Ultrastructural data on the scales of the dipnoan Protoptems annectens (Sarcopterygii, Osteichthyes)

The structure and the mineralization of the scales of the living dipnoan (lungfish) Protoptems annectens have been investigated by transmission electron microscopy (TEM). The thin and imbricated scales are composed of two layers: the squamulae and the basal plate. At the outer surface, the squamulae form isolated plates superficially ornamented with spines and concretions and made up of acellular bone. After demineralization, the squamulae show a heterogeneous organic matrix composed of thin randomly oriented collagen fibrils forming a loose network within which the concretions appear as electronlucent circular areas. Abundant and aggregated concretions are located within the spines. The crystallites are oriented by the collagen fibrils except in the concretions. Anchoring bundles composed of parallel collagen fibrils arise from the squamulae and connect the scales to the overlying dermis.
The basal plate, the most developed part of the scale, is made up of isopedine. Its main component consists of thick, closely packed collagen fibrils organized in a 'double twisted plywood-like structure'. Fibroblasts are present in the basal plate. Mineralization occurs only in few plies located beneath the squamulae. Mandl's corpuscles are found in front of the mineralization front. The mineral deposit is oriented by the collagen fibrils.
The scales of Protoptems annectens differ from the typical elasmoid scales of the teleosts by the peculiar structure of the squamulae, nevertheless they show enough structural characteristics to support the hypothesis that they can be considered as scales of the elasmoid grade, which have retained some plesiomorphic characteristics.     

Formation and structure of scales in the Australian lungfish,neoceratodus forsteri (Osteichthyes: Dipnoi)

The large elasmoid scales of the Australian lungfish, Neoceratodusforsteri, are formed within the dermis by unpigmented scleroblasts, growing within a collagenous dermal pocket below a thick glandular epidermis. The first row of scales, on the trunk of the juvenile lungfish, appears below the lateral line of the trunk, single in this species, at around stage 53. The scales, initially circular in outline, develop anteriorly and posteriorly from the point of initiation in the mid‐trunk region, and rows are added alternately below the line, and above the line, until they reach the dorsal or ventral midline, or the margins of the fins. Scales develop later on the ventral surface of the head, from a separate centre of initiation. Scales consist of three layers, all produced by scleroblasts of dermal origin. The outermost layer of interlocking plates, or squamulae, consists of a mineralised matrix of fine collagen fibrils, covered by unmineralised collagen and a single layer of cells. Squamulae of the anterior and lateral surfaces are ornamented with short spines, and the mineralised tissue of the posterior surface is linked to the pouch by collagen fibrils. The innermost layer, known as elasmodin, consists of bundles of thick collagen fibrils and cells arranged in layers. An intermediate layer, made up of collagen fibrils, links the outer and inner layers. The elasmoid scales of N. forsteri can be compared with scale types among other osteichthyan groups, although the cellsand canaliculi in the mineralised squamulae bear littleresemblance to typical bone. J.Morphol., 2012. © 2011 Wiley Periodicals, Inc.     

Cytoskeletal organization and collagen orientation in the fish scales

Summary Immunofluorescence and electron microscopy were used to analyze the relationships between the organization of collagen fibrils in elasmoid scales, and the orientation of microtubules and actin microfilaments in the scleroblasts producing this collagenous stroma. Attention was focused on the basal plate of the scales because of the highly ordered three-dimensional arrangement of the collagen fibrils in superimposed plies forming an acellular plywood-like structure. The collagen fibrils are synthesized by the scleroblasts forming a monolayered pseudo-epithelium, the hyposquama, at the lowest surface of the scale. Fully developed scales with a low collagen deposition rate were compared with regenerating scales active in fibrillogenesis. When an ordered array of the collagen fibrils is found, the innermost collagen fibrils are coaligned with microtubules and actin microfilaments. Thus, because of this coalignment, microtubules and actin microfilaments of the hyposquamal scleroblasts are subjected to consecutive alterations during the formation of the plies of the basal plate. The sequence of events when the collagen fibrils change their direction from one ply to the other in the basal plate is deduced from immunofluorescence and phase-contrast-microscopic observations. During the formation of the orthogonal plywood-like structure in the regenerating scales, first microtubules may change their curse with a rotating angle of about 90°; then, actin microfilaments are disorganized and reorganized by interacting mechanically with the microtubules with which they are coaligned. Collagen fibrils are synthesized in a direction that is roughly perpendicular to that of the preceding ply. The unknown signals inducing the change in direction of the cytoskeleton may be transmitted throughout the hyposquama via gap junctions.This work is dedicated to the memory of Jacques Escaig     

