Four‐dimensional analysis of early pelvic girdle development in Rana temporaria

A key event in vertebrate evolution is the linkage of the appendicular to the axial skeleton. The present study investigates the developmental dynamics of pelvic girdle morphogenesis in Rana temporaria up until metamorphosis, with respect to its functional and spatio‐temporal organization. The main questions to be addressed are: initial location and the number of elements contributing to pelvic girdle formation, mechanism of bridging between the pelvic anlage and the sacrum and arthrogenesis. Serial histological sections of specimens from Gosner Stages 30 to 41 were bright‐field microscopically examined and 3D‐reconstructed. 3D‐models were merged to 4D‐animations illustrating the complex developmental dynamics through time. The results reveal the initial formation of a single mesenchymal condensation located close to the appendicular skeleton, but far from the axial skeleton. In addition, our analysis detects a thin connective tissue strand in R. temporaria guiding the elongation of the ilium towards the sacrum. The 4D‐visualization allows novel insight into the ilio‐sacral bridging process and the reorientation of the pelvis. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

Agenesis of the scapula in Emx2 homozygous mutants

The shoulder and pelvic girdles represent the proximal bones of the appendicular skeleton that connect the anterior and posterior limbs to the body trunk. Although the limb is a well-known model in developmental biology, the genetic mechanisms controlling the development of the more proximal elements of the appendicular skeleton are still unknown. The knock-out of Pax1 has shown that this gene is involved in patterning the acromion, while the expression pattern candidates Hoxc6 as a gene involved in scapula development. Surprisingly, we have found that scapula and ilium do not develop in Emx2 knock-out mice. In the homozygous mutants, developmental abnormalities of the brain cortex, the most anterior structure of the primary axis of the body, are associated with important defects of the girdles, the more proximal elements of the secondary axis. These abnormalities suggest that the molecular mechanisms patterning the more proximal elements of the limb axis are different from those patterning the rest of appendicular skeleton. While Hox genes specify the different segments of the more distal part of the appendicular skeleton forming the limb, Emx2 is concerned with the more proximal elements constituting the girdles.     

3D reconstructions of quail–chick chimeras provide a new fate map of the avian scapula

Limbed vertebrates have functionally integrated postcranial axial and appendicular systems derived from two distinct populations of embryonic mesoderm. The axial skeletal elements arise from the paraxial somites, the appendicular skeleton and sternum arise from the somatic lateral plate mesoderm, and all of the muscles for both systems arise from the somites. Recent studies in amniotes demonstrate that the scapula has a mixed mesodermal origin. Here we determine the relative contribution of somitic and lateral plate mesoderm to the avian scapula from quail-chick chimeras. We generate 3D reconstructions of the grafted tissue in the host revealing a very different distribution of somitic cells in the scapula than previously reported. This novel 3D visualization of the cryptic border between somitic and lateral plate populations reveals the dynamics of musculoskeletal morphogenesis and demonstrates the importance of 3D visualization of chimera data. Reconstructions of chimeras make clear three significant contrasts with existing models of scapular development. First, the majority of the avian scapula is lateral plate derived and the somitic contribution to the scapular blade is significantly smaller than in previous models. Second, the segmentation of the somitic component of the blade is partially lost; and third, there are striking differences in growth rates between different tissues derived from the same somites that contribute to the structures of the cervical thoracic transition, including the scapula. These data call for the reassessment of theories on the development, homology, and evolution of the vertebrate scapula.     

Development and evolution of the muscles of the pelvic fin

Locomotor strategies in terrestrial tetrapods have evolved from the utilisation of sinusoidal contractions of axial musculature, evident in ancestral fish species, to the reliance on powerful and complex limb muscles to provide propulsive force. Within tetrapods, a hindlimb-dominant locomotor strategy predominates, and its evolution is considered critical for the evident success of the tetrapod transition onto land. Here, we determine the developmental mechanisms of pelvic fin muscle formation in living fish species at critical points within the vertebrate phylogeny and reveal a stepwise modification from a primitive to a more derived mode of pelvic fin muscle formation. A distinct process generates pelvic fin muscle in bony fishes that incorporates both primitive and derived characteristics of vertebrate appendicular muscle formation. We propose that the adoption of the fully derived mode of hindlimb muscle formation from this bimodal character state is an evolutionary innovation that was critical to the success of the tetrapod transition.     

