Neural Crest Does Not Contribute to the Neck and Shoulder in the Axolotl (Ambystoma mexicanum)

Background

A major step during the evolution of tetrapods was their transition from water to land. This process involved the reduction or complete loss of the dermal bones that made up connections to the skull and a concomitant enlargement of the endochondral shoulder girdle. In the mouse the latter is derived from three separate embryonic sources: lateral plate mesoderm, somites, and neural crest. The neural crest was suggested to sustain the muscle attachments. How this complex composition of the endochondral shoulder girdle arose during evolution and whether it is shared by all tetrapods is unknown. Salamanders that lack dermal bone within their shoulder girdle were of special interest for a possible contribution of the neural crest to the endochondral elements and muscle attachment sites, and we therefore studied them in this context.

Results

We grafted neural crest from GFP+ fluorescent transgenic axolotl (Ambystoma mexicanum) donor embryos into white (d/d) axolotl hosts and followed the presence of neural crest cells within the cartilage of the shoulder girdle and the connective tissue of muscle attachment sites of the neck-shoulder region. Strikingly, neural crest cells did not contribute to any part of the endochondral shoulder girdle or to the connective tissue at muscle attachment sites in axolotl.

Conclusions

Our results in axolotl suggest that neural crest does not serve a general function in vertebrate shoulder muscle attachment sites as predicted by the “muscle scaffold theory,” and that it is not necessary to maintain connectivity of the endochondral shoulder girdle to the skull. Our data support the possibility that the contribution of the neural crest to the endochondral shoulder girdle, which is observed in the mouse, arose de novo in mammals as a developmental basis for their skeletal synapomorphies. This further supports the hypothesis of an increased neural crest diversification during vertebrate evolution.     

Growth of the pectoral girdle of the Leopard frog, Rana pipiens (Anura: Ranidae)

This article describes the growth of the anuran pectoral girdle of Rana pipiens and compares skeletal development of the shoulder to that of long bones. The pectoral girdle chondrifies as two halves, each adjacent to a developing humerus. In each, the scapula and coracoid form as single foci of condensed chondrocytes that fuse, creating a cartilaginous glenoid bridge articulating with the humerus. Based on histological sections, both the dermal clavicle and cleithrum begin to ossify at approximately the same time as the periosteum forms around the endochondral bones. The dermal and endochondral bones of the girdle form immobile joints with neighboring girdle elements; however, the cellular organization and growth pattern of the scapula and coracoid closely resemble those of a long bone. Similar to a long bone epiphysis, distal margins of both endochondral elements have zones of hyaline, stratified, and hypertrophic cartilages. As a result, fused elements of the girdle can grow without altering the glenoid articulation with the humerus. Comparisons of anuran long bone and pectoral girdle growth suggest that different bones can have similar histology and development regardless of adult morphology.     

The Upper Devonian tetrapodomorph Gogonasus andrewsae from Western Australia: Reconstruction of the shoulder girdle and opercular series using X-ray Micro-Computed Tomography

The tetrapodomorph fish, Gogonasus andrewsae is a three dimensionally well-preserved sarcopterygian from the Gogo Formation (Frasnian, early Upper Devonian, ∼380 million years ago) in Western Australia. High-resolution X-ray Micro-Computed Tomography and 3D printouts were used to obtain a digital reconstruction of its shoulder girdle and opercular series. Our new findings show the opercular series in a close fit against the upper bones of the shoulder girdle only if the anocleithrum, supracleithrum and post-temporal are aligned more horizontally than in previous reconstructions. The lowermost subopercular bone also differs, in partly covering the clavicle of the shoulder girdle. The ascending process of the clavicle, and the ventral process of the anocleithrum, do not fit closely inside the cleithrum, and perhaps functioned for ligamentous attachment. A rugose area on the anocleithral process is in a similar relative position to the attachment of a muscle ligament on the shoulder girdle of various living actinopterygians. Our manipulation of 3D printouts permits testing of the morphological fit of extremely fragile acid-etched bones, and indicates a new way to investigate the constructional morphology of one or more mechanical units of the vertebrate skeleton. It is suggested that Micro-CT imaging, reconstruction, visualisation and 3D printing techniques will provide a rigorous new test leading to modification of previous reconstructions of extinct vertebrates that were based on graphical methods and 2D imaging.     

