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Light and TEM study of nonregenerated and experimentally regenerated scales of Lepisosteus oculatus (holostei) with particular attention to ganoine formation.

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THE ANATOMICAL RECORD 240:189-207 (1994)
Light and TEM Study of Nonregenerated and Experimentally
Regenerated Scales of Lepisosteus oculatus (Holostei) With
Particular Attention to Ganoine Formation
Laboratoire d’Anatomie comparee, Uniuersitk Paris 7, C N R S , U R A 1137, 75251 Paris
cedex 05, France
Background: The structure of nonregenerated and experimentally regenerated scales of the holostean fish Lepisosteus oculatus and
the events taking place before and during ganoine deposition on the scale
surface were studied. The aim of this study was to answer the question of
the origin of the ganoine in lepisosteids, the scales of which are devoid of
dentine, and to compare them to ganoine formation in polypterid scales
and to enamel formation in teeth.
Methods: Two adult specimens were used and the scale structure was
studied using light and transmission electron microscopy. Regeneration
was used as an alternative to the lack of developmental stages and to induce ganoine deposition on the scale surface.
Results: Nonregenerated scales are composed of a thick, avascular bony
plate capped by ganoine that is covered either by the epidermis or by dermal elements. The ganoine surface is separated from the covering soft tissues by an unmineralized layer, the ganoine membrane. During the first 2
months of regeneration, the bony plate forms. It differs from the bony plate
of nonregenerated scales only by its large, woven-fibered central region
and by the presence of numerous vascular canals. Shortly before ganoine
deposition, the osteoblasts cease their activity and an epithelial sheet
comes to contact them and spreads on the bony surface. This epithelial
sheet is connected to the epidermis by a short epithelial bridge only and is
composed of two layers: the inner ganoine epithelium (IGE),in contact with
the bone surface and composed of juxtaposed columnar cells that synthesize the ganoine matrix, preganoine; the outer ganoine epithelium (OGE),
composed of elongated cells, the surface of which is separated from the
overlying dermal space by a basal lamina. Isolated patches of preganoine
are deposited by the IGE cells in the upper part of the osteoid matrix of the
scale. The interpenetrated preganoine and osteoid matrices constitute an
anchorage zone between ganoine and bone. Preganoine patches fuse and a
continuous layer of preganoine is progressively synthesized by the IGE
cells. Preganoine progressively mineralizes to become ganoine.
Conclusions: The processes of ganoine formation are similar to those
known for the ganoine in the polypterid scales and to those described for
enamel deposition in teeth. Ganoine is enamel. o 1994 WiIey-Liss, Inc.
Key words: Lepisosteus oculatus, Scales, Scale regeneration, Ganoine formation, Transmission electron microscopy
The scales of the living lepisosteid fishes (Holostei) Williamson that exist only in the bone of holostean
are thick bony plates covered by a stratified, highly fishes (see the study by Sire and Meunier, 1994).
The structure and organization of the scales in lepimineralized, layer of ganoine, a name first given by
Williamson (1849). Among osteichthyan scales they sosteids have been known for a long time (Agassiz,
are of a primitive type and are classified a s ganoid
scales (along with, e.g., the polypterid scales) in which
they represent the lepidosteoid type (Goodrich, 1907).
Received February 2, 1994; accepted May 3, 1994.
They differ from the palaeoniscoid scales of the PolyAddress reprint requests to Dr. J.-Y. Sire, Universite Paris 7, Lapteridae in the lack of dentine, in the absence of vas- boratoire dAnatomie comparee, Case 7077, 2 Place, Jussieu, 75251
cular canals, and in the presence of canaliculi of Paris cedex 05, France.
1833-44; Williamson, 1849; Reissner, 1859; Hertwig,
1879; Klaatsch, 1890; Nickerson, 1893; Stephan, 1990;
Goodrich, 1907). The presence of the ganoine layer
early caught the attention of these authors, both with
regard to the source and nature of this outer layer and
to the relationships existing between the ganoid scales
and the scales of Selachians, Dipnoi, and Teleosteans.
Agassiz (1833-44), Reissner (1859), and Hertwig
(1879) and later Gross (1935) believed that ganoine is a
true enamel layer derived from the overlying epidermis, but Klaatsch (1890) cast doubt on this by showing
its dermal origin. Nickerson (1893) was the first to
study the scale development in Lepisosteus using 10
different fishes of various sizes. In fish presenting a
ganoine layer, he found that “the epidermis was distinctly separated from the scale by a thin layer of dermal scleroblasts seldom over two or three cells thick,”
which led him to the conclusion that the ganoine layer
“is secreted, not by epidermis, but by cells of dermal
origin. Hence i t is not enamel in the modern sense of
the term, but may better be known by the name of
ganoin.” Nickerson was thus compelled to admit that
osteoblasts give rise to the bone of the basal plate and
to the ganoine. Goodrich (1907) thought that to call the
outer layer true enamel was a fundamental error and
agreed with the conclusions of Nickerson (1893). Half a
century later, Kerr (1952) in the course of a re-examination of the calcified tissues of the scales of primitive
living actinopterygians remarked t h a t “ganoine does
resemble enamel, but only in being a very hard substance produced by a great reduction in organic content. It differs from enamel in being laid down in layers
throughout life, in being not necessarily formed in association with dentine, and in being possibly mesodermal in origin.” Kerr agreed with the conclusions of
Nickerson (1893) and Goodrich (1907). 0rvig (1967) in
his work on the phylogeny of tooth tissues concluded
that “Ganoin is thus a hard tissue which in structure,
and presumably also in mode of formation, shows
marked resemblance to the enameloid substances and
which also derives from the mesoderm.” To my knowledge, the last published work dealing with the formation of the ganoine in the scales of living Lepisosteus
species was by Thomson and McCune (1984). They
gave information on the histology and mode of growth
of scales, and they described that the “ganoine is
formed on the inner surface of a cell layer, apparently
mesenchymal in origin.”
Recent studies concerning the ganoine formation in
the scales of the polypterid Calamoichthys calabaricus,
another primitive actinopterygian fish, have clearly
demonstrated that ganoine in this fish is a n epidermal
product and, consequently, true enamel (Sire et al.,
1986,1987). The question now is to know whether the
ganoine that covers the scale of lepisosteids is also a n
epidermal product and therefore true enamel; the answer will close a discussion that has lasted more than
a century. The present work describes the process of
regeneration of the ganoid scale in the holostean fish
Lepisosteus oculatus, and it focuses mainly on the ganoine formation and on the relationships among epidermis, dermis, ganoine, and the upper surface of the
bony plate. Moreover, I have taken advantage of this
ultrastructural study to describe some additional details on nonregenerated scales of this fish species. Com-
parisons are made to ganoine formation in ganoid scales
of Polypteridae and to enamel formation in teeth.
Scale regeneration was induced in two Lepisosteus
oculatus (Winchell), the spotted gar, (225 and 250 mm
standard length) raised in a tank a t 25°C and fed daily
with small portions of frozen fish. The fishes were anaesthesized in MS-222, and eight nonregenerated scales
were dissected out on the posterior part of the left flank
with a scalpel and fine scissors. These scales were used
a s control, nonregenerated scales. The fishes rapidly
recovered from surgery and did not show infection. The
regenerating scales were similarly removed 1 , 2 , and 3
months after the first surgery to follow ganoine formation (Sire et al., 1987).Two to three regenerating scales
were removed for each sample.
