Globuli ossei in the long limb bones of Pleurodeles waltl (Amphibia,Urodela, Salamandridae)

To date, little is known about the structure of the cells and the fibrillar matrix of the globuli ossei, globular structures showing histochemical properties of an osseous tissue, sometimes found in the resorption front of the hypertrophied cartilage in many tetrapods, and easily observed in the long bones of the Urodele Pleurodeles waltl. Here, we present the results obtained from the appendicular long bones of metamorphosed juveniles and subadults using histological and histochemical methods and transmission electron microscopy. The distal part of the cone‐shaped cartilage contains a heterogeneous cell population composed of the typical “light” hypertrophic chondrocytes and scarce “dark” hypertrophic chondrocytes. The “dark” chondrocytes display ultrastructural characteristics suggesting that they probably undergo degeneration through chondroptosis. However, in the hypertrophic, calcified cartilage close to the erosion front by the marrow, several noninvaded chondrocytic lacunae retained cells that do not show any morphological characteristics of degeneration and that cannot be identified as regular chondrocytes or osteocytes. These modified chondrocytes that have lost their regular morphology, appear to be active in the terminal cartilage and synthesize collagen fibrils of a peculiar diameter intermediate between the Type I collagen found in bone and the Type II collagen characteristic of cartilage. It is suggested that the local occurrence of globuli ossei is linked to a low rate of longitudinal growth as is the case in the long bones of postmetamorphic urodeles. J. Morphol. 275:1226–1237, 2014. © 2014 Wiley Periodicals, Inc.     

The dorsal fin engine of the seahorse (Hippocampus sp.)

The muscles, fin ray joints, and supporting structures underlying the dorsal fin are described for two seahorse species: Hippocampus zosterae and Hippocampus erectus. A fan-shaped array of cartilaginous bones, the pterigiophores, form the internal supporting structure of the dorsal fin. Each pterigiophore is composed of a proximal radial that extends from a vertebra to the dorsal side of the animal, where it fuses to a middle radial. The middle radials fuse with each other to form a dorsal ridge upon which sit the spheroidal distal radials. Each distal radial articulates with a fin ray on its dorsal side and is attached to the dorsal ridge on its ventral side by a material that has been histologically identified as elastic cartilage. Together these connections form a two-axis joint that permits elevation, depression, and inclination of the ray. Each fin ray is actuated by two bilateral pairs of muscles, an anterior pair of inclinators, and a posterior pair of depressors. The anteriormost fin ray is actuated by three bilateral pair of muscles, the inclinators, the depressors, and a pair of elevator muscles that are positioned anterior to the inclinators. Preliminary examinations of the ray joints of the pectoral and anal fins of adult H. zostera and the pectoral fins of newborn H. erectus revealed structures similar to that seen in the dorsal fins. To further explore the structure and function of the dorsal fin gross dissections and simple functional tests were performed on H. erectus and H. barbouri and behavioral observations were made of all three species plus Hippocampus kuda.     

Differentiation and growth of bone ornamentation in vertebrates: A comparative histological study among the Crocodylomorpha

Bone ornamentation, that is, hollow (pits and grooves) or protruding (ridges) repetitive reliefs on the surface of dermal bones, is a frequent, though poorly studied and understood, feature in vertebrates. One of the most typical examples of this characteristic is given by the Crurotarsi, a taxon formed by the crocodilians and their closest allies, which generally display deep ornamentation on skull roof and osteoderms. However, the ontogenetic process responsible for the differentiation and development of this character remains controversial. This study was conducted to settle the question on histological and microanatomical evidence in several crurotarsan taxa. Observational and experimental data in extant and extinct crocodyliforms show that bone ornamentation is initially created, and later maintained during somatic growth (that is indefinite in crocodilians), by a complex process of bone remodeling comprising local resorption of superficial bone cortices, followed by partial reconstruction. The superficial reliefs of crocodilian dermal bones are thus permanently modified through pit enlargement, drift, stretching, shrinking, or complete filling. Ridges are also remodeled in corresponding ways. These processes allow accommodation of unitary ornamental motifs to the overall dimensions of the bones during growth. A parsimony optimization based on the results of this study, but integrating also published data on bone histology in non‐crocodyliform crurotarsans and some non‐crurotarsan taxa, suggests that the peculiar mechanism described above for creating and maintaining bone ornamentation is a general feature of the Crurotarsi and is quite distinct from that attributed by previous authors to other vertebrates. J. Morphol. 276:425–445, 2015. © 2014 Wiley Periodicals, Inc.     

