Senegalese sole bone tissue originated from chondral ossification is more sensitive than dermal bone to high vitamin A content in enriched Artemia

Several studies have evaluated the effects of dietary vitamin A (VA) on the incidence of skeletal deformities during early ontogeny of fish, but little is known about its effects on bones depending on their process of ossification (dermal or chondral). We examined the incidence of skeletal deformities along development (30 and 48 dph) by double staining technique, in dermal (haemal and caudal vertebral bodies) and chondral (neural and haemal spines, epural, parahypural and hypurals) bones in Senegal sole post metamorphosed larvae fed with different dietary VA levels (37 000, 44 666, 82 666 and 203 000 UI total VA kg^?1 DW) during Artemia feeding phase (6–37 dph, at 18°C). Results obtained in this study showed that dietary VA disrupted the skeletogenesis in Senegalese sole post metamorphosed larvae by increasing the incidence of skeletal deformities in the axial skeleton and caudal fin complex, which were dependent on both bone morphogenesis and ossification processes. Fish fed with the highest dietary VA content showed the highest incidence of skeletal deformities and its value increased along ontogeny. However, when we compared the incidence of deformities in skeletal structures considering their ossification process, most skeletal structures derived from chondral ossification showed a significant higher increase in deformity incidences in fish fed an excess of VA (44 666, 82 666 and 203 000 UI kg^?1 DW), however within chondral bones, hypurals deformity incidence only increased in sole larvae fed Artemia highest VA content. In contrast, this dietary dose‐response effect was only noted in dermal bones from fish fed the highest dose of VA (203 000 UI kg^?1 DW). In addition, the incidence of deformities in chondral bones increased even when the dietary imbalance of VA was corrected, whereas dermal bones were not affected at later ages. These results indicated that depending on the ossification process from which different skeletal structures are derived, bones might be differentially affected by high dietary VA content. Those directly originated from the connective tissue with a preliminary cartilage stage were more sensitive to dietary VA excess than those formed by intramembranous ossification.     

Synthesis and in vivo evaluation of [11C]tariquidar,a positron emission tomography radiotracer based on a third-generation P-glycoprotein inhibitor

The aim of this study was to develop a positron emission tomography (PET) tracer based on the dual P-glycoprotein (P-gp) breast cancer resistance protein (BCRP) inhibitor tariquidar (1) to study the interaction of 1 with P-gp and BCRP in the blood–brain barrier (BBB) in vivo. O-Desmethyl-1 was synthesized and reacted with [¹¹C]methyl triflate to afford [¹¹C]-1. Small-animal PET imaging of [¹¹C]-1 was performed in naïve rats, before and after administration of unlabeled 1 (15 mg/kg, n = 3) or the dual P-gp/BCRP inhibitor elacridar (5 mg/kg, n = 2), as well as in wild-type, Mdr1a/b^(?/?), Bcrp1^(?/?) and Mdr1a/b^(?/?)Bcrp1^(?/?) mice (n = 3). In vitro autoradiography was performed with [¹¹C]-1 using brain sections of all four mouse types, with and without co-incubation with unlabeled 1 or elacridar (1 μM). In PET experiments in rats, administration of unlabeled 1 or elacridar increased brain activity uptake by a factor of 3–4, whereas blood activity levels remained unchanged. In Mdr1a/b^(?/?), Bcrp1^(?/?) and Mdr1a/b^(?/?)Bcrp1^(?/?) mice, brain-to-blood ratios of activity at 25 min after tracer injection were 3.4, 1.8 and 14.5 times higher, respectively, as compared to wild-type animals. Autoradiography showed approximately 50% less [¹¹C]-1 binding in transporter knockout mice compared to wild-type mice and significant displacement by unlabeled elacridar in wild-type and Mdr1a/b^(?/?) mouse brains. Our data suggest that [¹¹C]-1 interacts specifically with P-gp and BCRP in the BBB. However, further investigations are needed to assess if [¹¹C]-1 behaves in vivo as a transported or a non-transported inhibitor.     