Nonregenerated and regenerated scales were fixed
for 2 hr a t room temperature in a mixture of 1.5%glutaraldehyde and 1.5%paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 0.1 ml of 0.5%
CaC12. Although decalcification removes the ganoine
layer completely (the organic matrix disappears along
with the mineral), it was necessary in order to facilitate sectioning and to leave the structure of the scale
intact. Some scales were not decalcified and they were
rinsed for 20 min in the buffer containing 10% sucrose.
The other samples were demineralized after a 2-hr fixation as above, by immersion for 7 days a t 4°C in the
fixative solution to which 0.1 M EDTA was added (pH
7.4). This solution was renewed daily. Decalcified samples were subsequently rinsed for 24 h r at 4°C in the
buffer containing 10%sucrose. Postfixation of both decalcified and undecalcified samples was performed for 2
h r at room temperature in 1% osmium tetroxide in cacodylate buffer containing 5% sucrose. Samples were
next dehydrated in a graded series of ethanol, then
embedded in Epon.
Sections (1 km thick) were stained with toluidine
blue in order to observe the organization of nonregenerated and regenerated scales and to select appropriate
areas for transmission electron microscopy (TEM).
Thin sections were contrasted with uranyl acetate and
lead citrate and examined at 80 kV with a Philips 201
electron microscope.
Nonregenerated Scales
The structure of the scales of Lepisosteus oculatus
(Fig. 1)is similar to that already known for other living
lepisosteid fishes (e.g., Nickerson, 1893; Goodrich,
1907). The scales are thick bony plates covered by ganoine. They do not possess any kind of dentine. The
bony plate contains numerous osteocytes and their processes, and it is characterized in the mature condition
by the scarcity of vascular canals and by the presence
of canaliculi of Williamson (Figs. 1 , 2 ) .The latter house
the cytoplasmic processes of the typical cells of
Williamson, which are located at the scale surface and
for which a nutritive function, probably compensating
for the lack of vascular canals, has recently been attributed (Sire and Meunier, 1994). Each scale is firmly
attached to the dermis and to its neighbour by large
Sharpey’s fibers issuing from the deep and lateral regions of the scales. This explains the difficulties en-
countered when trying to remove the scales without
injuring the fish. Ganoine is composed of several layers, which are deposited episodically, but these are not
visible on 1-pm-thick decalcified sections. However,
the rim of each ganoine layer is partially overlapped by
bone. On 1-pm-thick vertical sections, these layers appear as a series of short serrations (Fig. 3). The structure of the bony plate and of the cells of Williamson and
their canals in Lepisosteus oculatus has already been
described in a previous work (Sire and Meunier, 1994);
consequently the following descriptions concern only
the superficial region of the scale and the surrounding
soft tissues, dermis, and epidermis.
The epidermis is thick (250 pm on average) and is
characterized by the presence of numerous, large sacciform cells and some mucous cells (Figs. 1-3). Scarce
pigment cells (melanocytes) are also present. The basal
region of the epidermis shows a layer of more or less
regular cells, the basal layer. These plump cells show a
large nucleus and their cytoplasm is electron-dense
and contains bundles of microfilaments, numerous free
ribosomes, some mitochondria, and some cisternae of
the rough endoplasmic reticulum (RER) (Fig. 4).
Neighbouring cells are interdigitated and linked by
short desmosomes. The epidermis either covers the ganoine (Fig. 4) or is separated from i t by dermal elements (see below).
Where epidermis covers the ganoine, the basal layer
cells are in contact with a n unmineralized, 500-nmthick layer that borders the ganoine surface; I follow
Nickerson (1893) in calling this peculiar layer the ganoine membrane (Figs. 3-6). From the plasmalemma
of the basal layer cells toward the ganoine surface, the
ganoine membrane is composed of (1)a 50-nm-thick,
rather clear zone containing thin dark granules and
filaments, and close to this zone the plasmalemma of
the basal layer cells is electron-dense and some caveolae are fusing with it, (2) a 50-nm-thick, striated zone
with lines of dark granules parallel to the scale surface
separated by electron-lucent spaces, and (3)a 400-nmthick zone that is the main part of the ganoine membrane and is constituted of a homogeneous thin granular substance (Fig. 5). The inner surface of the latter
zone is irregular and underlined by a dark line. This
surface corresponds to the interface between the ganoine membrane and the (mineralized) ganoine matrix, which has been totally removed (mineral plus
organic matrix) after EDTA decalcification. Undecalcified samples reveal that the mineral crystals of the
ganoine are disposed perpendicularly to the ganoine
membrane (Fig. 6).
In several samples dermal elements are interposed
between the ganoine membrane and the epidermis
(Fig. 7). This dermal layer contains various cell types
(fibroblasts, capillary blood vessels, pigment cells) surrounded by a loose extracellular matrix in which scarce
striated collagen fibrils are visible. A 80-nm-thick
basal lamina separates the basal layer cells from the
dermal layer. The basal layer is composed of irregularly shaped cells that are separated by large intercellular spaces. These cells are linked by short desmosomes and they show numerous cytoplasmic extensions
(Fig. 8).The structure of the ganoine membrane is conserved when the basal layer cells are separated from its
upper surface (Fig. 9). The fibroblasts located in the
dermal space close to the ganoine membrane establish
short contacts with the outer surface of its upper zone
(Fig. 7, inset). On the borders of the ganoine layer,
osteoblasts are located on the outer surface of the ganoine membrane. They synthesize bone matrix that is
deposited on the surface of the ganoine membrane (Fig.
10). The bony material covers a small region of the
ganoine surface and it mineralizes. This deposit gives
rise to a ganoine serration covered by bone, a s described a t the light microscope level (cf. Fig. 3). The
ganoine membrane is conserved between the ganoine
and the bone during the processes of bone deposition
and mineralization, and it is still clearly visible after
decalcification (Figs. 11, 12). In contrast, the structure
of the ganoine membrane is modified when a new layer
of ganoine is deposited on the ganoine surface (Fig. 12)
and here i t disappears completely after EDTA decalcification.
The bony plate of the scale enlarges by addition of
new bone matrix on the lateral and deep surfaces only.
Laterally the osteoblasts are roughly organized into
one layer. They are 3-4 pm high cuboidal cells
equipped with numerous RER cisternae, Golgi saccules, and mitochondria (Fig. 13). Bundles of collagen
fibrils, issuing from the bone surface, cross the osteoblast layer and pass downward within the adjacent dermis. The unmineralized, -3-pm-thick layer of osteoid
is composed of a loose network of collagen fibrils,
40-50 nm in diameter (Fig. 14). The osteoblasts show
numerous cytoplasmic extensions that penetrate the
osteoid, a zone where some osteoblasts can be entrapped; these become osteocytes. The mineralization
progresses within the bone matrix following a rather
regular front and the mineral crystals are orientated
along the collagen fibrils (Fig. 14). On the lower surface of the deep region, the osteoblasts are rectangular,
elongated, 1- to 2-pm-thick cells showing some RER
cisternae but rare cytoplasmic extensions (Fig. 15). In
this region the osteoid is 1 pm thick.
Regenerated Scales
The main goal of this work being to study ganoine
formation, the first steps of scale regeneration are commented upon only briefly and not illustrated. The removal of some ganoid scales, even carefully done, implies injury to the tissues associated with the scale,
mainly due to sectioning of the Sharpey’s fibers and
capillary blood vessels. Wound healing of the dermis
generally requires half a month (the period is rather
variable) before the first elements of the regenerated
scales appear (Sire, unpub. data). Newly recruited osteoblasts (of unknown origin) accumulate in the middle
region of the regenerated dermis. They first synthesize
a loose woven-fibered collagenous network enclosing
bundles of fibrils of the dermis, capillary blood vessels,
osteoblasts, and sometimes pigment cells. This osseous
matrix starts to mineralize and constitutes the anlage
of the bony plate of the regenerating ganoid scale. It
increases by addition of bone matrix on its periphery.