Development and fine structure of the bony scutes in Corydoras arcuatus (Siluriformes,callichthyidae)

The development and the structure of the bony scutes have been studied in a growth series of the armored catfish Corydoras arcuatus using light and electron microscopy. Fibroblast-like cell condensations appear in the dermis, in the posterior region of the caudal peduncle, and these will constitute the scute papillae. Collagen bundles of the preexisting dermis colonized by the papilla cells are remodeled and incorporated in the papilla to form, in addition to newly synthesized woven-fibered bony material, the initium of the scute. This process of formation differs from that described for the dermal papilla of an elasmoid scale. During growth, the osteoblasts surrounding the scute constitute the scute sac in which the scute grows. Parallel-fibered bone is deposited on both sides of the initium, and osteoblasts are incorporated within the scute matrix. The remodeling and incorporation of collagen bundles of the preexisting dermis is maintained during growth only in the deep, anterior region of the scute. The posterior region and the upper surface of the scute are close to the epidermal-dermal boundary. When growth slows down in the upper part of the scute, a characteristic, well-mineralized tissue, composed of thin vertical fibrils and granules and devoid of typical striated collagen fibrils, is deposited on the scute surface. A new term, hyaloine, is introduced for this nonosseous, highly mineralized layer constituting the upper part of the scute. Hyaloine shows thin electron-dense lines, which probably correspond to periodic growth arrests. The structure and localization of the hyaloine are compared to other well-mineralized, similar tissues found on the surface of the dermal skeleton in lower vertebrates. © 1993 Wiley-Liss, Inc.     

The twisted collagen network of the box-fish scutes

The present article describes the three-dimensional arrangement of collagen fibrils in dermal plates of different species of Ostraciidae. These dermal plates or 'scutes' are transformed scales, which have a polygonal shape and form a rigid tiling. They are natural composites, associating a fibrous network with a mineral deposit lying at two different levels of the scute, the 'ceiling' and the 'floor', plus a set of similarly mineralized walls joining the two levels. The three-dimensional structure of the collagen network can be compared to that of 'plywood': fibrils align parallel within superposed layers of uniform thickness, and their direction changes from layer to layer. In the dermal plate, two types of plywood have been evidenced: (1) one lying between the two mineralized plates, where the orientation of fibrils rotates continuously, and (2) one under the lower plate, with thick layers of fibrils, each showing a constant orientation, but abrupt angular changes are observed at the transition from one layer to the following one. In oblique sections, both types of plywood reveal large series of arced patterns, testifying to a twisted arrangement of collagen fibrils, analogous to the arrangement of molecules or polymers in cholesteric liquid crystals. The network is reinforced by some collagen fibrils running unidirectionally and almost normally to the lamellate structure. Moreover in the overall organization of the scute, these plywood systems form a set of nested boxes. This original architecture is compared to the arrangement of the collagenous network previously described in most fish scales and in other extracellular matrices.     

Spatial Organization and Mineralization of the Basal Plate of Elasmoid Scales in Osteichthyans