Morphological novelty and modest developmental truncation in Barboides,Africa's smallest vertebrates (Teleostei: Cyprinidae)

Miniaturization, the evolution of extremely small adult body size, is widespread amongst animals and commonly associated with novel ecological, physiological, and morphological attributes. The phenotypes of miniaturized taxa are noteworthy because they combine reductions and structural simplifications with novel traits not developed in their larger relatives. Previous research on miniature cyprinid fishes (focused predominantly on South and South East Asian taxa of a single subfamily) has identified two distinct classes of miniature taxa: proportioned dwarves and developmentally truncated miniatures. Miniaturization has also occurred independently in the subfamily Cyprininae, particularly in African lineages. We investigate the skeletal anatomy of Barboides , a genus of miniature African cyprinids that includes Africa's smallest known species of vertebrates, to assess whether miniaturization has resulted in similar organismal outcomes in different lineages of the Cyprinidae. The skeleton of Barboides is characterized by the complete absence of a number of dermal and endochondral ossifications, and marked reduction in size and/or complexity of other skeletal elements, particularly those of the dermatocranium. Absent skeletal elements in Barboides include those which develop relatively late in the ossification sequence of the non‐miniature African relative ‘Barbus ’ holotaenia suggesting that their absence in Barboides can be explained by a simple scenario of developmental truncation. In contrast to this theme of loss and reduction, the os suspensorium of Barboides is enlarged and the outer arm distally trifid and associated with a novel bulbous muscle in males. An evaluation of the skeleton of Barboides provides further evidence for a link between developmental truncation and evolutionary morphological novelty in Cyprinidae. In the spectrum of miniature cyprinids ranging from proportioned dwarves with few bones missing to highly progenetic taxa with dozens of missing bones, the two species of Barboides range roughly in the middle showing that the extremes are connected by intermediate levels of truncatedness.     

First skeleton of the notoungulate mammal Notostylops murinus and palaeobiology of Eocene Notostylopidae

Here, we describe the first skeletal remains of Notostylops recovered from middle Eocene levels of the Sarmiento Formation, Patagonia, Argentina. The remains include two teeth of Notostylops murinus, the axis, vertebral bodies, a rib, a left humerus, both radii, two metapodials, two phalanges, the pelvis, a right femur, a right calcaneus and several broken bones. Radial bones are not fused to ulnas, and are shorter than the humerus, very generalized, with an oval head, a marked neck and a radial tubercle. The humerus and the femur show pronounced insertion structures. Our analysis suggests that the appendicular skeleton of Notostylops is too generalized and shares several features with that of terrestrial rodents as Sciuridae. Unlike the appendicular skeletons of cursorial or saltatorial mammals, which restrict mobility, the skeleton of Notostylops indicates the ability to make a variety of different movements, as would be expected for terrestrial, fossorial or arboreal mammals. This skeleton gives new information about the locomotor behaviour of notoungulates, particularly in their basal forms. The results will also allow the identification of isolated notoungulate bones and raise questions about the previous taxonomic assignment of postcrania to Pleurostylodon.     

The alleged early Miocene Auk Petralca austriaca is a Loon (Aves,Gaviiformes): restudy of a controversial fossil bird

We restudy the holotype specimen of the alleged fossil auk Petralca austriaca from early Miocene marine deposits of the Austrian locality Pucking, which was considered the earliest European representative of Alcidae (auks). The specimen is a partial skeleton consisting mainly of wing bones on two slabs. A recent re-preparation yielded new data on the skeletal morphology of Petralca, which allow more detailed comparisons with auks and loons. Our study shows that the taxon is clearly distinguished from auks in various skeletal features and can be confidently identified as a loon (Gaviiformes). Petralca resembles Colymboides in overall morphology of the wing bones, and clearly is a stem group representative of Gaviiformes. However, some features indicate that it is more closely related to the crown group taxon Gavia than to the stem group taxa Colymboides and Colymbiculus. Unusually thick bone walls of the limb bones indicate well-developed diving capabilities for Petralca austriaca.     