The capillary blood vessels entrapped within the bone
matrix organize and vascular canals are constituted.
Embedded osteoblasts become osteocytes and other cell
types die. When the scale has regained, approximately,
its initial shape, the growth of the bony plate slows
down and parallel-fibered bone matrix is deposited.
This stage precedes the deposition of ganoine and is
described in detail below.
End of bone deposit
Approximately 2 months after scale removal, the osteoblasts lining the surface of the regenerating scale of
Lepisosteus oculatus flatten and the deposition of OSseous matrix changes (Figs. 16, 17). At this stage the
upper surface of the bony plate is close to the epidermis. The central region of the scale shows a n irregular
aspect corresponding to the first steps of regeneration
and houses numerous vascular canals. The bony plate
is rich in osteocytes. Canaliculi of Williamson are visible only in the last deposited layers of parallel-fibered
bone (Figs. 16, 17). The deep surface of the scale is
connected to the dermis by means of Sharpey’s fibers.
During regeneration, “dermal teeth” ( = odontodes) can
develop and fix on the scale surface (Figs. 16, 171, but
they have not been observed on every sample. There
are no ligaments and the bases of the odontodes are
directly fixed into the bone matrix. The surface of the
scale is covered by a layer of osteoblasts and is separated from the epidermis by a 15-pm-wide dermal
space containing capillary blood vessels, fibroblasts,
pigment cells, and extracellular matrix (Figs. 17, 18).
The epidermis is already thick (200 pm in average) and
is mainly composed of sacciform cells and mucous cells
(Fig. 16, 17). In the basal region of the epidermis, the
basal layer is composed of interdigitated, closely juxtaposed cells connected by short desmosomes. The basal
layer differs from that described in the nonregenerated
scales (Fig. 4) in being composed of more juxtaposed
and closely interdigitated cells, the cytoplasm of which
containing numerous small vesicles. A 100-nm-thick
basal lamina underlines the basal layer a t the epidermal-dermal junction (Fig. 19).
The osteoblasts synthesizing the osseous matrix on
the lateral and deep surfaces of the bony plate are
roughly cuboidal cells, -6 pm high (Fig. 20). Their
cytoplasm is mainly occupied by a well-developed network of RER with numerous dilated cisternae, a n im-
anchoring fiber
bone matrix
bony plate
canaliculi of Williamson
epithelial bridge
Golgi region
ganoine membrane
inner ganoine epithelium
mineralization front
outer ganoine epithelium
pigment cell
rough endoplasmic reticulum
sacciform cell
Sharpey fiber
vascular canal
Figs. 1-1 5. Nonregenerated scales of Lepisosteus oculatus. Except
for Figures 6 and 14, all were obtained from EDTA decalcified scales.
Anterior part of the fish on the left.
Fig. 1. Posterior part of a scale schematically drawn from a l-pmthick section.
portant Golgi region with various types of secretory
granules, and mitochondria (Fig. 20). The apical region
of the osteoblasts shows some bundles of microfilaments and secretory granules and numerous cytoplasmic prolongations penetrating the osteoid matrix (Fig.
Fig. 2. Section (1pm thick) of the posterior part of a scale showing
its general organization and its localization in the skin. Ganoine has
been totally removed during the decalcification process and is represented only by an empty space (Ga) located between the bony plate
(BP) and epidermis (Ep).The asterisk indicates the enlarged region in
Figure 12. Bar = 100 pm; x 100.
Fig. 3. Upper region of a scale showing in detail the ganoine (Ga)
serrations. The ganoine surface is separated from the epidermis (Ep)
by a thick membrane, the ganoine membrane (GM). Arrowheads
point to the remnants of such a ganoine membrane a t the boundary
between a ganoine layer and the overlapping bone. Bar = 50 pm;
x 160.
Fig. 4. The cells of the basal epidermal layer (Ep) are in direct
contact with the ganoine membrane (GM) that covers the ganoine
(Ga). Arrowhead points to a desmosome. Bar = 1 km; x 15,000.
Fig. 5.Detail of the ganoine membrane (GM),which is composed of
three regions (1, 2, and 3). Note the thickening of the plasmalemma
(arrowhead) of the epidermal cell (Ep) facing the ganoine membrane.
An arrow points t o a caveolae fusing with the plasmalemma of the
epidermal cell. The open arrow indicates an electron-dense line a t the
deep surface of the ganoine membrane representing the place to
which the ganoine (Ga) matrix was attached before it was removed
along with the mineral during the decalcification process. Bar = 200
nm; X60,OOO.
Fig. 6. Undecalcified sample showing the localization of the mineral
crystals of the ganoine (Gab along the deep surface of the ganoine
membrane (GM),which is not mineralized. Bar = 200 nm; x 60,000.
Fig. 7.In this sample from another scale, the ganoine IGa) is separated from the epidermis (Ep)by a dermal layer (D)in which several
fibroblasts ( F )are located. The arrowhead indicates the basal lamina
forming the epidermal-dermal boundary. The ganoine membrane
(GM)still covers the ganoine. Bar = 500 nm; x 20,000. Inset: Higher
magnification of the region indicated by an asterisk on Figure 7. A
fibroblast (F)has established a contact (arrowhead) with the matrix
located at the upper surface of the gatiuitie iiieiiibrane (GM). Bar =
250 nm; x 40,000.
Fig. 8. Cell of the basal layer of the epidermis in the region where
the ganoine IS covered by dermal elements (D).The arrowhead points
to the epidermal-dermal boundary. Large intercellular spaces separate the cells which show cytoplasmic extensions. An arrow indicates
a series of desmosomes. Bar = 1 pm; x 10,500.
Figs. 2-8
Figs. 9-1 5.
21). In all the osteogenic regions, numerous bundles of
collagen fibrils emerge from the bone surface and penetrate into the adjacent dermis (Fig. 22). The osteoid is
-10 pm thick and the mineralization front is roughly
regular (Fig. 23). Some sites of mineral nucleation are
located a few microns off the mineralization front at
the level of membrane-bound vesicles (Fig. 23, inset).
The mineral crystals have invaded the anchoring bundles faster than the osteoid; there the mineral crystals
clearly follows the direction of the collagen fibrils (Fig.
23). In contrast, in the deep region where dermal collagen bundles are incorporated into the scale matrix
and constitute large Sharpey’s fibers, the mineral crystals have invaded the bundles later than in the surrounding osseous matrix (Fig. 24).
Close to the epidermis, in the upper region of the
scale the last-deposited collagen fibrils are perpendicular to the scale surface and to the osteoblast layer
(Fig. 25). These collagen fibrils have the same diameter
(50 nm on average) as those previously deposited in the
bony plate. The osteoblasts covering this region are
rectangular, elongated, 2-pm-thick cells with some cytoplasmic extensions located within the osteoid and
parallel to the vertical fibrils (Fig. 26). Deep within the
basal plate, some vascular canals progressively close,
due to a centripetal (osteonal) deposition of bony material. In rare examples the wall of a vascular canal
may be interrupted and the lumen is invaded progressively by extracellular matrix and cell debris (Fig. 27).
Epidermal contact on the scale surface
and ganoine deposition
The first elements of the ganoine matrix are deposited on the surface of the regenerating scales between
2-3 months after scale removal. This stage is characterized by the presence of an extensive epithelial sheet
covering a layer of unmineralized ganoine matrix,
preganoine, located on the scale surface (Fig. 28). The
epithelial sheet originates from the epidermis to which
it is connected by a thin epithelial bridge. Elsewhere
this epithelial sheet is largely separated from the basal
layer by a dermal space (Figs. 28,29A). The deposition
of the preganoine matrix begins on the surface of the
posterior region of the scale and progresses anteriorly.