Histology of the heterostracan dermal skeleton: Insight into the origin of the vertebrate mineralised skeleton

Living vertebrates are divided into those that possess a fully formed and fully mineralised skeleton (gnathostomes) versus those that possess only unmineralised cartilaginous rudiments (cyclostomes). As such, extinct phylogenetic intermediates of these living lineages afford unique insights into the evolutionary assembly of the vertebrate mineralised skeleton and its canonical tissue types. Extinct jawless and jawed fishes assigned to the gnathostome stem evidence the piecemeal assembly of skeletal systems, revealing that the dermal skeleton is the earliest manifestation of a homologous mineralised skeleton. Yet the nature of the primitive dermal skeleton, itself, is poorly understood. This is principally because previous histological studies of early vertebrates lacked a phylogenetic framework required to derive evolutionary hypotheses. Nowhere is this more apparent than within Heterostraci, a diverse clade of primitive jawless vertebrates. To this end, we surveyed the dermal skeletal histology of heterostracans, inferred the plesiomorphic heterostracan skeleton and, through histological comparison to other skeletonising vertebrate clades, deduced the ancestral nature of the vertebrate dermal skeleton. Heterostracans primitively possess a four‐layered skeleton, comprising a superficial layer of odontodes composed of dentine and enameloid; a compact layer of acellular parallel‐fibred bone containing a network of vascular canals that supply the pulp canals (L1); a trabecular layer consisting of intersecting radial walls composed of acellular parallel‐fibred bone, showing osteon‐like development (L2); and a basal layer of isopedin (L3). A three layered skeleton, equivalent to the superficial layer L2 and L3 and composed of enameloid, dentine and acellular bone, is possessed by the ancestor of heterostracans + jawed vertebrates. We conclude that an osteogenic component is plesiomorphic with respect to the vertebrate dermal skeleton. Consequently, we interpret the dermal skeleton of denticles in chondrichthyans and jawless thelodonts as independently and secondarily simplified. J. Morphol. 276:657–680, 2015. © 2015 The Authors Journal of Morphology Published by Wiley Periodicals, Inc.     

Biomechanics of vibration reception in the bullfrog,Rana catesbeiana

The opercularis system (OPS) of amphibians consists of an opercularis muscle that connects the shoulder girdle skeleton to the operculum, a movable element in the oval window of the otic capsule. The role of the OPS in reception of vibrations was examined in bullfrogs (Rana catesbeiana) tested in various postures that manipulated differential motion between the shoulder girdle (the origin of the opercularis muscle) and skull (including the inner ear). Amplitude and phase relationship of motions of the suprascapular cartilage of the shoulder girdle and the posterior skull were also measured during these tests. 1. Microphonic responses to vertical vibrations from 25-200 Hz were typically highest when frogs were in a normal, sitting posture with the head held off the vibrating platform. Responses from animals in which the head directly contacted the platform were often less (by up to 10 dB at certain frequencies). Responses from all test positions were highest at lower frequencies, especially between 50-100 Hz. 2. Suprascapular accelerations were typically highest in the normal, sitting posture, and at lower frequencies (50-75 Hz) were often greater than that of the vibrating platform by up to 8 dB. The shoulder girdle skeleton of the bullfrog is therefore readily affected by vertical substrate motion. 3. The amplitude of microphonic responses in the different test postures did not correspond well with head acceleration. Rather, response amplitude corresponded best with the absolute difference between shoulder and head motion. For example, in the normal posture, suprascapular motion was much greater than head motion, and responses were relatively high. If only the head was vibrated, head motion was high and shoulder motion low, and responses also were relatively high. If the head and body were vibrated together, their motions were similar, and responses to the same platform accelerations were often reduced. Phase differences between shoulder and head motions were small at the frequencies examined and may be of little functional significance. The importance of differences in shoulder and head motion suggests that the resulting differential motion of the operculum and inner ear fluids can produce waves that stimulate appropriate end organs (such as the saccule). 4. Removal of the opercularis muscle reduced responses up to 18 dB at certain frequencies in some of the test postures. The most significant reductions were observed in those postures with a significant difference between shoulder and head motion (such as the normal posture).(ABSTRACT TRUNCATED AT 400 WORDS)     