Developmental osteology of Sciaenops ocellatus and Cynoscion nebulosus (Teleostei: Sciaenidae), economically important sciaenids from the western Atlantic

The adult skeleton in members of the economically important Sciaenidae is well documented, but information on earlier developmental stages is sparse and often focused on a particular character complex. To generate information on skeletal development in sciaenid fishes, we investigated the ontogeny of the entire skeleton in the western Atlantic Sciaenops ocellatus (Red drum) and Cynoscion nebulosus (Spotted seatrout), which are the focus of successful captive rearing programmes within the southern United States. Development of the skeleton (excluding the basisphenoid and sclerotic bones) is complete in S. ocellatus and C. nebulosus at 14.4 mm SL and 13.5 mm SL, respectively. The basisphenoid did not appear until later in development (21.9 mm SL in S. ocellatus and 19.5 mm SL in C. nebulosus), while the sclerotic bones are not present in the material examined. No major differences are identified between the ossification sequences compiled for each species. Cynoscion nebulosus exhibited variation in the presence/absence of two elements, supraneural 1 and the coronomeckelian. Lastly, we compile and compare available information on skeletal development across members of the Sciaenidae and compare the sequence of ossification compiled for S. ocellatus to that available for Danio rerio and Salminus brasiliensis (entire skeleton), and Chanos chanos (cranium only).     

Proportions and variability in the Indian lorises: Bone weight observations onLoris tardigradus lydekkerianus andNycticebus coucang

Thirty uniformly prepared, disarticulated complete skeletons of adultLoris tardigradus lydekkerianus andNycticebus coucang have been weighed for preliminary study of the weight relations between different major skeletal parts. In these forms, combined relative weight of the precaudal vertebrae, ribs and sternum surpasses the averages for the rest of the skeletal parts. The weight of the skull equals one-fifth of the total skeletal weight inLoris andNycticebus. The weight of the skeletons of the limbs (including the shoulder girdle and the hip bones) contributes less than half of the total skeletal weight in lorises. The bones of the upper limbs are lighter than those of the lower limbs in the 2 series examined.Loris andNycticebus are distinguished by having the proportionately heaviest axial skeleton and skull and the lowest relative weights of the bones of the 4 limbs. The combined relative skeletal weight of the upper and lower limbs ranges between 45.34 and 50.01 inLoris andNycticebus. The relative weight of the skeletons of the hands and feet are almost similar to the corresponding weights in the 2 series.As far as the weights of the different skeletal parts are concerned, there is no relative asymmetry in the 2 series ofLoris andNycticebus examined.     

Suppression of Autophagy in Osteocytes Mimics Skeletal Aging

Bone mass declines with age but the mechanisms responsible remain unclear. Here we demonstrate that deletion of a conditional allele for Atg7, a gene essential for autophagy, from osteocytes caused low bone mass in 6-month-old male and female mice. Cancellous bone volume and cortical thickness were decreased, and cortical porosity increased, in conditional knock-out mice compared with control littermates. These changes were associated with low osteoclast number, osteoblast number, bone formation rate, and wall width in the cancellous bone of conditional knock-out mice. In addition, oxidative stress was higher in the bones of conditional knock-out mice as measured by reactive oxygen species levels in the bone marrow and by p66^shc phosphorylation in L6 vertebra. Each of these changes has been previously demonstrated in the bones of old versus young adult mice. Thus, these results demonstrate that suppression of autophagy in osteocytes mimics, in many aspects, the impact of aging on the skeleton and suggest that a decline in autophagy with age may contribute to the low bone mass associated with aging.     

Critical roles of the TGF-β type I receptor ALK5 in perichondrial formation and function, cartilage integrity, and osteoblast differentiation during growth plate development

