Cellular morphology and markers of cartilage and bone in the marine teleost Sparus auratus

ArticleinCell and Tissue Research 343(3):619-35 · March 2011with 22 Reads
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Abstract
Modifications have been characterised in terms of cellular organisation and the extracellular matrix (ECM) during bone ontogeny in the sea bream (Sparus auratus). During endochondral development, the agglomeration of matrix-secreting cells gives rise to chondrones; these chondrones frequently contain proliferating-cell-nuclear-antigen-positive cells, which subsequently become large collagen-II-positive cells with the characteristics of chondrocytes. Moreover, the matrix:cell ratio within the perichondrium increases, accompanied by a modification in ECM composition. Mineralisation of cartilage ECM is marked by a rapid fall in cell number, the switching off of collagen II transcription and the switching on of collagen X transcription, followed by collagen I transcription and bone mineralisation. The formation of dermal structures initiated upon the condensation of mesenchyme cells defines the future location of the dermal bone. Subsequent cellular differentiation gives rise to cells on the bone surface; these cells are positive for collagen I and osteonectin transcripts. The fish skeleton, with the exception of vertebrae, tends to comprise flattened bones that are covered by a monolayer of cells, the periosteum. A third type of tissue, present in gills, consists of chondrocyte-like cells embedded in a mineralised matrix resembling chondroid bone in mammals. The results suggest that the cellular organisation and ontogeny of endochondral and dermal bone in the sea bream are similar to those described in other vertebrates.

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    Among hatchery bred Sparus aurata, 5.4% of the offspring of a single parent pair showed severe skeletal deformations coupled with marked growth retardation and pathological changes in the swimbladder. This resulted from extreme proliferation, then hypertrophy, of the cuboidal epithelial cells of the gas gland and proliferation of the rete mirabile. This caused almost complete congestion of the swimbladder air space. The nature of the pathological changes in the swimbladder and the possibility of hereditary origin for both the skeletal and swimbladder deformations are discussed.
  • Article
    Small teleost fish, such as the medaka (Oryzias latipes), are attractive animal models to study the genetics underlying bone formation. In order to characterize specific marker genes for bone formation in medaka, we identified and analyzed the gene expression of collagen type10a1 and osteocalcin during embryonic and larval development. In mammals and chicken, Collagen type 10a1 is expressed in hypertrophic chondrocytes. Osteocalcin, on the other hand, is expressed in mature osteoblasts, which have started to produce mineralized bone matrix. In contrast to mammals and chicken, expression of collagen type 10a1 during medaka embryogenesis is not found in chondrocytes but instead is restricted to intramembranous and perichondral bone formation. Therefore, collagen type 10a1 expression marks early osteoblasts. Osteocalcin, on the other hand is expressed in mature osteoblasts in mineralizing intramembranous and perichondral bone.
  • Article
    The development of cartilaginous structures in cultured sea bream Sparus aurata larvae and the timing of their ossification was studied. In cultivated sea bream larvae the first cartilaginous structure to be identified was hypural 1 at 4.1 mm notochord length (LN). By 5.3 mm LN, prior to the onset of ossification, it was possible to distinguish the following cartilaginous structures: all 23 neural arches, all 13 haemal arches and two of the four pairs of parapophyses. The neural arches 1–4 and 15–23 were formed on the notochord and elongated dorsally, while neural arches 5–14 appeared on the dorsal side of the spinal cord and elongated ventrally. Initiation of ossification occurred at 5.7–6.0 mm standard length (LS) when the cartilaginous ontogeny of the vertebral column was completed. Ossification was coincident with dorsal flexion at the posterior end of the notochord and occurred in a sequential manner: (1) dorsoanteriorly, the cartilaginous neural arches and the centra were the first structures to ossify; (2) ventrad at the centre, at 7.0–7.5 mm LS; (3) posteriorly at 7.1 mm LS the hypural complex and urostyle (24th centrum) were ossified; and (4) dorsad at the centre (neural arches and spines).