All the stages of epithelial cell differentiation and of
ganoine formation are found between the epithelial
bridge, where the contact is established between the
epithelial cells and the bone surface (Fig. 29A), and the
Fig. 9. Detail of the ganoine membrane (GM) in the region invaded
by dermal elements (D). The ganoine membrane has conserved its
structure (compare to Fig. 5). Note the electron-dense granules and
filaments composing its upper layer (arrow).Bar = 200 nm; x 60,000.
Flg. 10. Lateral region corresponding to the future localization of a
ganoine serration (see Fig. 3). The ganoine membrane (GM)is covered
by dermal elements (D) one of them being an osteoblast (Ob) that is
depositing osteoid matrix ( 0 s ) .Bar = 1 pm; x 15,000.
Fig. 11. A ganoine serration is inserted between two successive layers of bony tissue (B1 and B2). The ganoine membrane (GM1)remains
on the surface of the ganoine layer ( G a l ) where a new bone layer is
deposited. Bar = 1 pm; X 10,500.
Fig. 12. Two ganoine serrations (Gal and Ga2l envelope the extremity of a bony layer (B2)(see framed region in Fig. 2). A t the boundary
between bone (B2) and ganoine (Ga2), collagen fibrils penetrate
roughly perpendicularly into the ganoine matrix. The structure of the
extremity of the epithelial sheet, at the posterior edge
of the scale, where a preganoine layer is already deposited (Fig. 29B). These stages are described in detail
At the light microscopic level, the recently deposited
preganoine is well defined in the 1-pm-thick sections
as a metachromatic layer located at the bone surface
(Fig. 30). The columnar, juxtaposed cells depositing
preganoine matrix are organized into an epithelial
sheet, which is separated from the epidermis by a dermal space containing fibroblasts, pigment cells, and
capillary blood vessels. At such a magnification it is
not easy to localize the epithelial bridge (Fig. 30). The
organization of the epidermis, as well as its thickness
(250 pm), are similar to those described in control
scales, the large sacciform cells and the mucous cells
being the prominent cell types (Fig. 30).
Epithelial bridge and first contact of the epithelium on the
bone surface. The epithelial cells first contact the os-
teoblasts lining the bone surface of the regenerating
scales via a short and narrow epithelial bridge, which
is -10 pm long and 10 pm wide (Figs. 29A, 30,31). It
is composed of elongated, irregularly-shaped cells
which possess a large nucleus with a clear nucleolus
(Fig. 31). The cells are close to one another and are
linked by short desmosomes (Fig. 32). Their cytoplasm
is electron-dense and occupied by numerous bundles of
filaments and free ribosomes, and by some mitochondria and some RER cisternae (Fig. 32). Some cytoplasmic extensions are seen within narrow intercellular
spaces. A continuous, 80 nm-thick basal lamina runs
along the epidermal-dermal boundaries, i.e., below the
epidermis, along each side of the epithelial bridge and
along the upper surface of the epithelial sheet (Fig. 31).
At the site of the first contact with the epithelial
cells, the dermal space contains cellular debris. The
osteoblasts adjacent to the epithelial cells show two
types of organization: either they are roughly rounded
cells equipped with numerous bundles of microfilaments and with a few dilated RER cisternae (Fig. 33),
or they have a necrotic aspect, characterized by a nucleus with undulated contours and dark condensations
of chromatin, and by dilated RER cisternae (Fig. 34).
As soon as they cover the bone, the epithelial cells
organize into a bilayered epithelial sheet (Figs. 29A,
29B, 35). Because this epithelial sheet is responsible
for the ganoine (enamel) formation, its productive inner layer, which is in contact with the scale surface,
ganoine membrane (GM1) is well conserved where it is covered by
bony material (B2) but not where it is covered by another layer of
ganoine (asterisk).Bar = 500 nm; x 22,500.
Fig. 13. Growing (marginal)region of a scale that is not covered by
ganoine. A layer of roughly cuboidal osteoblasts (Ob) deposits the
osteoid matrix ( 0 s ) .Anchoring fibers (AF) issuing from the bone matrix (B)cross the osteoblast layer and penetrate the dermis (D). Bar =
1 pm; ~ 9 , 0 0 0 .
Fig. 14. Mineralization front (MF) progressing into the osteoid matrix ( 0 s )in the lateral region of the scale. Cytoplasmic extensions of
the osteoblasts (Ob) penetrate the osteoid. Bar = 500 nm; x 20,000.
Flg. 15. Deep region of the basal plate. After decalcification the bony
region (B) is easily distinguishable from the unmineralized collagenous matrix of the osteoid (0s)lined by osteoblasts (Ob). Bar = 500
nm; ~ 2 0 , 0 0 0 .
Figs. 16-27. Two-month regenerated scales of Lepisosteus oculatus.
Except for Figures 23 and 24, all were obtained from EDTA decalcified scales.
Fig. 16. Posterior part of a scale schematically drawn from a l-Fmthick section. The bone surface is covered by a layer of osteoblasts and
a dermal space separates them from the epidermis.
Fig. 17. Section (1 pm thick) of the posterior part of a scale. At this
stage of regeneration the ganoine is not yet deposited. Note the presence of two odontodes (Od) and of several vascular canals (VC). Bar =
100 pm; x 100.
Fig. 18. The osteoid surface ( 0 s ) of the regenerating scale is covered
by a layer of osteoblasts (Obj, which is separated from the epidermis
(Ep) by a dermal space (D) containing some pigment cells (PC). Bar =
2 pm; ~ 4 , 5 0 0 .
Fig. 19. Cell of the basal layer of the epidermis in the region above
the scale. The roughly rectangular cells of this layer are linked by
desmosomes (arrowhead). They have an electron-dense cytoplasm
that contains scarce bundles of microfilaments, numerous free ribosomes, some mitochondria, and RER cisternae as well as Golgi saccules and numerous vesicles, 100 nm in diameter, and caveolae on the
deep plasmalemma (arrow).Bar = 1 pm; x 15,000.
Fig. 20. Osteoblasts covering the upper surface of the scale, where
they deposit the osteoid matrix (0s).They are equipped with a large
Golgi region ( G ) and their cytoplasm is largely occupied by a welldeveloped network of RER cisternae. Bar = 1 Fm; x 10,000.
Fig. 21. Detail of the apical region of a n osteoblast facing the scale surface. There are numerous
secretory granules (arrowheads) close to bundles of microfilaments, and cytoplasmic extensions (arrow)
penetrate the osteoid matrix ( 0 s ) . Bar = 500 nm; x 22,500.
Fig. 22. Surface of the scale in a marginal region showing anchoring fibers (AF) crossing the osteoblast
layer (Ob) and reaching the dermis (D). Bar = 1 pm; x 10,500.
will be called the inner ganoine epithelium (IGE) and
its outer layer, facing the dermis, will be called the
outer ganoine epithelium (OGE). The IGE is composed
of juxtaposed high, polarized cells (-12 pm high and 5
pm wide) perpendicular to the scale surface, whereas
the cells of the OGE are elongated (in average 4 pm
high and 15 pm wide) parallel to the scale surface. The
OGE cells have a n ovoid nucleus and their cytoplasm
contains some mitochondria, some cisternae of the
RER, a small amount of free ribosomes, and bundles of
microfilaments (Fig. 35). They are linked to one another and to the IGE cells by scarce, short desmosomes
and have cytoplasmic extensions penetrating the intercellular spaces. The IGE cells are closely juxtaposed
and connected to one another by desmosomes and gap
junctions. Their cytoplasm is rich in RER cisternae,
Golgi saccules, and mitochondria (Fig. 36). The Golgi
apparatus is located close to the rounded nucleus that
generally occupies a large part of the central region of
the cell. The apical region, which is in contact with the
osteoid, shows only small vesicles, free ribosomes, and
microfilaments (Fig. 37). The contact of the IGE cells
with the scale surface is established in the region
where the last deposited collagen fibrils of the bone
matrix are vertically oriented. Here the basal lamina
disappears and numerous cytoplasmic extensions of the
IGE cells penetrate the osteoid parallel to the last deposited vertical collagen fibrils (Fig. 37).