TGF-β has been implicated in the proliferation and differentiation of chondrocytes and osteoblasts. However, the in vivo function of TGF-β in skeletal development is unclear. In this study, we investigated the role of TGF-β signaling in growth plate development by creating mice with a conditional knockout of the TGF-β type I receptor ALK5 (ALK5^CKO) in skeletal progenitor cells using Dermo1-Cre mice. ALK5^CKO mice had short and wide long bones, reduced bone collars, and trabecular bones. In ALK5^CKO growth plates, chondrocytes proliferated and differentiated, but ectopic cartilaginous tissues protruded into the perichondrium. In normal growth plates, ALK5 protein was strongly expressed in perichondrial progenitor cells for osteoblasts, and in a thin chondrocyte layer located adjacent to the perichondrium in the peripheral cartilage. ALK5^CKO growth plates had an abnormally thin perichondrial cell layer and reduced proliferation and differentiation of osteoblasts. These defects in the perichondrium likely caused the short bones and ectopic cartilaginous protrusions. Using tamoxifen-inducible Cre-ER™-mediated ALK5-deficient primary calvarial cell cultures, we found that TGF-β signaling promoted osteoprogenitor proliferation, early differentiation, and commitment to the osteoblastic lineage through the selective MAPKs and Smad2/3 pathways. These results demonstrate the important roles of TGF-β signaling in perichondrium formation and differentiation, as well as in growth plate integrity during skeletal development.     

Heterochrony during skeletal development of Pseudis platensis (anura,hylidae) and the early offset of skeleton development and growth

The aquatic frog Pseudis platensis has a giant tadpole, long developmental time, and dissociated metamorphic events that include later offset of larval somatic morphologies. Moreover, when the tadpole metamorphoses, the young frog is nearly the size of an adult, suggesting that this species has low rates of postmetamorphic growth. Herein, we study the development of the skeleton during larval development up to the end of metamorphosis, which is denoted by the complete lost of the tail in P. platensis. Our study revealed heterochronic differences in skeletal development compared with that of most anurans; these involve the complete differentiation of skull bones and the extensive ossification of the postcranial skeleton before completion of metamorphosis. The skull of metamorphosing P. platensis has an ossified sphenethmoid and a fully formed plectral apparatus, thus differing with regard to the pattern observed in most anurans in which both developmental events take place during the postmetamorphic life. Despite the fact that the iliosacral articulation and the urostyle are present at the end of metamorphosis as in most anurans, ossification/calcification of carpus, tarsus, and limb epihyses during metamorphosis of P. platensis suggests that the postcranial skeleton lacks postmetamorphic growth. This study also includes a discussion of the pattern of development of the plectral apparatus, which allows us to propose a new hypothesis regarding pars externa plectri homology. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

A comparative view on mechanisms and functions of skeletal remodelling in teleost fish, with special emphasis on osteoclasts and their function

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). The cellular mechanisms of differentiation and activation of osteoclasts and the functions of teleost skeletal remodelling are described. Several characteristics, related to skeletal remodelling, distinguish teleosts from mammals. These characteristics include (a) the absence of osteocytes in most species; (b) the absence of haematopoietic bone marrow tissue; (c) the abundance of small mononucleated osteoclasts performing non‐lacunar (smooth) bone resorption, in addition to or instead of multinucleated osteoclasts; and (d) a phosphorus‐ rather than calcium‐driven mineral homeostasis (mainly affecting the postcranial dermal skeleton). Furthermore, (e) skeletal resorption is often absent from particular sites, due to sparse or lacking endochondral ossification. Based on the mode of skeletal remodelling in early ontogeny of all teleosts and in later stages of development of teleosts with acellular bone we suggest a link between acellular bone and the predominance of mononucleated osteoclasts, on the one hand, and cellular bone and multinucleated osteoclasts on the other. The evolutionary origin of skeletal remodelling is discussed and whether mononucleated osteoclasts represent an ancestral type of resorbing cells. Revealing the differentiation and activation of teleost skeletal resorbing cells, in the absence of several factors that trigger mammalian osteoclast differentiation, is a current challenge. Understanding which characters of teleost bone remodelling are derived and which characters are conserved should enhance our understanding of the process in fish and may provide insights into alternative pathways of bone remodelling in mammals.     