Staining and Histomorphometry of Microcracks in the Human Femoral Head

We developed staining techniques that permit identification and histomorphometric analysis of microcracks in the human femoral head 1) from thick, ground bone sections (100 μm) by prestaining with the Villanueva mineralized bone stain (MIBS), and 2) from plastic embedded, undecalcified thin bone sections (5-15 μm) by staining in gallocyanin chrome alum-Villanueva blood stain methods. Both methods represent a significant improvement in the stainability of the microcracks, cellular and tissue elements, and the simultaneous assessment of osteoid seams and tetracycline markers by histomorphometry. Shrinkage and other artifacts were minimized, which helped to clarify some of the uncertainties arising from artifacts resulting from some bone staining methods. Histomorphometric analyses of microcracks were conducted on thick, ground sections of subchondral and trabecular bone. Microcracks were more prevalent in the subchondral bone and osteochondral junction than in the more distant trabeculae. We have consistently localized microcrack areas in bone tissues prepared in these ways.     

Epigenetic mechanical factors in the evolution of long bone epiphyses

In developing vertebrate long bones in which endochondral ossification occurs, it is preceded or accompanied by perichondral ossification. The speed and extent of perichondral apposition relative to endochondral ossification varies in different taxa. Perichondral ossification dominates early long bone development in extinct basal tetrapods and dinosaurs, extant bony fish, amphibians, and birds. In mammals and lizards, perichondral and endochondral ossification proceed more synchronously. One of the most important epigenetic factors in skeletogenesis is mechanical loading caused by muscle contractions which begin in utero or in ovo . It has been previously shown that the stress distributions created perinatally in the chondroepiphysis during human skeletal development can influence the appearance of secondary ossification centres. Using finite element computer models representing bones near birth or hatching, we demonstrate that in vertebrates in which perichondral ossification significantly precedes endochondral ossification, the distribution of mechanical stresses in the ossifying cartilage anlagen tends to inhibit the appearance of secondary ossification centres in the ends of long bones. In models representing vertebrates in which endochondral ossification keeps pace with perichondral apposition, the appearance of secondary centres is promoted. The appearance of secondary centres leads to the formation of bony epiphyses and growth plates, which are most common in mammals and extant lizards. We postulate that genotypic factors influencing the relative speed and extent of perichondral and endochondral ossification interact with mechanical epigenetic factors early in development to account for many of the morphological differences observed in vertebrate skeletons.     

Growth and shaping of metacarpal and carpal cartilage anlagen: application of morphometry to the development of short and long bone. A study of human hand anlagen in the fetal period

A histological and morphometric analysis of human metacarpal and carpal anlagen between the 16th and 22nd embryonic weeks was carried out with the aim of studying the establishment of the respective anlage architecture. No differences in the pattern of growth were documented between the peripheral and central zones of the metacarpal epiphyses and those of the carpals. The regulation of longitudinal growth in long bone anlagen occurred in the transition zone between the epiphysis and the diaphysis (homologous to the metaphyseal growth plate cartilage in more advanced developmental stage of the bone). Comparative zonal analysis was conducted to assess the chondrocyte density, the mean chondrocyte lacunar area, the paired chondrocyte polarity in the orthogonal longitudinal and transverse planes, and the lacunar shape transformation in the metacarpal. In transition from epiphysis to diaphysis chondrocyte density decreased and mean lacunar area increased. No significant differences in the chondrocyte maturation cycle were observed between proximal/distal metacarpal epiphyses and the carpal anlagen. The number of paired chondrocyte oriented along the growth vector was significantly higher in both proximal/distal transition zones between epiphysis and diaphysis. Human metacarpals shared with experimental models (like mice and nonmammal tetrapods) an early common chondrocyte maturation cycle but with a different timing due to the slower embryonic and fetal developmental rate of human anlagen.     