  • Article
    Sensitivity of skeletal ontogenesis to temperature was assessed in Mozambican tilapia Oreochromis mossambicus and culture temperature manipulations (22, 27 and 32 C) were used to establish if age or length gave the most suitable metric for standardization. Oreochromis mossambicus larval growth was composed of two growth stanzas: an initial period of rapid growth, followed by a slower growth phase. Irrespective of culture temperature chondrogenesis occurred during the first rapid growth phase and ossification was initiated during the second slower growth phase. The sequence of events and rate at which ossification occurred was much more sensitive to temperature than chondrogenesis. Cumulative counts provide a useful developmental index for skeletal onto-genesis; overall, age (effective days-degrees) gave the best estimation of developmental status during chondrogenesis and the initiation of ossification, although standard length (as log 10 L S) was a better metric for completion of ossification. The timing of development of functionally important structures, such as Meckel's cartilage, the branchial arches, the centra and the cleithrum important for breathing, feeding and swimming, was well conserved at all temperatures and may be a good index of teleost developmental stages.
  • Article
    The morphology of scale osteoclasts in rainbow trout Oncorhynchus mykiss was characterized by light and scanning electron microscopy, and the effects of oestradiol-17β-treatment and sexual maturation on scale osteoclast morphology were investigated. The cells associated with resorption cavities could be distinguished morphologically as two types: symmetrical, compact cells lacking or having only a few cell processes, termed type 1 cells, and asymmetrical cells covered with folds and having several cell processes, termed type 2 cells. In adult sexually maturing fish, where scale resorption was high, type 1 cells were predominant. In juveniles and spawned adults where scale resorption was assumed to be relatively low, mostly type 2 cells were present. Oestradiol 17-β-treatment of juvenile rainbow trout increased the osteoclast activity, but did not affect the osteoclast morphology. Using light microscopy, the majority of the cells observed in, and closely associated with, the resorption cavities were mononucleated in both maturing and spawned fish. Occasionally, bi- and multinucleated osteoclasts were observed in the maturing, but not in the spawned fish. Light microscopic enzyme-histochemistry showed that the majority of the mononucleated cells, as well as the bi- and multinucleated ones, were tartrate resistant acid phosphatase positive in both groups of fish, thus implying that both type 1 and type 2 cells were osteoclasts. It is thus apparent that scale resorption in rainbow trout is carried out by two morphologically distinct osteoclast populations, representing different stages of osteoclast activity and/or different stages of osteoclast differentiation.
  • Article
    Full-text available
    In teleosts, a considerable part of the body calcium is found in the scales. Associated with the scales are osteoblasts and osteoclasts, and during periods of high calcium demand such as during sexual maturation or starvation, the scales can be resorbed and thereby act as an internal calcium reservoir. In mammalian bone tissue, the activity of an acid phosphatase (ACP) isoenzyme, tartrate resistant acid phosphatase (TRACP), can be used as a marker for osteoclastic activity. In the present study, an evaluation of TRACP as a marker for osteoclastic activity in teleost scales has been performed. ACP and TRACP was histologically localized at resorption sites around the edge of the scales as well as at resorption holes in the scales. The optimal conditions for biochemical measurements of ACP and TRACP activity were found to be pH 5.3, 10 mM paranitrophenylphosphate, incubated for 30 min at room temperature, and 10 mM tartrate added when required. Using TRACP as a marker, estradiol-17 (E2) was found to increase the proportion of scales being resorbed, as well as the number and size of resorption sites per scale. Also, the scales of E2-treated fish showed weaker staining for calcium. Together, the obtained data indicate that estradiol-17 induces osteoclastic activity in teleost scales, resulting in increased resorption of the scales. A period of refeeding following a period of starvation did not have detectable effects on the scale osteoclastic activity and scale resorption.
  • Article
    Fin-ray rudiments became visible at 9.0 mm TL in the dorsal and anal fins, at 8.0 mm TL in the pectoral and pelvic fins and 6.0 mm TL (size at extrusion) in the caudal fin. Completion of segmentation of soft rays in the dorsal and anal fins was attained by 14 mm TL and in all fins by 17 mm TL. Branching of soft rays in the respective fins started and was completed considerably later than the completion of segmentation, as well as ossification of the fin-supports. Morphological transformation from larva to juvenile was apparently completed by about 17 mm TL. Although the completion of basic juvenile structures was attained by transformation at that body size, succeeding morphological changes occurred between 17 mm and 32 mm TL. Newly-extruded larvae possessed one or two teeth on the lower pharyngeal and pharyngobranchials 3 and 4, but lacked premaxillary, dentary, palatine and prevomer teeth. The fish attained full development of gill rakers and gill teeth by 15 mm TL, the upper and lower pharyngeal teeth subsequently developing into a toothplate. Development of the premaxillary, dentary and palatine teeth was completed at about 30 mm TL, by which time loop formation of the digestive canal and the number of pyloric caeca had attained the adult condition. The developmental sequence of swimming and feeding functions during larval and early juvenile periods appeared to proceed from primitive functions to advanced or complex ones, from the ability to produce propulsive force to that of swimming with high maneuverability and from development of the irreducible minimum function of passing food into the stomach to the ability to actively capture prey via passive food acquisition with the gill rakers and gill teeth. The relationship of morphological development to the behavior and feeding activity of artificially-produced hatchlings is also discussed.