In Sarcopterygii (Latimeria, Neoceratodus, Protopterus, Leptdosiren)and Amiidae (Amia) collagen fibrils of the basal plate are packedin bundles whereas they remain isolated in Teleostei. The basalplate looks like plywood, a system of superimposed layers ofparallel fibers or fibrils the directions of which rotate witha regular angle in two successive layers. The double twistedplywood is constituted of two imbricate systems, the odd andthe even, where the rotation of the fibrillar directions isright-handed in Sarcopterygii and lefthanded in Amiidae andnumerous primitive Teleostei. The orthogonal plywood, with itstwo main orthogonal fibrillar directions, characterizes theevolved Teleostei and some more primitive ones. In most teleosteanspecies, as in Amia and Protopterus, mineralization of the basalplate in elasmoid scales involves Mandl's corpuscles that mineralizewithout contact with a pre-existing calcified tissue; they growand coalesce with the neighbouring ones and fuse to the mineralizingfront. Their shape is directly influenced by the local arrangementof the collagenous fibrils. In two teleostean families (Osteoglossidaeand Mormyridae) Mandl's corpuscles are completely lacking butspreading of mineralization in the basal plate has a peculiaraspect. Whatever that may be, the various characteristic organizationsof the skeletal tissues or isopedine that constitute the basalplate of osteichthyan elasmoid scales, all are varieties ofbone tissue that have undergone more or less important specializationlinked to the general regression of dermal ossifications andto functional adaptations.     

Scales in young Polypterus senegalus are elasmoid: new phylogenetic implications

A component of the basal plate which has a plywood-like organization similar to that of the elasmoid scales of teleosts is described in the scales of Polypterus senegalus for the first time. The origin and development of this structure is studied in young (50-117 mm, standard length) and adult (225 and 240 mm) specimens using light and electron microscopy. In 50 mm fish, the scales are imbricated and composed mainly of a succession of orthogonal collagen layers forming a plywood-like structure, the isopedin. The outer surface of the scale is ornamented locally by irregular patches of collagenous material. The layers are not mineralized, whereas the superficial patches are well calcified. The isopedin thickens until it has 12-15 layers and then stops growing (88 mm fish). It mineralizes irregularly from its upper part, and two vascular regions, surrounded by woven-fibered osseous material, form on the outer and deeper surfaces of the isopedin. These regions thicken while the vascular canals close by centripetal deposition of parallel-fibered osseous tissue. The outer region is the superficial part of the mature scale (called here osteodentin), which is covered by the ganoine deposited by the epidermal cells. The deeper part constitutes the definitive basal plate, composed of parallel-fibered osseous tissue. The results show 1) that the young ganoid scales of Polypterus senegalus have a structure similar to that of typical elasmoid scales; and 2) that the isopedin structure does not change during ontogeny and so represents a permanent record of the first ontogenetic stages. The phylogenetic implication of these results is that the elasmoid scales of teleosts arose by a process of paedomorphosis.     

Evidence for participation of the epidermis in the deposition of superficial layer of scales in zebrafish (Danio rerio): A SEM and TEM study

Comparative studies on scale structure and development in bony fish have led to the hypothesis that elasmoid scales in teleosts could be dental in origin. The present work was undertaken to determine whether the scales in zebrafish (Danio rerio), a species widely used in genetics and developmental biology, would be an appropriate focus for further studies devoted to the immunodetection of dental components or to the detection of the expression of genes coding for various dental proteins in fish scales. The superficial region of mature and experimentally regenerated scales and its relationships to the epidermal cover were studied in adult zebrafish using scanning (SEM) and transmission (TEM) electron microscopy. The elasmoid scales are relatively large, thin, and are located in the upper region of the dermis, close to the epidermis. In adults, the surface of the posterior region appears smooth at the SEM level and is entirely covered by the epidermis. During regeneration, the relationship of the epidermal cover to the scale surface is established within 4 days. This interface is easier to study in regenerating than in mature scales because the former are poorly mineralized. TEM revealed that: (1) the epidermis is in direct contact with the scale surface, from which it is separated only by a basement membrane-like structure, (2) there are no dermal elements at the scale surface except at the level of grooves issuing from the focus and crossing the scale surface radially, (3) the mineral crystals located in this superficial region are perpendicular to the scale surface, whereas those located deeper within the collagenous scale matrix are randomly disposed, and (4) when decalcified, the matrix of the superficial region of the scale appears devoid of collagen fibrils but contains thin electron-dense granules, some of which are arranged into layers. The continuous epidermal covering, the absence of dermal elements, as well as the fine structure of the matrix and its type of mineralization, strongly suggest that epidermal products, possibly enamel-like proteins, are deposited at the scale surface and contribute to the thickening of the upper layer in zebrafish scales. J. Morphol. 231:161–174, 1997. © 1997 Wiley-Liss, Inc.     