Formation of dermal skeletal and dental tissues in fish: a comparative and evolutionary approach

Osteichthyan and chondrichthyan fish present an astonishing diversity of skeletal and dental tissues that are often difficult to classify into the standard textbook categories of bone, cartilage, dentine and enamel. To address the question of how the tissues of the dermal skeleton evolved from the ancestral situation and gave rise to the diversity actually encountered, we review previous data on the development of a number of dermal skeletal elements (odontodes, teeth and dermal denticles, cranial dermal bones, postcranial dermal plates and scutes, elasmoid and ganoid scales, and fin rays). A comparison of developmental stages at the tissue level usually allows us to identify skeletogenic cell populations as either odontogenic or osteogenic on the basis of the place of formation of their dermal papillae and of the way of deposition of their tissues. Our studies support the evolutionary affinities (1) between odontodes, teeth and denticles, (2) between the ganoid scales of polypterids and the elasmoid scales of teleosts, and (3) to a lesser degree between the different bony elements. There is now ample evidence to ascertain that the tissues of the elasmoid scale are derived from dental and not from bony tissues. This review demonstrates the advantage that can be taken from developmental studies, at the tissue level, to infer evolutionary relationships within the dermal skeleton in chondrichthyans and osteichthyans.     

A new plant assemblage (microfossil and megafossil) from the Lower Old Red Sandstone of the Anglo-Welsh Basin: its implications for the palaeoecology of early terrestrial ecosystems

Lower Old Red Sandstone deposits penetrated by a series of cored boreholes near Newport (South Wales) have been sedimentologically logged, and recovered plant assemblages (microfossil and megafossil) investigated. Sedimentological logging indicates that the deposits are typical of the extensive terrestrial-fluviatile floodplain deposits of the Anglo-Welsh Basin. Palynomorph assemblages have been recovered from a number of horizons and comprise entirely terrestrial forms (spores and phytodebris). They essentially represent a single assemblage, belonging to the middle subzone of the micrornatus-newportensis sporomorph assemblage biozone, and indicate an Early Devonian (mid-Lochkovian) age. The new biostratigraphical data enables correlation with other Lower Old Red Sandstone deposits of the Anglo-Welsh Basin, and the deposits are assigned to the lower part of the St. Maughan's Group. A plant megafossil/mesofossil assemblage recovered from one of the spore-bearing horizons includes a zosterophyll assigned to Zosterophyllum cf. fertile. This is the earliest reported zosterophyll from the Anglo-Welsh Basin. The new palynological/palaeobotanical data provide important information on the palaeoecology and palaeobiogeography of the vegetation of the southeastern margin of the Old Red Sandstone continent during Lochkovian times. Palaeogeographical variation in the distribution of plant microfossils and megafossils is interpreted as reflecting differences between the flora of the lowland floodplain and inland intermontaine basins, although this is to a certain extent overprinted by variation due to localized differences in environmental conditions.     

A two-headed reptile from the Cretaceous of China

A malformed embryonic or neonate choristoderan reptile from the Lower Cretaceous Yixian Formation of northeastern China is described. The tiny skeleton exhibits two heads and two necks, with bifurcation at the level of the pectoral girdle. In a fossil, this is the first occurrence of the malformation known as axial bifurcation, which is well known in living reptiles.     