  • Article
    The development of the frontal bone and the formation of the first head scales are described during post-embryonic ontogeny of Hemichromis bimaculatus, using light and transmission electron microscopy. The frontal bone originates close to the cartilaginous taenia marginalis in a loose mesenchymal cell condensation (=primordium) lying 1 m from the epidermis with which it establishes no cell contacts. The anlage appears at 4.2 mm standard length (SL) in the form of the membranodermal component of the bone, and extends first over the brain and then over the eye; the neurodermal component forms later to surround the supraorbital canal. The first head scales appear at 10.0 mm SL in a dense cell condensation (papilla) adjoining the epidermal-dermal junction and, once formed, remain in this position. In both organs, the initial matrix is similarly composed of woven-fibred bone that soon mineralizes in a similar manner to other dermal elements. In some areas of the frontal bone, parallel-fibred bone is deposited unequally on both surfaces, whereas isopedine is deposited in scales on the deep surface only. Osteoblastic features confirm this eccentric growth. Differences in the shape, organization and localization of the mesenchymal condensations giving rise to the frontal bone and to the scale reflect the existence of two types of dermal cell condensations. Our data are compared with those available for the post-cranial dermal skeleton of fishes both from a developmental and structural viewpoint. Structural differences in the matrices of the frontal bone and scales are discussed in a phylogenetic perspective.
  • Article
    Zebrafish have recently become a model of choice among developmental biologists. This unique model enables both modern molecular and genetic studies to be carried out to identify genes involved in a wide variety of developmental processes. The success of the genetic approach depends largely on the application of an easy and effective screening method to identify interesting mutants. In order to develop a method for visualizing skeletal structures in zebrafish embryos that would be suitable for screening skeletal mutants, we investigated the use of the fluorescent chromophore calcein, which binds specifically to calcified skeletal structures. By using this method, we followed the development of the skeletal structures in zebrafish embryos from day 1 to day 21 postfertilization, and analyzed the effect of bone morphogenetic protein-2 (BMP2) on axial skeleton development. We found the development of the calcified skeletal structure to appear in a progressive fashion from head to tail. Calcified structures in the head (i.e., the jaw) developed first, which were then followed by the axial skeleton in the trunk. Interesting to note was that there appeared to be two domains in the calcification of vertebrae within the axial skeleton. The first three vertebrae were in the first domain; the rest being in the second domain. Compared with Alcian blue staining, we found that calcein staining indeed labels calcified skeletal structures, and, moreover, it is a more sensitive and inclusive method for visualizing skeletal structures. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed BMP2 in zebrafish notochord cells. We demonstrated that ectopic expression of BMP2 in notochord cells inhibited the development of the axial skeleton. Together, these results clearly demonstrated the sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants that have bone structure defects.
  • Article
    The skeleton is a single organ composed of >200 different elements spread throughout the body. These skeletal elements comprise two tissues: cartilage and bone. Both tissues contain specific cell type(s): chondrocytes in cartilage and osteoblasts and osteoclasts in bone. We are beginning to understand the genetic control of the differentiation and function of these cells through recent developments in mouse and human genetics, and also through the use of molecular biological and biochemical techniques. The most recent advances in terms of cell differentiation in the skeleton are presented in this review.
  • Article
    This paper presents transmission electron microscopical observations on the chondroid bone (CB) supporting the neurocraniad articulation facet of the upper pharyngeal jaws of juvenile specimens of Hemichromis bimaculatus (an acellular-boned teleost fish). Chondroid bone, a skeletal tissue morphologically intermediate between cartilage and bone, is composed of a dense mineralized collagenous matrix, resembling that of woven-fibred bone, and large chondrocyte-like cells. The latter vary considerably in their morphological features (functional cells, cells containing a large vacuole and degenerating cells). The CB is mineralized except for its upper layer. Mineralization is initiated in matrix vesicles. Clusters of apatite crystals coalesce at the mineralization front. Distally, the tissue grows by incorporation of cells which exhibit the features of osteoblasts, and which derive from less differentiated fibroblast-like cells located in the outermost layer of the tissue. Proximally, the CB is subjected to erosion by multinucleated clastic cells. The deposition of bone against the wall of lacunae which have been opened by clastic resorption may suggest a possible active involvement of the CB cells. Further studies should point out whether this bone substantially contributes to the acellular dermal dentigerous bone located below.