Structure of the skin of Agonus cataphractus (Teleostei)

The skin of the scuted teleost Agonus cataphractus has been investigated by histochemical methods, SEM and TEM. The anterior dorsal skin bears tubercles of epidermis overlying tiny ossifications (scutelets) superficial to the main scutes. The epidermis secretes a cuticular layer containing acidic non-sulphated glycoproteins, but there are no mucous goblet cells in the external skin. Non-mucous sacciform cells of two types are present in the epidermis, also numerous chloride cells. Scanning electron microscopy reveals variation in the microridge pattern of superficial epithelial cells, thought to relate to arrival at the surface and secretion of the cuticle. The major scutes overlap anteriorly, contrary to the normal arrangement of scales, indicating that they are secondary ossifications. The type of mineralization is similar to that of acellular bone. The scutes are set directly in the collagen of the dermis. They have a girdered structure with radial and cross bars, inserting on both faces of a thin plate. The interstices are occupied by unmineralized collagen, and extrinsic collagen bundles impinge on the bone. Non-mineralized parts of the dermis contain tracts of microfibrils in addition to collagen; these are best developed in the flexible gular skin and in the barbels and are interpreted as elastic tissue, although an amorphous component was not seen. The barbels have a core of connective tissue without a cartilaginous skeleton and bear taste buds and numerous chloride cells.     

Les relations isopedine - tissu osseux dans le post-temporal et les ecailJes de la ligne laterale de Latimeria chalumnae (Smith)

The scaly part of the posttemporal bone and the lateral-line scales have the same structure as other scales. However, the lateral line with its bony canal induces some local transformations in both structures. The posttemporal bone can be interpreted as a lateral-line scale whose "dermo"-component is extensively developed. With its composite structure, both osseous and scaly, the posttemporal bone affords an instance peculiarly well suited for studying the relationships between isopedine and bone. The observations demonstrate that isopedine whose superposed collagen layers make a double-twisted plywood, is a specialized structure which evolved from a bony tissue. The fibrous basal plate of a Latimeria scale is homologous to the osseous basal plate of a cosmoid scale.     

Corneal morphogenesis in the Indian garden lizard,Calotes versicolor (agamidae)

The present study traces corneal morphogenesis in a reptile, the lizard Calotes versicolor, from the lens placode stage (stage 24) until hatching (stage 42), and in the adult. The corneal epithelium separates from the lens placode as a double layer of peridermal and basal cells and remains bilayered throughout development and in the adult. Between stages 32– and 33+, the corneal epithelium is apposed to the lens, and limbic mesodermal cells migrate between the basement membrane of the epithelium and the lens capsule to form a monolayered corneal endothelium. Soon thereafter a matrix of amorphous ground substance and fine collagen fibrils, the presumptive stroma, is seen between the epithelium and the endothelium. Just before stage 34 a new set of limbic mesodermal cells, the keratocytes, migrate into the presumptive stroma. Migrating limbic mesodermal cells, both endothelial cells and keratocytes, use the basement membrane of the epithelium as substratum. Keratocytes may form up to six cell layers at stage 37, but in the adult stroma they form only one or two cell layers. The keratocytes sysnthesize collagen, which aggregates as fibrils and fibers organized in lamellae. The lamellae become condensed as dense collagen layers subepithelially or become compactly organized into a feltwork structure in the rest of the stroma. The basement membrane of the endothelium is always thin. Thickness of the entire cornea increases up to stage 38 and decreases thereafter until stage 41. In the adult the cornea is again nearly as thick as at stage 38.     

