Light and TEM study of nonregenerated and experimentally regenerated scales of Lepisosteus oculatus (holostei) with particular attention to ganoine formation.код для вставкиСкачать
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 JEAN-YVES SIRE Laboratoire d’Anatomie comparee, Uniuersitk Paris 7, C N R S , U R A 1137, 75251 Paris cedex 05, France ABSTRACT 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. s 1994 WILEY-LISS. INC 190 J.-Y. SIRE 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. MATERIALS AND METHODS 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. RESULTS 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- GANOINE FORMATION I N S C A L E S O F LEPZSOSTEUS 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 191 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. 192 J.-Y. SIRE 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- AF B BP CW D EB EP F G Ga GM IGE MF Ob oc Od OGE 0s PC PGa RER sc SF vc Abbreviations anchoring fiber bone matrix bony plate canaliculi of Williamson dermis epithelial bridge epidermis fibroblast Golgi region ganoine ganoine membrane inner ganoine epithelium mineralization front osteoblast osteocyte odontode outer ganoine epithelium osteoid pigment cell preganoine rough endoplasmic reticulum sacciform cell Sharpey fiber vascular canal 1 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. GANOINE FORMATION IN SCALES O F LEPISOSTEUS Figs. 2-8 193 194 J:Y. SIRE Figs. 9-1 5. GANOINE FORMATION IN SCALES OF LEPZSOSTEUS 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 195 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 below. 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 . 16 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. GANOINE FORMATION I N SCALES OF LEPZSOSTEUS 197 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. 23-27 199 GANOINE FORMATION IN SCALES OF LEPISOSTEUS Figs. 28-50. Three-month regenerated scales of Lepisosteus oculatus. Except for Figure 50, all were obtained from EDTA decalcified scales. 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. 82 -;\ -... -.&E: ... :.. . .._ ... .. .. 3s V6Z 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 . 200 J.-Y. SIRE 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). DISCUSSION 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. GANOINE FORMATION I N SCALES OF LEPISOSTECIS 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). tibodies. 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 202 J.-Y. SIRE Figs. 30-35. GANOINE FORMATION IN SCALES OF LEPZSOSTEUS 203 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 . 204 J.-Y. SIRE 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. GANOINE FORMATION IN SCALES OF LEPISOSTEUS FIgS. 41-50 205 206 J.-Y. SIRE 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 epidermis. GANOINE FORMATION IN SCALES OF LEPISOSTEUS ACKNOWLEDGMENTS 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, Universites P6/P7, Paris. LITERATURE CITED Abeloos, M. 1932 La Regeneration et les Problemes de la Morphogenese. Gauthiers-Villars, Paris. Agassiz, L. 1833-1844 Recherches sur les Poissons Fossiles. 1, XLIV, 188 pp. Neuchdtel. Butler, W.T. 1984 Matrix macromolecules of bone and dentin. Collagen Rel. Res., 4r297-307. Gayet, M., and F.J. 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