Lessons from the first dorsal fin in atheriniforms—A new mode of dorsal fin development and its phylogenetic implications

The median fins in extant actinopterygians are the product of millions of years of evolution. During this time, different developmental patterns for the dorsal and anal fins emerged leading to a high variation in median fin morphology and ontogeny. In this study, the development of anal and dorsal fins in atheriniforms is described and its consequences for the current phylogenetic hypothesis are discussed. Developmental series of five atheriniform species were investigated using clearing and staining as well as antibody staining. The skeletal elements of the second dorsal fin and the anal fin emerge in a bidirectional pattern. The first dorsal fin, however, arises separately in front of the second dorsal fin after this one is almost completely formed. The pterygiophores of the first dorsal fin, including the interdorsal pterygiophores, develop from caudal to rostral, but the fin‐spines of the first dorsal fin form in the opposite direction. This new mode of fin development has been found in all examined atheriniform species with two dorsal fins. Several morphological characters of atheriniforms, including interdorsal pterygiophores, are also found in one other taxon: the Mugiliformes. Thus, several dorsal fin characteristics may provide evidence for a closer relationship of these two taxa.     

Immunolocalization of myostatin (GDF-8) following musculoskeletal injury and the effects of exogenous myostatin on muscle and bone healing

The time course and cellular localization of myostatin expression following musculoskeletal injury are not well understood; therefore, the authors evaluated the temporal and spatial localization of myostatin during muscle and bone repair following deep penetrant injury in a mouse model. They then used hydrogel delivery of exogenous myostatin in the same injury model to determine the effects of myostatin exposure on muscle and bone healing. Results showed that a "pool" of intense myostatin staining was observed among injured skeletal muscle fibers 12-24 hr postsurgery and that myostatin was also expressed in the soft callus chondrocytes 4 days following osteotomy. Hydrogel delivery of 10 or 100 μg/ml recombinant myostatin decreased fracture callus cartilage area relative to total callus area in a dose-dependent manner by 41% and 80% (p<0.05), respectively, compared to vehicle treatment. Myostatin treatment also decreased fracture callus total bone volume by 30.6% and 38.8% (p<0.05), with the higher dose of recombinant myostatin yielding the greatest decrease in callus bone volume. Finally, exogenous myostatin treatment caused a significant dose-dependent increase in fibrous tissue formation in skeletal muscle. Together, these findings suggest that early pharmacological inhibition of myostatin is likely to improve the regenerative potential of both muscle and bone following deep penetrant musculoskeletal injury.     

A biomechanical study of the long bones in platyrrhines.

The long bones of 72 individuals of extant platyrrhines, belonging to 17 species (11 genera) were studied by regressions of length, diameters and curvature. Cross-sectional shapes at midshaft and axial and bending strength indicators were also calculated. Results show that forelimb bones scale faster than hindlimb bones, for both length and diameters. Curvature scales faster in the femur than in other bones. Strength indicators showed a high variability in the relative importance of axial and bending loadings. Results are consistent with field observations of locomotor behaviour, mainly as regards quadrupedalism versus suspensory locomotion.     

Global-scale genetic identification of hammerhead sharks: Application to assessment of the international fin trade and law enforcement

The future status of sharks is an issue of widespread conservation concern due to declines in many species in the face of high levels of exploitation to satisfy market demands for products, especially fins. Substantial declines in the large-bodied hammerhead sharks, Sphyrna lewini, S. mokarran and S. zygaena, even in regions where some management occurs, indicate that informed conservation measures are warranted for these circumglobally distributed species. Despite the importance of assessing shark catch and trade on a species-specific basis to detect potential overexploitation of individual species, achieving this goal for hammerheads has proven elusive due to difficulties in identification of their products. Here, we present the development and application of a diagnostic, streamlined, five-primer multiplex polymerase chain reaction assay utilizing species-specific primers based on nuclear ribosomal ITS2 for the three hammerhead species throughout their global distribution. Application of this assay to investigations of the fin market confirmed the presence of hammerhead fins in the international trade. A study of the world’s largest fin market in Hong Kong revealed a high concordance between specific Chinese-name trade categories and fins from these three species (“Bai Chun” with S. lewini, “Gui Chun” with S. zygaena and “Gu Pian” with S.␣mokarran), and clear species preferences. This concordance information allows the use of market records for monitoring species-specific trends in trade and exploitation rates. The assay is also proving useful for identification of shark body parts in U.S. fisheries law-enforcement activities. Screening of morphologically identified “ S. lewini” from globally distributed areas using this assay with subsequent whole ITS2 sequencing suggests a cryptic species closely related to S. lewini occurs off the SE USA coast.     