Ganoine production. The first elements of the preganoine matrix are located in a region of the osteoid that
is not far from the IGE surface and often close to the
cytoplasmic extensions of the IGE cells (Fig. 38). The
preganoine matrix is generally deposited as roughly
rounded patches (150-300 nm in diameter). They are
composed of electron-dense material surrounding a
clear zone. At a distance from the IGE cell plasmalemma, the patches of preganoine enlarge to reach 0.5-
0.8 pm in diameter by addition of new material. Some
of them are located close to the mineralization front
(Fig. 39). In marginal regions of the scale, the osteoid is
often thin and the mineralization front is then located
near to the scale surface. Here, preganoine patches
grow in close contact with the plasmalemma of the IGE
cells. The cell membrane of the IGE cells surrounding
the preganoine patches shows thickenings and caveolae (Fig. 40 and inset). The largest patches of preganoine can reach 1.5 pm in diameter. Electron-dense
fibrils, 15 nm thick, radiate from a central area with a
loose granular appearance (Fig. 40, inset). The patches
fuse to form a preganoine layer that thickens by the
addition of new organic matrix at its surface, directly
below the IGE cells (Fig. 41). The matrix of the preganoine layer is mainly composed of 15 nm-diameter
fibrils that are oriented roughly perpendicular to the
IGE cell membrane. At the level of the mineralization
front, the preganoine matrix is intermingled with the
osteoid; the limit between the basal plate of the scale
and its ganoine layer is thus constituted by a 2-pmthick layer of mixed tissue composed of both preganoine and bone matrices (Fig. 42).
The IGE cells a t the stage when they produce the
layer of preganoine are 10-pm-high, regularly arranged
cells (Figs. 29B, 43). Their cytoplasm is darker than that
of the OGE cells. The latter have numerous mitochondria and show cytoplasmic extensions. The roughly
rounded nucleus of the IGE cells is centrally located and
the cytoplasm is equipped with numerous RER cisternae, mitochondria, Golgi saccules, and small vesicles
(Fig. 43). The apical region of the IGE cells does not
possess cytoplasmic organelles but is rich in small vesicles, some of which have an electron-dense content
(Figs. 44,45).Moreover, this region is characterized by
the presence of numerous, large bundles of microfilaments (Figs. 43-45). They emanate from large desmo-
Figs. 23-27
Figs. 28-50. Three-month regenerated scales of Lepisosteus oculatus. Except for Figure 50, all were obtained from EDTA decalcified
Fig. 28. Posterior part of a scale schematically drawn from a l-pmthick section. The bone surface in the marginal region is now covered
by a layer of preganoine matrix, itself covered by a n epithelial sheet
that is separated from the epidermis by a dermal space. The epithelial
sheet is linked to the epidermis by a n epithelial bridge (arrow, EB).
Fig. 29. A. Detail of the region of the epithelial bridge and of the
first contact of the epithelial sheet on the scale surface. B. Detail of
the region covered by a thick layer of preganoine.
-... -.&E: ...
... .. ..
Fig. 23. The mineralization front (MF) is located a t a distance from
the osteoblasts (Ob) and there is a wide osteoid layer ( 0 s ) in which
some sites of mineral nucleation are seen (arrowheads). Compare to
similar regions of nonregenerated scales (Fig. 14).Arrow indicates a n
anchoring fiber in which the mineralization progresses more rapidly.
Bar = 1 pm; x 14,000.Inset: Detail of the osteoid layer showing the
presence of small vesicles (arrowheads), some of which present mineral crystals (arrows). Bar = 500 nm; x 30,000.
Fig. 24. Mineralization of the deep surface of the bony plate of the
scale in a region where collagen bundles (asterisks) are incorporated.
The mineralization front (MF) is irregular and the mineral crystals
invade the bone matrix surrounding the bundles before the bundles
themselves. Bar = 2 pm; x 6,000.
Fig. 25. Upper surface of the central region of the scale. Here, the
orientation of the last deposited collagen fibrils of the osteoid (0s)are
mainly perpendicular to the scale surface (arrows). Bar = 1 pm;
x 10,000.
Fig. 26. Detail of perpendicularly oriented fibrils among which osteoblast (Ob)cell extensions (arrow) penetrate the osteoid (0s).Bar =
500 nm; x 30,000.
Fig. 27. Part of a vascular canal in the central region of the scale.
The wall of the canal is interrupted (on the right) and the lumen of the
canal is invaded by extracellular matrix. Deposit of osteoid matrix
has occurred along the cells constituting the wall of the canal. Bar =
1 pm; ~ 7 , 0 0 0 .
somes that connect the plasmalemma of neighbouring
cells (Fig. 46) and some of them appear to cross the
apical region from one side to the other (Fig. 44).
The layer of preganoine thickens and reaches -10
km. At that time the preganoine matrix deposition
comes to its terminal phase (Fig. 44). Mineralization
takes place throughout the preganoine layer and the
amount of mineral progressively increases to reach a
high level of mineralization (called hypermineralization compared to the lesser degree of mineralization of
bone). By this maturation process, preganoine becomes
ganoine. As soon as the deposition of the preganoine
matrix stops, the first elements of the ganoine membrane (which remains unmineralized) appear between
the IGE cells and the ganoine surface (Fig. 47). This
still thin (50 nm) anlage of the ganoine membrane is
composed of a granular, electron-dense layer located
close to the IGE cell plasmalemma, which itself presents numerous hemidesmosomes. The process of maturation of the preganoine results in the progressive
disappearance of its organic matrix as observed in a
series of decalcified samples. In the first stages of maturation, the decalcified ganoine matrix looks rather
hazy compared to the stages preceding the mineralization (compare Figs. 47 and 44), then it appears even
more loose (Fig. 48)) and finally i t disappears when the
maturation of the ganoine is finished (Fig. 49). At the
same time, the ganoine membrane thickens (Fig. 481,
reaches its definitive thickness of 500 nm (Fig. 491, and
progressively acquires its normal organization when
ganoine is completely formed (Fig. 50).
Experimental Regeneration of Lepisosteus Scales
The present study using experimental regeneration
and light and TEM observations of the scales in Lepisosteus oculatus confirms and improves our knowledge
on the structure of the ganoid scales in lepisosteids and
brings new data on the origin of the ganoine. It is
clearly demonstrated that experimental regeneration
is a useful means to provoke ganoine production. Previous comparative studies devoted to the development
and regeneration of the ganoid scales in the polypterid
Calamoichthys calabaricus (Sire et al., 1987; Sire,
1989) and of the elasmoid scales in the teleost Hemichromis bimaculatus (Sire and Geraudie, 1983, 1984)
have shown that the processes involved during scale
regeneration largely follow the normal ontogenetic
pattern, as previously suggested by Abeloos (1932) and
Goss (1969). The use of experimental regeneration of
the scales presents several advantages: (1)regeneration needs a shorter time than normal development, (2)
adult or juvenile fish can be used instead of larvae or
juveniles, (3) i t is not necessary to obtain reproduction
and this is particularly useful when artificial reproduction has not yet succeeded or is difficult a s is the case
for lepisosteids or polypterids, and (4) the regenerated
scales reach the same size a s the controls and consequently, since the fish are adults or juveniles, there are
larger amounts of tissue than in comparable stages in
ontogeny. Nevertheless, slight differences can be observed in the organization or in the structure of regenerated and nonregenerated scales. Generally this is the
result of rapid growth and not the consequence of different developmental processes. One can thus claim
that the processes involved in regeneration in Lepisosteus scales (especially their upper part) reflect those
occurring during ontogeny.