Appendicular skeletal morphology in minute salamanders,genus Thorius (amphibia: Plethodontidae): Growth regulation,adult size determination,and natural variation

Patterns of growth and variation of the appendicular skeleton were examined in Thorius, a speciose genus of minute terrestrial plethodontid salamanders from southern Mexico. Observations were based primarily on ontogenetic series of each of five species that collectively span the range of adult body size in the genus; samples of adults of each of seven additional species provided supplemental estimates of the full range of variation of limb skeletal morphology. Limbs are generally reduced, i.e., pedomorphic, in both overall size and development, and they are characterized by a pattern of extreme variation in the composition of the limb skeleton, especially mesopodial elements, both within and between species. Fifteen different combinations of fused carpal or tarsal elements are variably present in the genus, producing at least 18 different overall carpal or tarsal arrangements, many of which occur in no other plethodontid genus. As many as four carpal or tarsal arrangements were observed in single population samples of each of several; five tarsal arrangements were observed in one population of T. minutissimus. Left-right asymmetry of mesopodial arrangement in a given specimen is also common. In contrast, several unique, nonpedomorphic features of the limb skeleton, including ossification of the typically cartilaginous adult mesopodial elements and ontogenetic increase in the degree of ossification of long bones, are characteristic of all species and distinguish Thorius from most related genera. They form part of a mechanism of determinate skeletal growth that restricts skeletal growth after sexual maturity. Interspecific differences in the timing of the processes of appendicular skeletal maturation relative to body size are well correlated with interspecific differences in mean adult size and size at sexual maturity, suggesting that shifts in the timing of skeletal maturation provide a mechanism of achieving adult size differentiation among species. Processes of skeletal maturation that confer determinate skeletal growth in Thorius are analogous to those typical of most amniotes – both groups exhibit ontogenetic reduction and eventual disappearance of the complex of stratified layers of proliferating and maturing cartilage in long bone epiphyses – but, unlike most amniotes, Thorius lacks secondary ossification centers. Thus, the presence of secondary ossification centers cannot be used as a criterion for establishing determinate skeletal growth in all vertebrates.     

Angiotensin II induced catabolic effect and muscle atrophy are redox dependent

Angiotensin II (Ang II) causes skeletal muscle wasting via an increase in muscle catabolism. To determine whether the wasting effects of Ang II were related to its ability to increase NADPH oxidase-derived reactive oxygen species (ROS) we infused wild-type C57BL/6J or p47^phox−/− mice with vehicle or Ang II for 7 days. Superoxide production was increased 2.4-fold in the skeletal muscle of Ang II infused mice, and this increase was prevented in p47^phox−/− mice. Apocynin treatment prevented Ang II-induced superoxide production in skeletal muscle, consistent with Ang II increasing NADPH oxidase derived ROS. Ang II induced loss of body and skeletal muscle weight in C57BL/6J mice, whereas the reduction was significantly attenuated in p47^phox−/− animals. The reduction of skeletal muscle weight caused by Ang II was associated with an increase of proteasome activity, and this increase was completely prevented in the skeletal muscle of p47^phox−/− mice. In conclusion, Ang II-induced skeletal muscle wasting is in part dependent on NADPH oxidase derived ROS.     

Can mechanical forces be responsible for novel bone development and evolution in fishes?

Mechanical forces influence the induction, growth and maintenance of the vertebrate skeleton. Using the zebrafish, Danio rerio, we explore the hypothesis that mechanical forces can ultimately lead to the generation of skeletal evolutionary novelties by modifications of the mechano‐responsive molecular pathways. Locomotion and feeding in zebrafish larvae begin early in ontogeny and it is likely that forces incurred during these behaviours affect subsequent skeletal development. We provide two case studies in which our hypothesis is being tested: the kinethmoid and intermuscular bones. The kinethmoid is a synapomorphy for the order Cypriniformes and is intricately linked to the bones of the protrusible upper jaw. It undergoes chondrogenesis within a ligament well after muscular forces are present within the head. Subsequent ossification of the kinethmoid occurs at sites of ligamentous attachment, leading us to believe that mechanical forces are involved. Unlike the kinethmoid, which has evolved only once, intermuscular bones have evolved several times during teleostean evolution. Intermuscular bones are embedded within the myosepta, the collagenous sheets between axial muscles. The effect of mechanical forces on the development of these intermuscular bones is experimentally tested by increasing the viscosity of the water in which larval zebrafish are raised. Since locomotion in high viscosity requires greater muscular forces, we can directly test the influence of mechanical forces on the development of intermuscular bones. Using developmental techniques paired with outgroup comparison for the kinethmoid, and direct experimentation for intermuscular bones, our case studies provide complementary insights into the effects of mechanical forces on the evolution of skeletal novelties in fishes.     