Perlecan displays variable spatial and temporal immunolocalisation patterns in the articular and growth plate cartilages of the ovine stifle joint

Perlecan is a modular heparan sulphate and/or chondroitin sulphate substituted proteoglycan of basement membrane, vascular tissues and cartilage. Perlecan acts as a low affinity co-receptor for fibroblast growth factors 1, 2, 7, 9, binds connective tissue growth factor and co-ordinates chondrogenesis, endochondral ossification and vascular remodelling during skeletal development; however, relatively little is known of its distribution in these tissues during ageing and development. The aim of the present study was to immunolocalise perlecan in the articular and epiphyseal growth plate cartilages of stifle joints in 2-day to 8-year-old pedigree merino sheep. Perlecan was prominent pericellularly in the stifle joint cartilages at all age points and also present in the inter-territorial matrix of the newborn to 19-month-old cartilage specimens. Aggrecan was part pericellular, but predominantly an extracellular proteoglycan. Perlecan was a prominent component of the long bone growth plates and displayed a pericellular as well as a strong ECM distribution pattern; this may indicate a so far unrecognised role for perlecan in the mineralisation of hypertrophic cartilage. A significant age dependant decline in cell number and perlecan levels was evident in the hyaline and growth plate cartilages. The prominent pericellular distribution of perlecan observed indicates potential roles in cell-matrix communication in cartilage, consistent with growth factor signalling, cellular proliferation and tissue development.     

Cloning and embryonic expression of six wnt genes in the medaka (Oryzias latipes) with special reference to expression of wnt5a in the pectoral fin buds

WNTs are secreted signaling molecules which control cell differentiation and proliferation. They are known to play essential roles in various developmental processes. Wnt genes have been identified in a variety of animals, and it has been shown that their amino acid sequences are highly conserved throughout evolution. To investigate the role of wnt genes during fish development from the evolutionary viewpoint, six medaka wnt genes (wnt4, wnt5a, wnt6, wnt7b, wnt8b and wnt8-like) were isolated and their embryonic expression was examined. These wnt genes were expressed in various tissues during embryonic development, and most of their expression patterns were conserved or comparable to those of other vertebrates. Thus, these wnt genes may be useful as molecular markers to investigate development and organogenesis using the medaka. Focus was on wnt5a, which was expressed in the pectoral fin buds, because its expression pattern was particularly comparable to that in tetrapod limbs. Its detailed expression pattern was further examined during pectoral fin bud development. The conservation and diversification of Wnt5a expression through the evolutionary transition from fish fins to tetrapod limbs is discussed.     

Amphioxus tails: source and fate of larval fin rays and the metamorphic transition from an ectodermal to a predominantly mesodermal tail

It was previously discovered that tail fin rays of larval amphioxus are long ciliary rootlets in posterior epidermal cells. This work describes the heretofore unknown origin and fate of these organelles in the Florida amphioxus (Branchiostoma floridae). In late embryos, epidermal cells at the posterior end of the body increase in height, thus producing a tail fin. One ciliary rootlet in each cell elongates and also rotates through about 90°, soon becoming oriented parallel to the long axis of the cell and running continuously from the apical to the basal plasma membrane. During the subsequent growth of the larval tail, the rootlets and epidermal cells housing them reach lengths up to 120 μm. At metamorphosis, the rootlets become vacuolated and rapidly decrease in length along with the height of the tail epidermis. Contemporaneously, abundant extracellular dermal matrix accumulates in the sagittal plane of the body to produce a predominantly dermal tail fin. Throughout postmetamorphic life, the posterior epidermal cells, now without ciliary rootlets, thinly cover a largely dermal tail flange. Thus, the specialized morphology of the amphioxus tail fin is generated by two different cellular mechanisms, involving different cell populations (ectodermal and mesodermal), at different life‐history stages.     