Vertebrate Axial and Appendicular Patterning: The Early Development of Paired Appendages

Determination of paired fin or limb number, identity and positionare key issues in vertebrate development and evolution. Phylogeniesincluding fossil data show that paired appendages are uniqueto jawed vertebrates and their immediate ancestry; that suchfins evolved first as a single pair in an anterior location;that appendicular endoskeletons are primitively AP asymmetric;and that pectoral and pelvic fins primitively differ. It isconjectured that Hox gene expression patterns along the lateralplate mesoderm establish boundaries that contribute to localisationof AP levels at which signals initiate outgrowth from the bodywall. Such regionalisation may be regulated independently ofthat in the paraxial mesoderm and axial skeleton. When combinedwith current hypotheses of Hox gene phylogenetic and functionaldiversity, these data suggest a new model of fin/limb developmentalevolution. This coordinates body wall outgrowth regions withprimitive boundaries established in the gut, and the fundamentalnon-equivalence of pectoral and pelvic structures.     

Dlx5 regulates chondrocyte differentiation at multiple stages

Endochondral ossification, in which cartilaginous templates are progressively replaced by marrow and bone, represents the dominant mode of development of the axial and appendicular skeleton of vertebrates. Chondrocyte differentiation within the cartilaginous core of these skeletal elements is tightly regulated, both spatially and temporally. Here, we describe the expression of Dlx5 in the cartilaginous core of limb skeletal elements in chicken and mouse embryos. We find that Dlx5 is one of the earliest genes expressed in condensing limb mesenchyme that will give rise to the limb skeleton. Later, when proliferating and differentiating chondrocytes are found in spatially distinct regions of the cartilaginous model, Dlx5 is expressed in the zone of hypertrophy and in proliferating chondrocytes that are poised to differentiate. Consistent with this pattern of expression, we show that forced expression of Dlx5 potentiates early and late chondrocyte differentiation and inhibits proliferation in cultured cells. Examination of the limbs of mutant Dlx5 mouse embryos revealed that they displayed a delay in chondrocyte maturation compared with wild type littermates. Taken together, our data reveal a positive role for Dlx5 during multiple stages of chondrocyte differentiation and, along with previous studies of Dlx5 and osteogenesis, identify Dlx5 as a general regulator of differentiation in the mouse skeleton.     

Chondrogenesis in mouse limb bud in vitro. I. Effect of the ectoderm

The role of the ectoderm in the chondrogenesis of mouse limb bud mesoderm was investigated in vitro at several developmental stages by analysis of the evolution of DNA content, the accumulation of sulfated glycosaminoglycans and histochemical procedures. Young limb buds or the undifferentiated apex of older buds (stages 17 and 19 of Theiler's table) from which the ectoderm had been removed with trypsin treatment initiated a large chondrogenesis but not morphogenesis. When the ectoderm was present, these limb buds showed a polarized proximal to distal outgrowth and differentiated skeletal primordia. Mesodermal cells of stage 20 limb bud apex were able to differentiate autopodial skeletons with or without the presence of the ectoderm: cartilaginous areas of the limb skeleton seem determined at this developmental stage. These results, which show the importance of the ectoderm in limb bud morphogenesis, are compared with results obtained using other methods with mouse or bird buds.     

On the serial homology of the pectoral and pelvic girdles of tetrapods

While fore‐ and hindlimbs are commonly assumed to be serially homologous, the serial homology of the pectoral and pelvic girdles is more ambiguous. We investigate the degree to which a common history, developmental program, and gene network are shared between the girdles relative to the rest of the appendicular skeleton. Paleontological data indicate that pectoral appendages arose millions of years before pelvic appendages. Recent embryological and genetic data suggest that the anatomical similarity between the fore‐ and hindlimbs arose through the sequential, derived deployment of similar developmental programs and gene networks, and is therefore not due to ancestral serial homology. Much less developmental work has however been published about the girdles. Here, we provide the first detailed review of the developmental programs and gene networks of the pectoral and pelvic girdles. Our review shows that, with respect to these programs and networks, there are fewer similarities between pelvic and pectoral girdles than there are between the limbs. The available data therefore support recent hypotheses that the anatomical similarities between the fore‐ and hindlimbs arose during the fin‐to‐limb transition through the derived co‐option of similar developmental mechanisms, while the phylogenetically older pectoral and pelvic girdles have remained more distinct since their evolutionary origin.     