  • Article
    The present study evaluated the effects of increasing levels of dietary retinol on diet utilisation, growth, mortality, retention of vitamin A1 plus its conversion to vitamin A2 in liver and whole body of juvenile sunshine bass. In addition, early indications of disturbed intestinal cell kinetics, as well as liver heat-shock protein 70 (HSP70) responses were included. Feeding juvenile sunshine bass a purified, casein-based diet with no added retinol resulted in significantly lower growth. Feeding diets containing from 509 to 40 516 μg retinol kg−1 resulted in equal growth, survival, feed efficiency, condition factors as well as liver and carcass indices. However, dietary retinol levels were reflected in both liver and whole body in a linear manner, but with no retention in the groups fed a deficient or 509 μg retinol kg−1 diet. The conversion of vitamins A1 to A2 in all groups depended on the dietary level of vitamin A1. Fish fed the 40 516 μg retinol kg−1 diet had reduced conversion of vitamins A1 to A2 and altered distribution of proliferating cell nuclear antigen (PCNA)-positive cells along their intestinal folds and crypts. The resistance to temperature stress and expression of liver HSP70 appeared to be efficient in all dietary treatments, but fish fed the 1614 μg retinol kg−1 diet showed the greatest increase in HSP70 following heat shock, compared to the other groups. Based on growth, mortality, retention and conversion of vitamins A1 and A2, distribution of PCNA-positive cells and increase in HSP70, we conclude that the requirement for optimal growth and health of sunshine bass is greater than 509 μg retinol kg−1 diet and less than 40,516 μg retinol kg−1 diet.
  • Article
    The osteological ontogeny of Pagellus erythrinus (Linnaeus, 1758) and its temperature-induced plasticity is studied. The ontogeny of the skeleton follows the pattern described in all other Sparidae studied so far: the onset of the ontogeny of the vertebral column and the caudal fin is at 4.3 mm total length (TL) and the completion at 17.6 and 15.4 mm TL, respectively. Dorsal and anal fin development appears at about 5.2 mm TL and completes at 7.5 mm TL. The pelvic are the last fins to develop (7.5-11.5 mm TL). Although the pectorals are the only fins present before first feeding, their ontogeny is completed only after 12.0 mm TL. Temperature affects the developmental rate of the skeleton giving as much as 10.9% of differentiation in total length between 16 and 21°C (caudal lepidotrichia), 9.2% between 16 and 18°C (caudal lepidotrichia) and 6.7% between 18 and 21°C (pelvic lepidotrichia). The role of temperature in developmental plasticity is enhanced by the induction of abnormalities mainly in the area of the caudal fin, which reached (in total) 75% occurrence in the higher temperature regime (23°C) tested. The results of the skeletal ontogeny are compared with the pattern of development of the Sparidae, and are discussed in respect to the role of temperature in the developmental plasticity of fish. © 2004 Elsevier B.V. All rights reserved.
  • Article
    The osteological development of dorsal, anal and paired fins and the squamation in common dentex, Dentexdentex (Linnaeus, 1758), was studied under extensive larval rearing conditions. The ontogeny of the fins was determined in three stages: (1) the yolk-sac and the early larval stage (up to 6.7 mm in vivo total length, TL), in which the majority of the elements of the pectoral fins and the predorsals, dorsal and anal proximal pterygiophores formed; (2) the late larval stage (up to 11.6 mm TL), in which the majority of the pectoral lepidotrichia, the lower extrascapulae, all the elements of the pelvic fins, as well as the lepidotrichia, spines and the distal radials of the rest fins developed; and (3) the metamorphosis stage (up to 24.0 mm TL), when the upper extrascapulae formed and the pectoral lepidotrichia attained their full count. The ontogeny of squamation was carried out during the metamorphosis stage; it started at 13.4 mm TL with laterally symmetric points on the middle of each side of the body, followed by one ventral centre (15.6 mm TL) which was located anterior to the pelvic fins, and two bilateral pairs of centres which appeared on the head (17.8 and 21.2 mm TL). The results are discussed from a functional viewpoint and in comparison with the fin and scale ontogeny of other teleosts.