In addition to the new information on the origin of
the ganoine and on the ganoine membrane structure
(these data are discussed below), the experimental regeneration of Lepisosteus scales has brought other data
worthy of interest.
The anlage of the regenerated scale is mainly constituted of collagen bundles belonging to the pre-existing
dermis, which are embedded in bony material; the bony
plate then grows by addition of osseous tissues around
this initium. Such a phenomenon has recently been
described (Sire, 1993) during the formation of the scute
anlage in a n armoured catfish, Corydoras arcuatus. In
this teleost, the main part of the scute is constituted, as
in a Lepisosteus scale, of a thick bony plate, the anlage
of which appears in the deep region of the dermis. Such
a n incorporation of collagen bundles does not happen in
polypterid scales, where the anlage develops in the upper region of the dermis (Sire, 1989).
In Lepisosteus, the deeper formation of the regenerating scale anlage in the dermis, compared to the more
superficial formation of polypterid scales, is probably
related to the lack of dentine. Polypterid scales possess
a layer of “dentine”; the scale anlage forms in the upper
region of the dermis, close to the epidermis, and ganoine starts to be deposited when the bony plate develops (Sire, 1989, 1990). In Lepisosteus, the ganoine is
deposited later on the scale surface, only when the bony
plate is well developed. The presence or absence of
odontoblast precursors in the upper layers of the dermis could determine the localization (superficial or
deep) of the scale anlage in polypterids and Lepisosteus,
respectively. In Lepisosteus, the deep localization of the
osteoblast precursors in the dermis would not allow
interactions with the epidermal cells to develop until
the bony surface of the scale reaches the upper region
of the dermis, when ganoine is deposited.
Capillary blood vessels and various cell types including pigment cells are entrapped within the bony plate
during the early stages of regeneration. In well-developed regenerating scales this phenomenon results in
the formation of several vascular canals, a feature that
does not exist in normal scales and that is undoubtedly
related to the rapid deposition of the bony tissues during the first steps of regeneration. In the last studied
stage of regeneration (3 months after scale removal),
the vascular canals show osteonal bone deposition and
some have breaks in their walls. The centripetal ossification, leading progressively to the closure of the vascular canals, could be a n indication that these canals
are no longer necessary. Regenerated scales contain
typical canaliculi of Williamson, some of which reach
the remaining vascular canals. Given that the complex
of Williamson, typical of holostean bone and present in
normal scales of Lepisosteus (Sire and Meunier, 1994),
most likely has a nutritive function, it may well be that
this complex takes over the role of the vascular canals.
In regenerated scales, the connection of canaliculi of
Williamson with the vascular canals could support 0rvig’s hypothesis (1951) that the network of canaliculi of
Williamson appears to have been established in association with canals before the horizontal vascular canal
system disappeared.
20 1
i t does not show the typical bell shape as in tooth development, and it is covered by dermal elements. The
Based upon sequential observations, but not on enamel organs, in Lepisosteus scale and in tooth, and
markers, the present study definitively elucidates now their components, are homologous: IGE and OGE corthe problem of the origin of the cells depositing ganoine respond to the inner and outer dental epithelia (IDE
in lepisosteid scales. The ganoine matrix is exclusively and ODE) in tooth development. The columnar organisecreted by a n epithelium, called here the inner ga- zation and the cytoplasmic content of the IGE cells
noine epithelium, which is epidermal in origin and (ameloblasts) are comparable to that known for tooth
equivalent to the inner dental epithelium involved in ameloblasts.
The early deposits of the ganoine matrix in Lepisostooth enamel secretion. Moreover, the preganoine matrix is collagen-free and progressively matures to be- teus (and also in polypterid scales) are morphologically
come a highly mineralized tissue, ganoine that conse- similar to the globular patches of early enamel matrix
quently is enamel. This finding closes a century and a in the teeth of the urodele, Triturus pyrrhogaster
half of controversy (starting with Agassiz, 1833-44) on (Kogaya et al., 1992). Moreover, this newt enamel is
the origin of the ganoine in the scales of the lepisos- reported to be composed of two layers: a n outer layer of
teids, and i t confirms our previous results demonstrat- true enamel and a n inner layer interpreted as a mixed
ing that ganoine is a n epidermal product in polypterid form of dentine-enamel matrices (Kogaya et al., 1992).
scales (Sire e t al., 1986, 1987). In view of the results A similar organization has been described herein for
obtained in both types of rhombic scales (palaeoniscoid the interface between bone and ganoine, which shows a
and lepidosteroid), we can reasonably assume that ga- 2-pm-thick region composed of interpenetrated bone
noine has a similar origin in all the living and fossil and ganoine matrices. I consider this region as a n anactinopterygian fishes, at least. As a n enamel, ganoine chorage of the preganoine within the osteoid, and there
differs from tooth enamel in being stratified, in being is no reason to consider it as a special layer. Ganoine is,
deposited in the absence of dentine (in lepisosteids), in on the contrary, totally different from the so-called
being not always covered by epidermal cells but some- enameloid, a hard tissue containing ectodermally or
times by dermal elements, and in never being exposed mesenchymally derived collagen fibrils, which covers
to the external medium. Probably ganoine is also the teeth of most fishes (e.g., Herold, 1974 in a pike;
slightly different from tooth enamel in the protein con- Shellis and Miles, 1976, in a n eel; Prostak and Skobe,
tent of its organic matrix, but this can be confirmed 1986 in a cichlid; see also Kawasaki and Fearnhead,
only by immunohistochemistry using appropriate an- 1983 for a review).
Ganoine Membrane
Two main reasons can explain the doubts of previous
In nonregenerated and regenerated scales, a thick
authors about the origin of the ganoine in lepisosteid
scales: (1) the exclusive use of the light microscope, organic layer, here called the ganoine membrane, alwhich does not allow precise localization of the limit ways covers the definitively formed ganoine. The term
between epidermis and ganoine surface, and (2) the is due to Nickerson (1893). The presence of this memfrequent presence of dermal elements between the ga- brane has long been known in polypterid and lepisosnoine surface and the epidermis, as shown in the teid scales and was named “enamel membrane” by
present study. The existence of such a dermal space Hertwig (1879) and “structureless membrane” by Kerr
was the main reason to suppose that the epithelial (1952). In polypterids such a n “intermediate layer”, or
layer, which deposits ganoine matrix, was of dermal “organic layer,” has also been described on the ganoine
origin (e.g., Nickerson, 1893; Kerr, 1952; Thomson and surface of the scales (Zylberberg et al., 1985)and of the
lepidotrichia (Geraudie, 1988). The thickness and fine
McCune, 1984).