Pectoral fin and girdle development in the basal actinopterygians Polyodon spathula and Acipenser transmontanus

The pectoral fins of Acipenseriformes possess endoskeletons with elements homologous to both the fin radials of teleosts and the limb bones of tetrapods. Here we present a study of pectoral fin development in the North American paddlefish, Polyodon spathula, and the white sturgeon, Acipenser transmontanus, which reveals that aspects of both teleost and tetrapod endoskeletal patterning mechanisms are present in Acipenseriformes. Those elements considered homologous to teleost radials, the propterygium and the mesopterygial radials, form via subdivision of an initially chondrogenic plate of mesenchymal cells called the endoskeletal disc. In Acipenseriformes, elements homologous to the sarcopterygian metapterygium develop separately from the endoskeletal disc as an outgrowth of the endoskeletal shoulder girdle that extends into the posterior margin of the finbud. As in tetrapods, the elongating metapterygium and the metapterygial radials form in a proximal to distal order as discrete condensations from initially nonchondrogenic mesenchyme. Patterns of variation seen in the Acipenseriform fin also correlate with putative homology: all variants from the "normal" fin bauplan involved the metapterygium and the metapterygial radials alone. The primary factor distinguishing Polyodon and Acipenser fin development from each other is the composition of the endoskeletal extracellular matrix. Proteoglycans (visualized with Alcian Blue) and Type II collagen (visualized by immunohistochemistry) are secreted in different places within the mesenchymal anlage of the fin elements and girdle and at different developmental times. Acipenseriform pectoral fins differ from the fins of teleosts in the relative contribution of the endoskeleton and dermal rays. The fins of Polyodon and Acipenser possess elaborate endoskeletons overlapped along their distal margins by dermal lepidotrichia. In contrast, teleost fins generally possess relatively small endoskeletal radials that articulate with the dermal fin skeleton terminally, with little or no proximodistal overlap.     

Age estimation of adolescent and young adult male and female skeletons II, epiphyseal union at the upper limb and scapular girdle in a modern Portuguese skeletal sample

This study completes previously reported ages for timing of epiphyseal union in the postcranial skeleton in a recent sample, with data from the scapula, clavicle, humerus, radius, and ulna. A sample of 121 individuals between the ages of 9 and 29 (females = 65, males = 56) was derived from the Lisbon documented skeletal collection. Epiphyseal union was scored at 16 anatomical locations, using a three-stage scheme: 1) no union; 2) partial union; and 3) completed union, all traces of fusion having disappeared. In the upper limb, the epiphyses of the elbow are the first to fuse at around 11 to 15 years of age, followed by those of the shoulder and wrist. In the scapular girdle, the coracoid area is the first to fuse, followed by the glenoid surface and remaining epiphyses, with the medial clavicle fusing last, by the age of 25-27. There is a sex difference in maturation, with females showing an advance relative to males of about 2 years in the upper limb. Sex differences in maturation are less noticeable in the scapular girdle, but data suggest that females are also ahead of males. Results suggest overall similar age ranges for stages of union as previous dry bone observations, but some studies show significant divergences which seem to derive from methodological issues. Although some radiographic reference standards provide comparable age ranges, they should probably be avoided when aging skeletal remains.     

A LATE MESOZOIC BIRD FROM INNER MONGOLIA

Otogornisgenghisigen.etsp.nov.HolotypePectoralgirdleandwingelements.(IVPPV96O7)HorizonandLocalityYijinhoIuoFormation(EarlyCretaceous);Chaibu-Sumi,otog-qi,Yikezhao-meng,InnerMongolia.DiagnosisAmedium-sizedbird.Longbonesthick-waIled.Acromionofscapulaprotrudingforward.Coracoidwithexpandedheadandbroadp1ate-shapeddistalend.Humerusrelativelystrongandshorterthanradiusandulna,pneumaticfossaabsent,capitalgrooveprominent,deltoidcrestnotdeve1oped,olecrana1fossasha1low,su1cusform.scapu1otricipit…     

A new actinolepid arthrodire (Class Placodermi) from the Lower Devonian Sevy Dolomite, East-Central Nevada

A new genus and species of actinolepid arthrodire, Aleosteus eganensis , is described from the Lower Devonian (Late Emsian) Sevy Dolomite in the Egan Range of east-central Nevada. A cladistic analysis of the family Actinolepidae is presented for the first time and shows the Baltic Actinolepis species form the primitive sister-group of all other actinolepids. Rapid evolution of the Actinolepidae during the Lochkovian is reflected in the dispersal of the family around the Old Red Sandstone Continent at this time followed by the development of endemic faunas through the Lower Devonian and into the Middle Devonian.     