Fine Structure of the Developing Scale in the Cichlid Hemichromis bimaculatus (Pisces,Teleostei, Perciformes)

The study of the formation and structure of the early teleost scale and its associated cells has been carried out on Hemichromis bimaculatus fry using in toto staining with alizarin and transmission electron microscopy techniques. Results of the study show very rapid scale formation in Hemichromis. The papilla of the scale differentiates a little in advance of the bone scale formation. No epidermal cells are involved in the constitution of the scale pocket made up of scleroblasts. In Hemichromis, as in other teleost scales, the osseous layer is the first one to be secreted by, presumably, only the scleroblasts. Then the scleroblasts specialize in their functions. Superficial ones are involved in the formation of osseous circuli; marginal scleroblasts are responsible for growth in diameter of the scale; while deep scleroblasts allow the scales to thicken owing to the progressive addition of collagen fibrils organized in a “plywood-like” structure which constitutes the fibrillary plate of the scale. Mineralization occurs very rapidly within the osseous layer in the form of hydroxyapatite-like crystal deposits. The fibrillary plate is not yet mineralized in Hemichromis at the stages studied here, but presumably is later. Results obtained in Hemichromis are discussed against similar data available in the literature on teleost scale formation.     

A morphological study of regeneration of the goldfish elasmoid scales

Studies were carried out on the model of regeneration of mechanically removed elasmoid scales. Regenerating scales were morphologically analyzed using light and electron microscopy. It was found that the cells responsible for regeneration of the elasmoid scale plates could be classified as osteogenic elements. Little differentiated preosteoblasts were detected in the connective tissue of dermis on day 3 of regeneration, while partially calcified plates underlaid osteoblasts on day 7. The scale cover was fully restored on day 14 and it took two days for each bone plate to be formed. Osteocytes, fully differentiated osteogenic elements, were found in the deepest regions of newly formed scales.     

Morphogenesis and histology of large scales of batoids (Elasmobranchii)

Large scales occur only in three families of batoids. Though they have a wide spectrum of different shapes they all serve protective functions in bottom-dwelling species. The crown consists of enameloid, orthodentine and osteodentine. Three different types of basal plates occur: (a) thin basal plate consisting of acellular bone; (b) basal plate which is secondarily thickened; it consists of massive acellular bone and thin denteons which surround the vascular canals; (c) basal plate which is secondarily thickened, consisting of a peculiar type of microspongy bone which has never been found in other elasmobranchs. The scales have either one or several crown elements. None of the scales, however, belongs to a growing type. All large scales were probably replaced regularly. It is the first time that dentine was found within the basal plate of an elasmobranch scale.     

The notochordal sheath in amphioxus--an ultrastructural and histochemical study

The notochord and notochordal sheath of 10 adult amphioxus were investigated ultrastructurally and histochemically. The notochord in amphioxus consists of parallel notochordal cells (plates) and each plate consists of parallel thicker and thinner fibrils and numerous profiles of smooth endoplasmic reticulum situated just beneath the cell membrane. Histochemical staining shows that the notochordal plates resemble neither the connective tissue notochordal sheath nor the typical muscular structure myotomes. The notochordal sheath has a complex three-layered organization with the outer, middle and inner layer The outer and middle layer are composed of collagen fibers of different thickness and course, that correspond to collagen type I and collagen type III in vertebrates, respectively, and the inner layer is amorphous, resembles basal lamina, and is closely attached to the notochord by hemidesmosome junctions. These results confirm the presence of collagen fibers and absence of elastic fibers in amphioxus.     

A morphological study of regeneration of the goldfish elasmoid scales

Studies were carried out on the model of regeneration of mechanically removed elasmoid scales. Regenerating scales were morphologically analyzed using light and electron microscopy. It was found that the cells responsible for regeneration of the elasmoid scale plates could be classified as osteogenic elements. Little differentiated preosteoblasts were detected in the connective tissue of dermis on day 3 of regeneration, while partially calcified plates underlay osteoblasts on day 7. The scale cover was fully restored on day 14 and it took two days for each bone plate to be formed. Osteocytes, fully differentiated osteogenic elements, were found in the deepest regions of newly formed scales.     