  • Article
    Full-text available
    The ontogenetic development of the chondrocranium of Ohrid trout Salmo letnica was studied from hatching until 92 days post-hatching (dph). Most of the samples were in toto trypsin cleared and stained, some specimens were used for serial histological sectioning. The serial histological sections of fish specimens at the age of 92 dph were used for a graphical recon- struction of the cartilaginous neurocranium. A chronological evaluation of the formation of the cartilaginous skull in the early development of S. letnica was performed. In order to investigate to what degree the ontogeny of the Ohrid trout is unique, the results were compared with data of the development of other salmonids, as well as some non-salmonid teleosts. The development of the cartilaginous structures of the Ohrid trout was found to be similar to that of other salmonids. Most of the cartilage structures of the neurocranium and the viscerocranium are present at the moment of hatching of this species. A fully developed chondrocranium was observed at the age of 92 dph, when the first signs of cartilage resorption could also be observed.
  • Article
    Full-text available
    Hedgehog (Hh) signalling has been implicated in the development of osteoblasts and osteoclasts whose balanced activities are critical for proper bone formation. As many mouse mutants in the Hh pathway are embryonic lethal, questions on the exact effects of Hh signalling on osteogenesis remain. Using zebrafish, we show that there are two populations of endochondral osteoblasts with differential sensitivity to Hh signalling. One, formed outside the cartilage structure, requires low levels of Hh signalling and fails to differentiate in Indian hedgehog mutants. The other derives from chondrocytes and requires higher levels of Hh signalling to form. This latter population develops significantly earlier in mutants with increased Hh signalling, leading to premature endochondral ossification, and also fails to differentiate in Indian hedgehog mutants, resulting in severely delayed endochondral ossification. Additionally, we demonstrate that the timing of first osteoclast activity positively correlates to Hh levels in both endochondral and dermal bone.
  • Article
    Full-text available
    Basic research in pattern formation is concerned with the generation of phenotypes and tissues. It can therefore lead to new tools for medical research. These include phenotypic screening assays, applications in tissue engineering, as well as general advances in biomedical knowledge. Our aim here is to discuss this emerging field with special reference to tools based on zebrafish developmental biology. We describe phenotypic screening assays being developed in our own and other labs. Our assays involve: (i) systemic or local administration of a test compound or drug to zebrafish in vivo; (ii) the subsequent detection or "readout" of a defined phenotypic change. A positive readout may result from binding of the test compound to a molecular target involved in a developmental pathway. We present preliminary data on assays for compounds that modulate skeletal patterning, bone turnover, immune responses, inflammation and early-life stress. The assays use live zebrafish embryos and larvae as well as adult fish undergoing caudal fin regeneration. We describe proof-of-concept studies on the localised targeting of compounds into regeneration blastemas using microcarriers. Zebrafish are cheaper to maintain than rodents, produce large numbers of transparent eggs, and some zebrafish assays could be scaled-up into medium and high throughput screens. However, advances in automation and imaging are required. Zebrafish cannot replace mammalian models in the drug development pipeline. Nevertheless, they can provide a cost-effective bridge between cell-based assays and mammalian whole-organism models.
  • Article
    Resorption and remodelling of skeletal tissues is required for development and growth, mechanical adaptation, repair, and mineral homeostasis of the vertebrate skeleton. Here we review for the first time the current knowledge about resorption and remodelling of the skeleton in teleost fish, the largest and most diverse group of extant vertebrates. Teleost species are increasingly used in aquaculture and as models in biomedical skeletal research. Thus, detailed knowledge is required to establish the differences and similarities between mammalian and teleost skeletal remodelling, and between distantly related species such as zebrafish (Danio rerio) and medaka (Oryzias latipes).
  • Article
    Intramembranous and chondral bone formation by osteoblasts is found in all vertebrates. The genetic network controlling osteoblast differentiation is highly conserved and regulated by a small number of key factors, including the zinc-finger transcription factor Osterix. Expression analysis of osterix in the teleost model medaka revealed a highly restricted expression in skeletal regions. For in vivo imaging, we generated transgenic medaka expressing mCherry under control of the osterix promoter. We show that the transgene becomes expressed in early osteoblasts, which have not yet mineralized bone matrix, and remains high in matured and mineralizing osteoblasts. Life imaging of transgenic larvae provided insight into the appearance and behavior of early osteoblasts during development of the teleost cranium, vertebrae, and caudal fin. In summary, osterix-mCherry transgenic medaka enable us to analyze osteoblasts during different maturation phases in vivo and represent a unique tool to study osteoblast behavior in vertebrate embryos and adults.