Development of the dermal skeleton in Alligator mississippiensis (Archosauria, Crocodylia) with comments on the homology of osteoderms

The dermal skeleton (=exoskeleton) has long been recognized as a major determinant of vertebrate morphology. Until recently however, details of tissue development and diversity, particularly among amniotes, have been lacking. This investigation explores the development of the dermatocranium, gastralia, and osteoderms in the American alligator, Alligator mississippiensis. With the exception of osteoderms, elements of the dermal skeleton develop early during skeletogenesis, with most initiating ossification prior to mineralization of the endoskeleton. Characteristically, circumoral elements of the dermatocranium, including the pterygoid and dentigerous elements, are among the first to form. Unlike other axially arranged bones, gastralia develop in a caudolateral to craniomedial sequence. Osteoderms demonstrate a delayed onset of development compared with the rest of the skeleton, not appearing until well after hatching. Osteoderm development is asynchronous across the body, first forming dorsally adjacent to the cervical vertebrae; the majority of successive elements appear in caudal and lateral positions. Exclusive of osteoderms, the dermal skeleton initiates osteogenesis via intramembranous ossification. Following the establishment of skeletal condensations, some preossified spicules become engorged with many closely packed clusters of chondrocyte-like cells in a bone-like matrix. This combination of features is characteristic of chondroid bone, a tissue otherwise unreported among nonavian reptiles. No secondary cartilage was identified in any of the specimens examined. With continued growth, dermal bone (including chondroid bone) and osteoid are resorbed by multinucleated osteoclasts. However, there is no evidence that these cells contribute to the rugose pattern of bony ornamentation characteristic of the crocodylian dermatocranium. Instead, ornamentation develops as a result of localized concentrations of bone deposited by osteoblasts. Osteoderms develop in the absence of osteoblastic cells, osteoid, and periosteum; bone develops via the direct transformation of the preexisting dense irregular connective tissue. This mode of bone formation is identified as metaplasia. Importantly, it is also demonstrated that osteoderms are not histologically uniform but involve a range of tissues including calcified and uncalcified dense irregular connective tissue. Between taxa, not all osteoderms develop by homologous processes. However, it is concluded that all osteoderms may share a deep homology, connected by the structural and skeletogenic properties of the dermis.     

Histology of “placoderm” dermal skeletons: Implications for the nature of the ancestral gnathostome

The vertebrate dermal skeleton has long been interpreted to have evolved from a primitive condition exemplified by chondrichthyans. However, chondrichthyans and osteichthyans evolved from an ancestral gnathostome stem‐lineage in which the dermal skeleton was more extensively developed. To elucidate the histology and skeletal structure of the gnathostome crown‐ancestor we conducted a histological survey of the diversity of the dermal skeleton among the placoderms, a diverse clade or grade of early jawed vertebrates. The dermal skeleton of all placoderms is composed largely of a cancellar architecture of cellular dermal bone, surmounted by dermal tubercles in the most ancestral clades, including antiarchs. Acanthothoracids retain an ancestral condition for the dermal skeleton, and we record its secondary reduction in antiarchs. We also find that mechanisms for remodeling bone and facilitating different growth rates between adjoining plates are widespread throughout the placoderms. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Nature of the turtle shell: Morphogenetic causes of bone variability and its evolutionary implication