犬齿窝与人类中面部骨骼的演化

犬齿窝是包括现代人类在内的许多人族成员面部骨骼的重要性状,但在分类学上的意义仍存在争议。有学者认为该性状是一个发生于基础面形的近祖性状,除了一些例外,在灭绝和现生的大猿及人属中都存在。另有学者认为,犬齿窝是仅存在于智人及其直系祖先的衍生性状,在发育上与颧齿槽突嵴有关。这种关系并非总是成立,在智人中存在着明显的差异：弧型颧齿槽突嵴和直斜型颧齿槽突嵴与犬齿窝有时共存,有时不共存。我们由此推测,犬齿窝的发生和形态与上颌窦的前部发育有关,颧齿槽突嵴的形态与鼻窦的侧面发育有关。在人类演化的过程中,犬齿窝经历了不同的变形,比如上颌沟(如南方古猿非洲种、傍人粗壮种)、上颌小窝(如傍人粗壮种)、上颌沟(如匠人)或犬齿窝缺如(如傍人埃塞俄比亚种、傍人鲍氏种、肯尼亚扁脸人、人属鲁道夫种)。犬齿窝消失的原因各类群并不相同,如中新世和早更新世人属以及中更新世人属(如人属海德堡种/人属罗得西亚种、人属尼安德特种)。人属罗得西亚种具有弱化的犬齿窝,不具备演化为智人的可能,因此被排除在智人的演化支之外。     

The phylogenetic distribution,anatomy and histology of the post‐cloacal bones and adnexa of geckos

Post‐cloacal bones of gekkotans may be present as a single (medial) pair, two pairs (medial and lateral), or may be lacking. We, herein, demonstrate that the presence of a single medial pair is the ancestral condition for the Gekkota, that the lateral pair is of sporadic occurrence within and between families, except for the Eublepharidae where it is universal, and that absence is also of sporadic occurrence except for the Sphaerodactylidae where it is the ancestral condition. Adult male Tokay geckos (Gekko gecko) possess only the medial pair of bones, and these exhibit a regionally‐specific expression of woven, fibrolamellar, and lamellar bone, and an enclosed medullary cavity. Females and small juvenile males lack bony elements but exhibit a conspicuous band of dense connective tissue located about the anterior and lateral margins of the cloacal sacs. As males grow and attain sexual maturity, the medial post‐cloacal bones condense in this band of dense connective tissue, and are thus shown to be dermal ossifications, similar to osteoderms but with muscular associations (although this is also known for crocodylians). Based upon ontogenetic data we set forth a scenario to explain the loss of the medial post‐cloacal bones in various lineages. Differential staining of the cloacal sacs failed to reveal any specialized glandular structures. Investigation of the post‐cloacal spurs shows them to be associated with cellular connective tissue of a type similar to that found in the vicinity of the medial post‐cloacal bones. This suggests that the lateral post‐cloacal bones may also be dermal bones, but histological evidence is needed to corroborate this. J. Morphol. 277:264–277, 2016. © 2015 Wiley Periodicals, Inc.     

Tenascin-C and the development of articular cartilage

In comparison to the vast literature on articular cartilage structure and function, relatively little is known about how articular cartilage forms during embryo-genesis and is endowed with unique phenotypic properties, most notably the ability to persist and function throughout postnatal life. In this minireview, we summarize recent studies from our laboratory suggesting that the extracellular matrix protein tenascin-C is involved in the genesis and function of articular chondrocytes. These and other data have led us to propose that tenascin-C may be part of in vivo mechanisms whereby articular chondrocytes develop at the epiphysis of long bone models, remain functional throughout postnatal life, and avoid the endochondral ossification process undertaken by the bulk of chondrocytes located in the metaphysis and diaphysis of skeletal models.