Limb development in the gekkonid lizard gonatodes albogularis: A reconsideration of homology in the lizard carpus and tarsus

Despite the attention squamate lizards have received in the study of digit and limb loss, little is known about limb morphogenesis in pentadactyl lizards. Recent developmental studies have provided a basis for understanding lizard autopodial element homology based on developmental and comparative anatomy. In addition, the composition and identity of some carpal and tarsal elements of lizard limbs, and reptiles in general, have been the theme of discussions about their homology compared to non‐squamate Lepidosauromorpha and basal Amniota. The study of additional embryonic material from different lizard families may improve our understanding of squamate limb evolution. Here, we analyze limb morphogenesis in the gekkonid lizard Gonatodes albogularis describing patterns of chondrogenesis and ossification from early stages of embryonic development to hatchlings. Our results are in general agreement with previous developmental studies, but we also show that limb development in squamates probably involves more chondrogenic elements for carpal and tarsal morphogenesis, as previously recognized on the grounds of comparative anatomy. We provide evidence for the transitory presence of distal carpale 1 and intermedium in the carpus and tibiale, intermedium, distal centralia, and distal tarsale 2 in the tarsus. Hence, we demonstrate that some elements that were believed to be lost in squamate evolution are conserved as transitory elements during limb development. However, these elements do not represent just phylogenetic burden but may be important for the morphogenesis of the lizard autopodium. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Developmental genetic basis for the evolution of pelvic fin loss in the pufferfish Takifugu rubripes

Paired appendages were a key developmental innovation among vertebrates and they eventually evolved into limbs. Ancient developmental control systems for paired fins and limbs are broadly conserved among gnathostome vertebrates. Some lineages including whales, some salamanders, snakes, and many ray-fin fish, independently lost the pectoral, pelvic, or both appendages over evolutionary time. When different taxa independently evolve similar developmental morphologies, do they use the same molecular genetic mechanisms? To determine the developmental genetic basis for the evolution of pelvis loss in the pufferfish Takifugu rubripes (fugu), we isolated fugu orthologs of genes thought to be essential for limb development in tetrapods, including limb positioning (Hoxc6, Hoxd9), limb bud initiation (Pitx1, Tbx4, Tbx5), and limb bud outgrowth (Shh, Fgf10), and studied their expression patterns during fugu development. Results showed that bud outgrowth and initiation fail to occur in fugu, and that pelvis loss is associated with altered expression of Hoxd9a, which we show to be a marker for pelvic fin position in three-spine stickleback Gasterosteus aculeatus. These results rule out changes in appendage outgrowth and initiation genes as the earliest developmental defect in pufferfish pelvic fin loss and suggest that altered Hoxd9a expression in the lateral mesoderm may account for pelvis loss in fugu. This mechanism appears to be different from the mechanism for pelvic loss in stickleback, showing that different taxa can evolve similar phenotypes by different mechanisms.     

Vertebrate aristaless-related genes

Aristaless-related genes, a subset of the Paired-related homeobox genes, have in the past few years emerged as a group of regulators of essential events during vertebrate embryogenesis. One group of aristaless-related genes has been linked to the morphogenesis of the craniofacial and appendicular skeleton by their expression patterns and by the phenotypes of natural and artificial mouse mutants. Expression and function in the nervous system characterise a second group, and a third group, the Pitx genes, have been shown to have many different roles, including functions in the pituitary, left-right determination and limb development.     

Musculoskeletal morphology of the pelvis and pelvic fins in the lungfish Protopterus annectens