  • Article
    Full-text available
    Otoliths and the homologous otoconia in the inner ear are essential for balance. Their morphogenesis is less understood than that of other biominerals, such as bone, and only a small number of their constituent proteins have been characterized. As a novel approach to identify unknown otolith proteins, we employed shotgun proteomics to analyze crude extracts from trout and catfish otoliths. We found three proteins that had not been associated previously with otolith or otoconia formation: 'Secreted acidic cysteine rich glycoprotein' (Sparc), an important bone protein that binds collagen and Ca(2+); precerebellin-like protein, which contains a C1q domain and may associate with the collagenous otolin-1 during its assembly into a framework; and neuroserpin, a serine protease inhibitor that may regulate local protease activity during framework assembly. We then used the zebrafish to investigate whether Sparc plays a role in otolith morphogenesis. Immunodetection demonstrated that Sparc is a true constituent of otoliths. Knockdown of Sparc expression in morphant zebrafish resulted in four principal types of defective otoliths: smaller, extra and ectopic, missing and fused, or completely absent. Smaller size was the predominant phenotype and independent of the severity of otic-vesicle defects. These results suggested that Sparc is directly required for normal otolith growth.
  • Article
    The comparative ultrastructure of fish bone osteogenesis and resorption induced by scale removal was described in the osteocytic (cellular-boned) Carassius auratus and the anosteocytic (acellular-boned) Tilapia macrocephala. Osteocytes, present in osteocytic bone, were lacking in anosteocytic bone. In osteocytic bone the osteoblast secreted a collagenous preosseous matrix in which it became enmeshed and then was termed a preosteocyte. When the preosseous matrix mineralized, the preosteocyte was termed an osteocyte and was completely surrounded by bone. In anosteocytic bone the osteoblasts receded from the mineralizing front and never became trapped as osteocytes. During resorption, types A and B resorptive cells, present in both bone types, invaded the matrix and demineralized the osseous zone. These cells were characterized by large amounts of granular endoplasmic reticulum and intracellular inclusions containing crystal-like material. Although functionally similar to mammalian osteoclasts, these cells lacked a characteristic ruffled border and were not multinucleated. The osteocytes of cellular bone did not appear to be involved during demineralization.
  • Article
    The structure and distribution of cartilage and related tissues in the dorsal fin, caudal fin and vertebrae of teleosts were studied in 11 species. With the exception of Zellknorpel, all the tissues previously described in teleost heads were present in the trunk and fins, although they were found in smaller quantities. The distribution of the supporting tissues indicates that they serve different functions. Hyaline cartilage was restricted to vertebral and fin bones undergoing endochondral ossification, fibro/cell-rich cartilage acted as an articular tissue, and hyaline-cell cartilage and its subtypes formed flexible and resilient supports in the caudal fin. Mucous connective tissue was packed as a space-filler around neurovascular bundles in fin rays, and chondroid bone was found beneath articular surfaces. The differences between cranial, and trunk and fin supporting tissues may reflect developmental as well as functional differences between the cranial and postcranial skeleton.
  • Article
    The mineralized scale of the freshwater sunfish Lepomis macrochirus (bluegill) contains a Gla protein. The protein was identified in extracts of scale by a new colorimetric assay for Gla-containing proteins. The protein was purified by gel filtration chromatography followed by reversed phase high performance liquid chromatography (HPLC). Several tests establish the identity of scale Gla protein and bone Gla protein (BGP). First, the proteins exhibit identical mobilities on electrophoresis and by reversed phase HPLC. Second, they have identical amino-terminal amino acid sequences. Finally, identical peptides are generated by proteolytic digestion. The 45-residue amino acid sequence of the bone Gla protein from L. macrochirus has a high sequence homology with swordfish, as well as homology to mammalian bone Gla protein. The BGP of bluegill shares with swordfish BGP a truncated NH2 terminus and an extended COOH terminus. These features may be unique to fish, as they have not been observed in terrestrial vertebrates. The bluegill BGP is the first vitamin K-dependent protein to contain a non-gamma-carboxylated residue to the NH2-terminal side of all of its Gla residues. In all other vitamin K-dependent proteins, Gla always appears to the NH2-terminal side of the first Glu. The implications of this result are discussed. The bluegill rib bone is curiously enriched in BGP, as are other mineralized tissues of this species. One hypothesis is that this may be due to the acellular nature of the bone in this species. The abundance of BGP in the bones of this fish may provide clues to the unknown function of this bone protein.