Elektronenmikroskopische Untersuchungen anAnaitides mucosa (Annelida,Polychaeta). Cuticula und Cilien,Schleimzellen und Schleimextrusion

Two components, a basal cuticle and an epicuticle, make up the cuticle ofA. mucosa. The basal cuticle consists of collagen fibrils, which are arranged in about 20 layers. The orientation of the fibrils changes rectangularly from one layer to the next. Fine filaments interweave the basal cuticle. The epicuticle, which is covered by a layer of electron dense material, is composed of irregularly arranged thin filaments. Branched microvilli of the epidermal cells penetrate the cuticle. Bacteria are found in the basal cuticle. Dorsally each segment has a band of densely packed smooth cilia. Laterally and partly ventrally aggregates of cilia are observed. These cilia exhibit apically artificial swellings. At least six different mucous cells are observed in the epidermis, morphologically distinguishable by the structure of the secretion products. Mucus is secreted via exocytosis through cuticular pores. During this process the mucus might expand. The secreted mucus consists of filamentous subunits.     

Minute tubercles on the skin surface of larvae in the Korean endemic bitterling, Rhodeus pseudosericeus (Pisces, Cyprinidae)

The morphology and distribution of the minute tubercles on the skin surface of larvae in Korean bitterling, Rhodeus pseudosericeus, were observed during larval development. Just after hatching, the epidermis of the larvae consists of a thin single cell layer having smaller basophilic flat or round‐flattened basal cells. As the larvae grow, the epidermis contains more small flat cells and large epidermal cells that are round or hemisphere‐shaped. These large unicellular epidermal cells, called minute tubercles, consist of more or less homogeneous cytoplasm that is PAS (Periodic acid‐Schiff method) positive. They are more densely distributed in the wing‐like yolk sac projection. Vestigial minute tubercles occur in the body region and the caudal fin‐fold region. These minute tubercles grow in number and height from 6 to 8 days after hatching onward. However, they become reduced in height and number as the larvae develop. At day 31 after hatching (i.e. free‐swimming stage), minute tubercles no longer exist on the larval skin. The sequence of occurrence and gradual disappearance of these cell structures are described and histologically documented for comparative purposes of beta, taxnomomic and environmental studies.     

The Osteichtyes,from the Paleozoic to the extant time,through histology and palaeohistology of bony tissues

The aim of this short review is to emphasize the richness of the comparative histological studies on both fossil and extant Osteichthyes. Some selected examples in both Sarcopterygii (excluding tetrapods) and Actinopterygii show how it is possible to improve our knowledge on bone biology of extinct species but also to obtain new data on their palaeobiology or on their paleobiogeography. After a brief survey of the organization of bony tissues in osteichthyes, we review some examples of skeletal peculiarities in the following extinct and extant taxa: the histological structure of polypterid scales that suggests a hypothesis on the possible age and the biogeographical history of this basal actinopterygian taxon; the ossified lung of the fossil coelacanthids, with a discussion on its potential function; the histological organization of the sarcopterygian derived elasmoid scales (of Eusthenopteron sp., Latimeria sp. and Neoceratodus sp.). These comparative palaeohistological and histological data provide the basis of a general discussion of the evolutionary trends of bony tissues and their derivatives in Osteichthyes.     

Particular structure of the anterior third of the human true vocal cord.

The histological aspects of the true vocal cord mucosa change in the anterior third compared with the posterior two thirds. The anterior third is characterized by an epithelium where the ridges, marked in the posterior two thirds, are very slight or even absent. The underlying basement membrane, which is thin in the posterior two thirds, here appears particularly thick. At the ultrastructural level in this area, beneath a normally thickened basal lamina, a thick layer of finely granulated electron-dense material, interspersed with thin and randomly scattered collagen fibrils and proteoglycan filaments, is detectable. Beneath this thickened basement membrane, a layer of small undulated collagen fibril bundles with very numerous interspersed oxytalan fibres is found. The collagen fibrils, small in diameter (30-40 nm), seem to continue with the collagen fibrils of the basement membrane. In this layer numerous blood vessels with a very thick, delaminated basement membrane are also observed. The underlying area is characterized by the vocal cord ligament, composed by large compact collagen fibril bundles with interspersed elastic fibres. The particular features of the thick basement membrane, the thick-walled and delaminated vessels and the modular distribution of the elastic system together may well form the basic structure enabling the functional integration of the vocal ligament into the overlying mucosa and the underlying vocal muscle.