The West African lungfish (Protopterus annectens) performs benthic, pelvic fin‐driven locomotion with gaits common to tetrapods, the sister group of the lungfishes. Features of P. annectens movement are similar to those of modern tetrapods and include use of the distal region of the pelvic fin as a “foot,” use of the fin to lift the body above the substrate and rotation of the fin around the joint with the pelvis. In contrast to these similarities in movement, the pelvic fins of P. annectens are long, slender structures that are superficially very different from tetrapod limbs. Here, we describe the musculoskeletal anatomy of the pelvis and pelvic fins of P. annectens with dissection, magnetic resonance imaging, histology and 3D‐reconstruction methods. We found that the pelvis is embedded in the hypaxial muscle by a median rostral and two dorsolateral skeletal projections. The protractor and retractor muscles at the base of the pelvic fin are fan‐shaped muscles that cup the femur. The skeletal elements of the fin are serially repeating cartilage cylinders. Along the length of the fin, repeating truncated cones of muscles, the musculus circumradialis pelvici, are separated by connective tissue sheets that connect the skeletal elements to the skin. The simplicity of the protractor and retractor muscles at the base of the fin is surprising, given the complex rotational movement those muscles generate. In contrast, the series of many repeating segmental muscles along the length of the fin is consistent with the dexterity of bending of the distal limb. P. annectens can provide a window into soft‐tissue anatomy and sarcopterygian fish fin function that complements the fossil data from related taxa. This work, combined with previous behavioral examination of P. annectens, illustrates that fin morphologies that do not appear to be capable of walking can accomplish that function, and may inform the interpretation of fossil anatomical evidence. J. Morphol. 275:431–441, 2014. © 2013 Wiley Periodicals, Inc.     

Noggin antagonism of BMP4 signaling controls development of the axial skeleton in the mouse

The interaction between bone morphogenetic proteins (BMPs) and their antagonist, Noggin, is critical for normal development. Noggin null mice die at birth with a severely malformed skeleton that is postulated to reflect the activity of unopposed BMP signaling. However, the widespread expression and redundancy of different BMPs have made it difficult to identify a specific role for individual BMPs during mammalian skeletal morphogenesis. Here, we report the effects of modifying Bmp4 dosage on the skeletal development of Noggin mutant mice. The reduction of Bmp4 dosage results in an extensive rescue of the axial skeleton of Noggin mutant embryos. In contrast, the appendicular skeletal phenotype of Noggin mutants was unchanged. Analysis of molecular markers of somite formation and somite patterning suggests that the loss of Noggin results in the formation of small mispatterned somites. Mis-specification and growth retardation rather than cell death most likely account for the subsequent reduction or loss of axial skeletal structures. The severe Noggin phenotype correlates with Bmp4-dependent ectopic expression of Bmp4 in the paraxial mesoderm consistent with Noggin antagonizing an auto-inductive feed-forward mechanism. Thus, specific interactions between Bmp4 and Noggin in the early embryo are critical for establishment and patterning of the somite and subsequent axial skeletal morphogenesis.     

Retracted: Evolution and development of the homocercal caudal fin in teleosts

The vertebrate caudal skeleton is one of the most innovative structures in vertebrate evolution and has been regarded as an excellent model for functional morphology, a discipline that relates a structure to its function. Teleosts have an internally‐asymmetrical caudal fin, called the homocercal caudal fin, formed by the upward bending of the caudal‐most portion of the body axis, the ural region. This homocercal type of the caudal fin ensures powerful and complex locomotion and is thought to be one of the most important evolutionary innovations for teleosts during adaptive radiation in an aquatic environment. In this review, we summarize the past and present research of fish caudal skeletons, especially focusing on the homocercal caudal fin seen in teleosts. A series of studies with a medaka spontaneous mutant have provided important insight into the evolution and development of the homocercal caudal skeleton. By comparing developmental processes in various vertebrates, we propose a scenario for acquisition and morphogenesis of the homocercal caudal skeleton during vertebrate evolution.     

Swim-training changes the spatio-temporal dynamics of skeletogenesis in zebrafish larvae (Danio rerio)

Fish larvae experience many environmental challenges during development such as variation in water velocity, food availability and predation. The rapid development of structures involved in feeding, respiration and swimming increases the chance of survival. It has been hypothesized that mechanical loading induced by muscle forces plays a role in prioritizing the development of these structures. Mechanical loading by muscle forces has been shown to affect larval and embryonic bone development in vertebrates, but these investigations were limited to the appendicular skeleton. To explore the role of mechanical load during chondrogenesis and osteogenesis of the cranial, axial and appendicular skeleton, we subjected zebrafish larvae to swim-training, which increases physical exercise levels and presumably also mechanical loads, from 5 until 14 days post fertilization. Here we show that an increased swimming activity accelerated growth, chondrogenesis and osteogenesis during larval development in zebrafish. Interestingly, swim-training accelerated both perichondral and intramembranous ossification. Furthermore, swim-training prioritized the formation of cartilage and bone structures in the head and tail region as well as the formation of elements in the anal and dorsal fins. This suggests that an increased swimming activity prioritized the development of structures which play an important role in swimming and thereby increasing the chance of survival in an environment where water velocity increases. Our study is the first to show that already during early zebrafish larval development, skeletal tissue in the cranial, axial and appendicular skeleton is competent to respond to swim-training due to increased water velocities. It demonstrates that changes in water flow conditions can result into significant spatio-temporal changes in skeletogenesis.     