The influence of muscle type and dystrophin deficiency on murine expression profiles

The phenotypic differences among Duchenne muscular dystrophy patients, mdx mice, and mdx^5cv mice suggest that despite the common etiology of dystrophin deficiency, secondary mechanisms have a substantial influence on phenotypic severity. The differential response of various skeletal muscles to dystrophin deficiency supports this hypothesis. To explore these differences, gene expression profiles were generated from duplicate RNA targets extracted from six different skeletal muscles (diaphragm, soleus, gastrocnemius, quadriceps, tibialis anterior, and extensor digitorum longus) from wild-type, mdx, and mdx^5cv mice, resulting in 36 data sets for 18 muscle samples. The data sets were compared in three different ways: (1) among wild-type samples only, (2) among all 36 data sets, and (3) between strains for each muscle type. The molecular profiles of soleus and diaphragm separate significantly from the other four muscle types and from each other. Fiber-type proportions can explain some of these differences. These variations in wild-type gene expression profiles may also reflect biomechanical differences known to exist among skeletal muscles. Further exploration of the genes that most distinguish these muscles may help explain the origins of the biomechanical differences and the reasons why some muscles are more resistant than others to dystrophin deficiency. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Judith N. Haslett, Peter B. Kang These authors contributed equally to this work.     

AMPK-dependent and -independent coordination of mitochondrial function and muscle fiber type by FNIP1

Mitochondria are essential for maintaining skeletal muscle metabolic homeostasis during adaptive response to a myriad of physiologic or pathophysiological stresses. The mechanisms by which mitochondrial function and contractile fiber type are concordantly regulated to ensure muscle function remain poorly understood. Evidence is emerging that the Folliculin interacting protein 1 (Fnip1) is involved in skeletal muscle fiber type specification, function, and disease. In this study, Fnip1 was specifically expressed in skeletal muscle in Fnip1-transgenic (Fnip1^Tg) mice. Fnip1^Tg mice were crossed with Fnip1-knockout (Fnip1^KO) mice to generate Fnip1^TgKO mice expressing Fnip1 only in skeletal muscle but not in other tissues. Our results indicate that, in addition to the known role in type I fiber program, FNIP1 exerts control upon muscle mitochondrial oxidative program through AMPK signaling. Indeed, basal levels of FNIP1 are sufficient to inhibit AMPK but not mTORC1 activity in skeletal muscle cells. Gain-of-function and loss-of-function strategies in mice, together with assessment of primary muscle cells, demonstrated that skeletal muscle mitochondrial program is suppressed via the inhibitory actions of FNIP1 on AMPK. Surprisingly, the FNIP1 actions on type I fiber program is independent of AMPK and its downstream PGC-1α. These studies provide a vital framework for understanding the intrinsic role of FNIP1 as a crucial factor in the concerted regulation of mitochondrial function and muscle fiber type that determine muscle fitness.     

Osteology of the Southern Ocean daggertooth (<Emphasis Type="Italic">Anotopterus vorax</Emphasis>) and status of the <Emphasis Type="Italic">Anotopterus</Emphasis> genus in the Aulopiformes order

The skeletal structure of the Southern Ocean daggertooth (Anotopterus vorax) has been studied for the first time. It generally corresponds to the A. pharao skeleton, as it has been previously described. The discrepancies pertain to the authors’ other various data on both the details of the structures of branchial arches, maxillae, cranium, and axial skeleton and the traits of the Anotopterus genus, which have not been described earlier. They include the availability of a considerable number of bony ribs (epineurals and epicentrals) attached to the neurocranium bones (epiotic, intercalar, and exooccipital) and some elongated and discontinuous lateral keel bones located on both sides of the central keel bone. Cladistic analysis has been performed with the finding that the A. vorax is united with the A. pharao and the Polymerichthys fossil genus into one clade, which takes an intermediate status between the representatives of the Alepisauridae and Paralepididae families. The assumption has been made about the necessity for the restitution of the family status of Anotopteridae.