In Lepisosteus scales the processes that precede the structure of this layer are roughly the same in the lepganoine deposition on the scale surface need further isosteid and polypterid scales, but the layer differs
comment, because these largely differ from what has slightly in polypterids by the presence of a vertical stribeen described in polypterid scales but recall what is ation (Zylberberg et al., 1985). Hertwig (1879) assumed
known for tooth development. In Lepisosteus, the epi- the ganoine membrane is a basement membrane, Nickdermis comes into contact with the scale surface erson (1893) saw it a s entirely distinct from a basement
through a n epithelial sheet that is linked to the epi- membrane, and Zylberberg et al. (1985) hesitated bedermis only by a short epithelial bridge; in polypterids tween considering it a s a basement membrane with a
the epidermis comes in direct contact with the bone thick lamina densa or as a special structure developed
surface (Sire e t al., 1987; Sire, 1989). In Lepisosteus the in relationship to the ganoine.
The present study shows that (1)the ganoine memepithelial sheet covering the scale is composed of two
layers, the outer ganoine epithelium (OGE) and the brane is a n epidermal product, ( 2 ) it is formed when
inner ganoine epithelium (IGE), whereas in poly- ganoine is mineralizing but it does not mineralize itpterids the basal epidermal layer cells differentiate self, (3) it persists on the surface of the ganoine layer
and constitute only one layer, called the inner epider- even when secondarily overlapped by dermal elements,
mal layer (IEL), which synthesizes ganoine matrix and (4) it mineralizes when bone or a new layer of
(Sire et al., 1987). In both IGE and IEL, the differenti- ganoine is deposited above it. This implies that the
ated cells are true “ameloblasts” synthesizing ganoine. protein content or organization of the ganoine memIn Lepisosteus, the bilayered organization of the epi- brane has been modified to allow mineral to penetrate
thelial sheet recalls the classical enamel (or enameloid) the matrix.
These data support the hypothesis that the ganoine
“bell” organ described in tooth development; however
the “enamel organ” of the scale covers a large surface, membrane is a special structure formed in relationship
In Lepisosteus Scales, Ganoine Is Enamel
Figs. 30-35.
Fig. 36. Cells of the inner ganoine epithelium (IGE) before preganoine production. They are columnar, juxtaposed cells equipped with
numerous mitochondria and RER cisternae. An arrowhead points to a
gap junction. Bar = 1 pm; ~ 8 , 0 0 0 .
Fig. 37. Detail of the apical region of the IGE cells. The basal lamina
has disappeared and cytoplasmic extensions (arrowheads) penetrate
the osteoid matrix (0s) along the vertically oriented collagen fibrils.
Bar = 500 nm; ~20,000.
Fig. 38. First elements of the preganoine matrix (arrowheads) deposited within the osteoid matrix by the IGE cells. Bar = 200 nm;
x 60,000.
Fig. 39. The first deposited patches of preganoine (PGa) are in contact with the osteoid matrix (0s) and reach to the mineralized bone
matrix (B). Bar = 1 pm; x 10,500.
Fig. 30. Section (1pm thick) of the posterior part of a scale showing
the darkly stained (metachromatic) preganoine layer (PGa) deposited
on the bone surface (B) by an epithelial sheet issuing from the epidermis (Ep) via an epithelial bridge (EB). Bar = 50 pm; x 250.
Fig. 31. General view of the epithelial bridge (EB)and of the area of
first contact of the epithelial sheet with the osteoid (0s)of the scale
surface. Arrowheads point to the basal lamina a t the epidermal-derma1 boundary. Bar = 5 pm; ~ 3 , 0 0 0 .
Fig. 32. The cells of the epithelial bridge are interdigitated and
sometimes linked to one another by short desmosomes (arrowhead).
The cytoplasm is occupied mainly by free ribosomes, RER cisternae,
Golgi regions (G), and bundles of filaments. Bar = 1 pm; x 14,000.
Fig. 33. Region where the cells of the epithelial layer (Ep) are in
first contact with the osteoblasts. This rounded osteoblast (Ob)shows
numerous bundles of filaments (arrowhead) and a few mitochondria
and RER cisternae. Bar = 1 pm; x 10,000.
Fig. 34. Same region as in Figure 33. In this case the osteoblast (Ob)
has a necrotic appearance characterized by the swollen nucleus and
the dilated RER cisternae. Note that a cell of the epithelial sheet (Ep)
is in direct contact with the osteoblast (arrowhead). Bar = 1 pm;
x 10,500.
Fig. 35. The epithelial sheet covering the scale surface is separated
from the epidermis by a dermal space and is composed of two layers:
the outer ganoine epithelium (OGE) and the inner ganoine epithelium (IGE), which is in direct contact with the osteoid matrix (0s).
Bar = 2 pm; ~ 4 , 5 0 0 .
Fig. 40. Lateral region of the scale surface on which the preganoine is first deposited. Here, the
mineralized bone matrix is close to the scale surface and the patches of preganoine grow directly along
the plasmalemma of the IGE cells. Note the thick, electron-dense aspect of the plasmalemma facing the
preganoine patches. Bar = 1 pm; x 14,000. Inset: Detail of a preganoine patch showing the radial
organization of its matrix. Bar = 500 nm; x 30,000.
to the presence of the hypermineralized ganoine. Zyl- even though the space between epidermis and ganoine
berberg et al. (1985) have shown that the electron- is completely invaded by dermal elements, bone depodense parts are rich in mucosubstances (proteogly- sition on the ganoine surface occurs in the marginal
cans); it is now well admitted that proteoglycans
possess numerous properties (Hay, 1991) including prevention of mineralization (Glimcher, 1976; Butler,
1984). The upper filamentous network of the ganoine
Fig. 41. Preganoine patches have fused and a preganoine layer is
membrane probably contains adhesive substances pro- formed. This then thickens by addition of new material on its surface.
viding for fixation of either the basal layer cells of the Note that the matrix of the preganoine (PGa) is deposited perpendicepidermis or the fibroblasts. Further studies involving ularly to the scale surface. Bar = 1 pm; x 15,000.
Fig. 42. Detail of the boundary between the bone matrix (B) and the
immunochistochemical detection of the protein content preganoine
matrix (PGa)showing the interpenetration of the tissues.
are needed. Meanwhile, I suggest that the ganoine Bar = 500 nm; x 22,000.
Fig. 43. Inner ganoine epithelium cell (IGE) during the stage of
membrane allows the soft tissues to stick on its superficial side. This structure has probably developed in preganoine (PGa)production. The cytoplasm is rich in RER cisternae.
apical region facing the preganoine layer is more or less devoid of
relation to the presence of a hard (enamel)-soft inter- The
organelles. Bar = 1 pm; x 10,500.
face to prevent movements of the epidermis or dermal
Fig. 44. Terminal phase of the preganoine deposition. Arrow points
elements during swimming. A similar function as a n to a bundle of filaments located in the apical region of the IGE cell.
"antislip pad" has been suggested for the ganoine mem- Bar = 1 pm; ~ 7 , 0 0 0 .
Fig. 45. Detail of the apical region of an IGE cell showing vesicles
brane in polypterid scales (Zylberberg et al., 1985; with
electron-dense content (arrowheads) and bundles of filaments
Geraudie, 1988).
(arrows). Bar = 250 nm; x 40,000.
Ganoine Deposition Is a Periodical Phenomenon
At given times (the regulation of which is still unknown but certainly in relation to epidermal-dermal
interactions), epidermis withdraws from the ganoine
surface and dermal elements invade the space on the
entire surface of the Lepisosteus scale. Later the epidermis comes back toward the ganoine surface, whereupon the dermal elements withdraw or are removed.