  • Article
    An ultrastructural study by transmission electron microscopy (TEM) of the vertebrae of embryonic, larval, juvenile and mature medaka shows that each vertebra consists of a core of notochordal cells surrounded by a sheath of bone. The vertebral bone lacks either fully or partially embedded cells in the matrix throughout development. Bone matrix is secreted by a layer of cells that lies over the outer surface of the vertebral bone. During the early stages of osteogenesis, these cells secrete bone matrix all around themselves. However, because of the gradual flow of the newly synthesized bone matrix through intercellular spaces, matrix-producing cells do not become trapped in their own secretion. In later stages of osteogenesis, these cells secrete matrix only toward the already-deposited bone. This polarized matrix secretion allows the osteoblasts to stay always on the bone surface and never to become trapped in the matrix as osteocytes.
  • Article
    Full-text available
    Among vertebrates, some teleosts are unique in having bone which lacks osteocytes embedded in the matrix. The fate of cells that secrete the matrix of these acellular bones has not been investigated thoroughly. Histological and fluorescent microscopic analysis of the vertebral bone of Oryzias latipes demonstrated that acellularity is not a secondary appearance of an early cellular bone during ontogeny. Vertebral bone is devoid of cells embedded in the matrix throughout development. Cells that secrete bone matrix do not become trapped in their own secretion. Instead, they always remain as a surface layer over the outer surface of the bone. Fluorescent microscopic visualization of tetracycline injected into growing fish demonstrated that bone was only deposited by osteoblasts lining the outer surface of the bone; no deposition of bone took place on the inner surface.
  • Article
    Bone growth and calcium metabolism were studied in marine acellular-boned toadfish (Opsanus tau) by tetracycline and by45Ca labeling over a 26-day period. The uptake of tracer in vertebrae was initially faster than in the jaws, but the percent retention of45Ca on a gram ash basis in these tissues was not significantly different after 4–6 hours. This was the time at which microdensitometric analysis of contact autoradiographs of jaw sections indicated that peak45Ca uptake had occurred on all cortical bone surfaces. No loss of45Ca from bone surfaces was detected during the next 14 days. The differences in45Ca uptake in these tissues appeared to be related to the relative surface areas of bone rather than to differences in mineral content per unit volume of bone. Attempts to measure appositional bone growth by the separation of two tetracycline labels administered at an interval of 18 days were unsuccessful, and only a single subperiosteal fluorescent band 8–9 μ thick was observed. This was probably the result of a very low rate of excretion of tetracycline from the blood, together with a very low appositional rate of bone growth of only 0.2–0.3 μ/day. The fall in the plasma radioactivity was almost inversely proportional to time since injection. No diffuse labeling could be detected, due presumably to the absence of osteocytes, in lacunae and canaliculi which, in cellular bone, permits diffusion of calcium ions from extracellular fluid to bone crystals deep within the skeleton. The diffuse component, if present, was less than one-fifth that expected in cellular bone.
  • Article
    An immunohistochemical assay for proliferating cell nuclear antigen (PCNA) identifies cells in all active phases of the cell cycle. In this study, PCNA methodology, which was developed primarily for mammalian tissues, was adapted to three small fish species, medaka (Oryzias latipes), guppy (Poecilia reticulata), and western mosquitofish (Gambusia affinis) that are used in carcinogenesis bioassays and environmental sentinel studies. Our study showed that PCNA can be identified in routinely processed, paraffin embedded specimens of these fishes. Optimum staining conditions were dependent on fixative, primary antibody, antigen retrieval processing, and protein blocking reagent. Best results were achieved using 10% neutral buffered formalin as the fixative, clone PC10 as the primary antibody, and a combination of powdered milk and bovine serum albumin as a protein block. Except for medaka specimens, antigen retrieval was not required for specimens preserved in 10% neutral buffered formalin, but was required for the other fixatives tested. In whole fish specimens, PCNA marked cells in normally proliferating tissues such as testis, ovary, primary filament epithelium of the gill, hematopoietic tissues, thymus, retina and alimentary tract. The study demonstrated the successful application of mammalian-based PCNA technology to these aquatic species. Further applications of the assay will aid in understanding the role of cell proliferation in normal, diseased, and toxicant-affected tissues of aquatic animals.