Microanatomical diversity of amniote ribs: an exploratory quantitative study

Bone microanatomical diversity in extant and extinct tetrapods has been studied extensively, using increasingly sophisticated quantitative methods to assess its ecological, biomechanical and phylogenetic significance. Most studies have been conducted on the appendicular skeleton, and a strong relationship was found between limb bone microanatomy and habitat preferences. Few comparative studies have focused on the microanatomy of the axial skeleton and its ecological signal. In the present study, we propose the first exploratory study of the microanatomical diversity of amniote ribs. Our comparative sample comprises 155 species of extant amniotes and encompasses the taxonomic, ecological, and body size diversity of this group. We standardized our sampling location to the midshaft of mid‐dorsal ribs. Transverse sections were obtained from classical petrographic methods, as well as by X‐ray microtomography. Most of the microanatomical and size characters of the ribs display a phylogenetic signal, which is an expected result and is also observed in amniote limb bones and vertebrae. We found a significant relationship between rib cortical thickness, global compactness, and lifestyle. As for the vertebrae, the development of the spongiosa in the medullary region appears to be strongly correlated with size. Even though an ecological signal was found in the inner structure of the ribs, additional work is needed to document the intra‐individual variability of the rib microanatomy along the rib cage and within a single element.     

Developmental basis of limb length in rodents: evidence for multiple divisions of labor in mechanisms of endochondral bone growth

SUMMARY Mammals are remarkably diverse in limb lengths and proportions, but the number and kind of developmental mechanisms that contribute to length differences between limb bones remain largely unknown. Intra- and interspecific differences in bone length could result from variations in the cellular processes of endochondral bone growth, creating differences in rates of chondrocyte proliferation or hypertrophy, variation in the shape and size of chondrocytes, differences in the number of chondrocytes in precursor populations and throughout growth, or a combination of these mechanisms. To address these questions, this study compared cellular mechanisms of endochondral bone growth in cross-sectional ontogenetic series of the appendicular skeleton of two rodent species: the mouse ( Mus musculus ) and Mongolian gerbil ( Meriones unguiculatus ). Results indicate that multiple cellular processes of endochondral bone growth contribute to phenotypic differences in limb bone length. The data also suggest that separate developmental processes contribute to intraspecific length differences in proximal versus distal limb bones, and that these proximo-distal mechanisms are distinct from mechanisms that contribute to interspecific differences in limb bone length related to body size. These developmental "divisions of labor" are hypothesized to be important features of vertebrate limb development that allow (1) morphology in the autopods to evolve independently of the proximal limb skeleton, and (2) adaptive changes in limb proportions related to locomotion to evolve independently of evolutionary changes in body size.     

Sensitive Windows of Skeletal Development in Rabbits Determined by Hydroxyurea Exposure at Different Times throughout Gestation

The critical periods of axial skeletal development in rats and mice have been well characterized, however the timing of skeletal development in rabbits is not as well known. It is important to have a more precise understanding of this timing of axial skeletal development in rabbits due to the common use of this species in standard nonclinical studies to assess embryo–fetal developmental toxicity. Hydroxyurea, a teratogen known to induce a variety of fetal skeletal malformations, was administered to New Zealand White rabbits as a single dose (500 mg/kg) on individual days during gestation (gestation day,GD 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 19) and fetal external, visceral, and skeletal morphology was examined following cesarean sections on GD 29. A wide range of fetal skeletal effects was observed following hydroxyurea treatment, with a progression of malformations from anterior to posterior structures over time, as well as from proximal to distal structures over time. The sensitive window of axial skeletal development was determined to be GD 8 to 13, while disruption of appendicular and cranio‐facial skeletal development occurred primarily from GD 11 to 16 and GD 11 to 12, respectively. The results of this study provide a better understanding of the critical developmental window for different segments of the rabbit skeleton, which will aid in the design of window studies to investigate teratogenicity in rabbits.