The existence of numerous layers of ganoine suggests
that this to-and-fro movement persists throughout the
life span of the fish. An overlap of the ganoine surface
by dermal elements is also known in polypterid scales
where it is limited to the marginal regions (Sire et al.,
1987; Sire, 1989). In the scales of Lepisosteus oculatus,
Fig. 46. Same place, detail showing a bundle of filaments (arrow)
linked to a large desmosome. Arrowheads point to vesicles containing
a n electron-dense material. Bar = 500 nm; x 20,000.
Fig. 47. Beginning of the maturation stage of the preganoine (PGa)
characterized by the appearance of the first elements of the ganoine
membrane (GM). Bar = 250 nm; x 45,000.
Fig. 48. Advanced stage of maturation of the preganoine (PGa).The
ganoine membrane (GM) thickens; the preganoine matrix has been
partially removed during the decalcification process. Bar = 250 nm;
x 45,000.
Flg. 49.Final stage of maturation of the preganoine, which now has
become ganoine. The matrix has been totally removed by the decalcification process. The ganoine membrane (GM) is thick but has not
reached its definitive organization (compare with Fig. 5). Bar = 250
nm; ~45,000.
F g 50. Same stage, undecalcified sample. The electron-lucent zone
between the ganoine membrane and the epidermis is probably an
artifact of sectioning due to the presence of mineral. Bar = 250 nm;
x 45,000.
FIgS. 41-50
regions only, as in the polypterid scales. This alternating deposition of ganoine and bone layers on the
lateral regions of the scale gives rise to the typical
ganoine serrations (already described by Thomson and
McCune, 1984, and much earlier in fossils by Goodrich,
1907). Such a succession of ganoine and bone (or dentine) layers has also been described in other elements
of the cranial (gular plate) and postcranial (dorsal
spines, lepidotrichia, pectoral plates) dermal skeleton
of polypterids (Meunier, 1980; Geraudie, 1988). In
these elements the overlap of layers of ganoine and
osseous tissues could also result from a n alternate
presence of epidermis and dermis on the ganoine
surface. This phenomenon has similarly been interpreted by 0rvig (1977) in his theory on the ontogeny
of the “odontocomplexes” in the dermal skeleton of
primitive fossil osteichthyan fish (see also Orvig,
1978a,b). During the evolutionary processes that led to
the scales of the living lepisosteids and polypterids, the
odontocomplexes have evolved to represent now the
stratified ganoine; the overlap by osseous tissues that
primitively concerned all the ganoine surface being
confined to the lateral regions only. In this line of
thought, it appears that the processes have been more
simplified in polypterid scales, in which the epidermis
always remains in contact with the ganoine, than in
Lepisosteus scales in which the epidermis periodically
retracts from the ganoine surface. In Lepisosteus scales
the processes (and the tissue interactions that control
them) are more reminiscent of those involved in the
formation of the odontocomplexes a s defined by 0rvig
(1977) and close to what is known for tooth development.
An intriguing question deals with the presence of
odontodes (dermal teeth) on the scale surface. The term
“odontode” was introduced by 0rvig (1967) to describe
special hard tissue units composed of a layer of dentine
surrounding a pulp cavity and covered by a layer of
hard tissue (enameloid or enamel). Such dermal teeth
are known in numerous fossil fishes and in living
sharks, lepisosteids, polypterids, and certain teleosts
(e.g., siluroids, denticipids, xiphiids), but see 0rvig
(1977) for a detailed review. Odontodes develop on the
scale surface in young living lepisosteids before deposition of the ganoine, but they are transient structures
that leave no traces of their existence in adult scale
(Nickerson, 1893). Their presence in the regenerated
scales of Lepisosteus demonstrates that, at least in the
first stages, the processes of regeneration recall those
involved during the normal development. Moreover,
recent observations of the scale structure in fossil
Lepisosteus sp. from the Upper Cretaceous and Lower
Tertiary of South America have shown clearly the presence of partially resorbed odontodes at the bony plateganoine boundary (Gayet and Meunier, 1993). This
suggests that the presence of odontodes a t the scale
surface of lepisosteids is the plesiomorphic condition
that has been conserved only in the first stages of scale
ontogeny in living lepisosteids.
The possibility of developing odontodes during scale
ontogeny in Lepisosteus could be explained by the existence of two different populations of mesenchymal
cells: one, deeply located in the dermis, is responsible
for the bony scale formation as discussed above; the
other, superficially located, can interact with the epidermis to form the dentine component of the odontodes.
The odontoblastic cell population is not programmed to
give an extensive dentine layer, as, e.g., in polypterid
scales, but to give dental units only, the odontodes,
which are present in the first stages of scale development and regeneration. According to 0 r v i g (19771, in
fossil actinopterygian fishes, odontodes are normal
components of the dermal skeleton and they have been
subjected to a successive phyletic reduction process
that results in a vestigial development. The question of
their persistence in the living actinopterygian remains
to be settled.
Scales of living lepisosteids do not normally possess
dentine, but this does not prevent ganoine being deposited directly upon the bone by epidermal cells. In mammalian tooth development, it is well known that dentine formation is a prerequisite for enamel deposition
after epidermal-dermal interactions (Lumsden, 1987,
1988).Even if the ancestral scales possessed dentine,
the osseous basal plate in the lepisosteids is not a tissue
of mixed origin (dentine and bone) but typically cellular bone (Orvig, 1951; Sire and Meunier, 1994). Such a
deposition of ganoine on a bony tissue has also been
observed in the lepidotrichia of the pectoral fin in the
polypterids (Geraudie, 1988). However, the nature of
the “dentine” layer located below the ganoine in the
scales of living polypterids has to be reconsidered (Sire,
1989). We can only assume here that the presence of
dentine is no more a prerequisite to induce epidermal
cells to secrete ganoine, and this does not prevent cooperation between epidermal cells and osteoblasts. Indeed, several observations can be interpreted in terms
of epidermal cell-osteoblast interactions: (1)before the
epidermal cells come into contact with them, the osteoblasts deposit collagen fibrils perpendicular to the cell
surface, instead of in the classical parallel network.
This organization of the bone matrix favours the penetration of the first components of the ganoine matrix
within the osteoid to allow a solid anchorage between
both matrices. (2) A similar phenomenon has been described in polypterid scales (Sire e t al., 1987), shortly
before the epithelial sheet covers the scale surface, dermal elements withdraw, or are eliminated, from the
scale surface. Thus epidermal cells can establish direct
contacts with the osteoblasts, which allows the epithelial sheet to cover the scale rapidly over a great surface.
(3)When a layer of ganoine is definitively formed, the
epidermal sheet withdraws from the ganoine surface
and the space is invaded by dermal elements.
Vascular canals are rare in the mature scales of Lepisosteus, which compensate this fact by housing canaliculi of Williamson (Sire and Meunier, 1994). Polypterid
scales, in contrast, have numerous vascular canals,
some of which crossing the scale to bring nutritive elements to the adjacent epidermis (Sire et al., 1987;
Sire, 1989). In the Lepisosteus scale, if the function of
the canaliculi of Williamson is probably to bring nutritive elements into the bony tissue of the scale (Sire and
Meunier, 1994), they do not contribute to nourish the
epidermis. The withdrawing of the epidermis from the
ganoine surface allows some capillary blood vessels to
bring nutritive elements to the basal surface of the
I thank Dr. Mary Whitear (Tavistock, U.K.) and Dr.
Ann Huysseune (Risjkuniversiteit, Gent, Belgium) for
reviewing the manuscript. I also thank Mrs. FranGoise
Allizard for her excellent technical assistance. TEM
observations and photography were done a t the Centre
Interuniversitaire de Microscopie Electronique, CIME,
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nonregenerated, oculatus, lepisosteus, holostei, scales, experimentale, formation, light, attention, ganoine, particular, tem, stud, regenerated
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