  • Article
    Bone resorption by mononucleated cells was studied in the acellular bone of a teleost fish (Oreochromis niloticus) by histological and enzyme histochemical observations and by transmission electron microscopy. Bone resorbing cells (osteoclasts) were identified by their location at the sites of bone resorption, their frequent association with a band of concentrated activity of tartrate-resistant acid phosphatase at the bone surface and by the presence or lack of certain enzymes. Tartrate-resistant acid phosphatase was used as a marker for osteoclasts, and alkaline phosphatase as a marker for osteoblasts. Osteoclasts in O. niloticus are not multinucleated; however, during intense bone resorption, they form cell aggregations that resemble multinucleated giant cells in mammals. Conversely, during less intense bone degradation, osteoclasts are flat, have long narrow cytoplasmic processes and resemble the bone-lining cells of mammals. All bone-resorbing cells in O. niloticus are mononucleated and lack a ruffled border. Similarities to and differences from bone resorption by mononucleated cells in mammals are discussed.
  • Article
    Contact, a new zebrafish transforming growth factor-beta (TGF-beta) member is most closely related to mouse GDF5 and to human CDMP-1 responsible, when mutated, for limb brachypodism phenotype and Hunter-Thompson syndrome, respectively. Contact exhibits a dynamic spatial expression pattern in the pharyngeal arches and the pectoral fin buds that much prefigures cartilage formation. Within the fin buds, contact expression is detected in the proximal mesenchyme from which the endoskeleton will develop. Exogeneously applied retinoic acid (RA) induces duplication of the pectoral fin rudiment in zebrafish embryos as well as contact expression along the proximal margin of the fin mesenchyme showing that both endoskeleton and exoskeleton can be duplicated.
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    Full-text available
    The principal role of the skeleton is to provide structural support for the body. While the skeleton also serves as the body's mineral reservoir, the mineralized structure is the very basis of posture, opposes muscular contraction resulting in motion, withstands functional load bearing, and protects internal organs. Although the mass and morphology of the skeleton is defined, to some extent, by genetic determinants, it is the tissue's ability to remodel--the local resorption and formation of bone--which is responsible for achieving this intricate balance between competing responsibilities. The aim of this review is to address bone's form-function relationship, beginning with extensive research in the musculoskeletal disciplines, and focusing on several recent cellular and molecular discoveries which help understand the complex interdependence of bone cells, growth factors, physical stimuli, metabolic demands, and structural responsibilities. With a clinical and spine-oriented audience in mind, the principles of bone cell and molecular biology and physiology are presented, and an attempt has been made to incorporate epidemiologic data and therapeutic implications. Bone research remains interdisciplinary by nature, and a deeper understanding of bone biology will ultimately lead to advances in the treatment of diseases and injuries to bone itself.
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    To provide basic data about bone resorbing cells in the skeleton during the life cycle of Danio rerio, larvae, juveniles, and adults (divided into six age groups) were studied by histological procedures and by demonstration of the osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP). Special attention was paid to the lower jaw, which is a standard element for fish bone studies. The presence of osteoclasts at endosteal surfaces of growing bones of all animals older than 20 days reveals that resorption is an important part of zebrafish skeletal development. The first bone-resorbing cells to form are mononucleated. They appear in 20-day-old animals concurrently in the craniofacial skeleton and vertebral column. Mononucleated osteoclasts are predominant in juveniles. Regional differences characterize the appearance of osteoclasts; at thin skeletal elements (neural arches, nasal) mononucleated osteoclasts are predominant even in adults. Multinucleated bone-resorbing cells were first observed in 40-day-old animals and are the predominant osteoclast type of adults. Both mono- and multinucleated osteoclasts contribute to allometric bone growth but multinucleated osteoclasts are also involved in lacunar bone resorption and repeated bone remodeling. Resorption of the dentary follows the pattern described above (mononucleated osteoclasts precede multinucleated cells) and includes the partial removal of Meckel's cartilage. Bone marrow spaces created by resorption are usually filled with adipose tissue. In conclusion, bone resorption is primarily subjected to the demands of growth, the appearance of mono- and multinucleated osteoclasts is site- and age-related, and bone remodeling occurs. The results are discussed in relation to findings in other teleosts and in mammals.