The turtle shell is characterized by a high degree of conservatism of the fundamental model and, at the same time, a high variability at the individual level. The components of the bony shell vary in origin. The costal and neural plates of the carapace are modified elements of the axial skeleton (ribs and neural arches) and the plastral plates are transformed dermal ossifications of the shoulder girdle and gastralia, peripheral, pygal, and suprapygal plates are similar to osteoderms of other reptiles. The variability of the structure of particular parts of the turtle shell is manifested differently. Most anomalies have been recorded in the caudal part of the carapace. The plastron is relatively stable in morphology. Variations in the bony shell structure are observed in (1) unusual shape and size of plates combined with normal number of plates, (2) presence of additional plates, and (3) absence of regular plates. Based on the morphogenetic characteris-tics, anomalies are subdivided into (1) variations caused by changes in the number of elements of the axial skeleton or their contacts with the dermis (neurals and costals); (2) variations due to changes in the number of horny scutes (peripherals); (3) variations connected with irregular osteogeny or disturbed growth of bones     

Regulation of skeletogenic differentiation in cranial dermal bone

Although endochondral ossification of the limb and axial skeleton is relatively well-understood, the development of dermal (intramembranous) bone featured by many craniofacial skeletal elements is not nearly as well-characterized. We analyzed the expression domains of a number of markers that have previously been associated with endochondral skeleton development to define the cellular transitions involved in the dermal ossification process in both chick and mouse. This led to the recognition of a series of distinct steps in the dermal differentiation pathways, including a unique cell type characterized by the expression of both osteogenic and chondrogenic markers. Several signaling molecules previously implicated in endochondrial development were found to be expressed during specific stages of dermal bone formation. Three of these were studied functionally using retroviral misexpression. We found that activity of bone morphogenic proteins (BMPs) is required for neural crest-derived mesenchyme to commit to the osteogenic pathway and that both Indian hedgehog (IHH) and parathyroid hormone-related protein (PTHrP, PTHLH) negatively regulate the transition from preosteoblastic progenitors to osteoblasts. These results provide a framework for understanding dermal bone development with an aim of bringing it closer to the molecular and cellular resolution available for the endochondral bone development.     

Bone microstructures and mode of skeletogenesis in osteoderms of three pareiasaur taxa from the Permian of South Africa

The extinct parareptilian clade of pareiasaurs was in the past often presented to constitute a morphocline from larger, less armoured forms to smaller, well armoured forms, indicating that the osteoderm cover became an increasingly prominent aspect in the post‐cranial skeleton of these animals. Here, we describe microanatomical and microstructural aspects of osteoderms of the three pareiasaur taxa Bradysaurus, Pareiasaurus and Anthodon from the Permian of South Africa. A generalized mode of osteoderm formation, consistent with intramembraneous skeletogenesis, is hypothesized to be present in all pareiasaurs. Few characters are shared between pareiasaur dermal armour and turtle shell bones and osteoderms. Otherwise, there is strong evidence from microanatomy and histology (i.e. absence of structures that formed via metaplasia of dermal tissue) that indicates nonhomology between pareiasaur dermal armour and the armour of living eureptiles. Analysis with bone profiler revealed no clear connection between bone compactness and lifestyle in the amniote osteoderm sample.     

Chondrogenesis and ossification of the lissamphibian pectoral girdle

Knowledge of amphibian shoulder development is requisite for further understanding of gnathostome pectoral girdle evolution. Fish and amniotes share few pectoral girdle elements, but modern amphibians exhibit a unique combination of traits that bridge the morphological gap between these two groups. I analyzed patterns of chondrogenesis, ossification, and bone histology of the pectoral girdles of two anuran species (Xenopus laevis and Bombina orientalis) and two urodele species (Ambystoma mexicanum and Desmognathus aeneus) to provide new insight into the evolution of the tetrapod pectoral girdle. Comparisons reveal the following: 1) variation in the pattern of chondrogenesis among the anuran species analyzed correlates to variation in adult pectoral girdle morphology; 2) morphologically similar pectoral skeletons do not necessarily have similar patterns of bone histology; and 3) the urodele and anuran pectoral girdles included herein share a common morphology despite differences in patterns of chondrogenesis.