterized
by the organization of the mineral crystals
perpendicular to the scale surface. Using
TEM, Schönbörner et al. (’79) described this
superficial layer as poor in collagen fibrils
and called it the external limiting layer.
Detailed studies of this layer in cichlids have
shown that it is thick, devoid of collagen
fibrils, and it develops preferentially around
the numerous anchoring fibers that form at
the scale surface and reach the epidermaldermal boundary (Sire, ’85, ’86). Moreover,
calcified spherules, containing substances
that are probably epidermal in origin, have
been seen to contribute to the periodical
thickening of the limiting layer (Sire, ’88). In
zebrafish, the limiting layer is thinner than
in cichlid scales (Sire, ’85), but the other
characteristics are similar: absence of collagen fibrils; matrix composed of thin granules, some being arranged in layers; and
mineral crystals perpendicular to the scale
surface. Recently, a similar matrix also has
been described to constitute a thick superficial layer in the scutes of armoured catfish.
This layer was called hyaloine (Sire, ’93).
Moreover, the first elements of the matrix
deposited in the limiting layer of the zebrafish scale, and the mineralization process, look similar to those described for gan-
oine in ganoid scales (Sire, ’95), namely,
patches of matrix and mineral crystals with
radial or urchin-like organization are deposited first. Then, these patches fuse with one
another as matrix is added to form a layer in
which the mineral crystals are oriented perpendicularly to the scale surface. Also, the
layers of granules within the limiting layer
strongly support the existence of a periodical deposition of the matrix, as is the case in
the limiting layer of cichlid scales (Sire, ’85,
’88), in the hyaloine of armored catfish (Sire,
’93), and for the deposition of ganoine on the
surface of the ganoid scales (Sire, ’94).
Evolutionary implications
All the comparative data presented above
show that: (1) the limiting layer at the scale
surface in zebrafish is similar in structure to
the limiting layers in other elasmoid scales
and to the hyaloine in callichthyid scutes,
and (2) the matrix deposit and the mineral
organization in the limiting layer of zebrafish scales look similar to ganoine deposit in ganoid scales. Thus, on the one hand,
the limiting layer of the zebrafish scale can
be considered as representative of this layer
in elasmoid scales in teleosts, and on the
other hand, it appears closer to the ganoine
of the scales in primitive actinopterygians
than previously suspected.
Given: (1) that it is widely believed that
elasmoid scales are derived from ganoid-like
(rhombic) scales (Schultze, ’77; Reif, ’82;
Smith and Hall, ’90), (2) that the elasmoid
scales, at least in part, are probably derived
from ‘‘odontodal’’ tissues that covered these
rhombic scales (Sire, ’89a, ’90), (3) that in
zebrafish scales, epidermal substances are
deposited in the limiting layer, and (4) that
this layer forms like ganoine, then the limiting layer of the elasmoid scales can be
derived from ganoine covering the dermal
skeleton of the more plesiomorphic actinopterygians, and it should contain enamel-like
proteins.
ACKNOWLEDGMENTS
We are indebted to Mary Whitear (Tavistok, UK) and Ann Huysseune (Gand, Belgium) for their constructive remarks and
English corrections. We thank Olivier Babiar for his technical assistance in rearing
zebrafish. TEM and the photographic work
have been done at the CIME (Centre Interuniversitaire de Microscopie Electronique,
Universités P6/P7, Paris).
FINE STRUCTURE OF ZEBRAFISH SCALE
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