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The production of blood cells (haematopoiesis) occurs in the limb bones of most tetrapods but is absent in the fin bones of ray-finned fish. When did long bones start producing blood cells? Recent hypotheses suggested that haematopoiesis migrated into long bones prior to the water-to-land transition and protected newly-produced blood cells from harsher environmental conditions. However, little fossil evidence to support these hypotheses has been provided so far. Observations of the humeral microarchitecture of stem-tetrapods, batrachians, and amniotes were performed using classical sectioning and three-dimensional synchrotron virtual histology. They show that Permian tetrapods seem to be among the first to exhibit a centralised marrow organisation, which allows haematopoiesis as in extant amniotes. Not only does our study demonstrate that long-bone haematopoiesis was probably not an exaptation to the water-to-land transition but it sheds light on the early evolution of limb-bone development and the sequence of bone-marrow functional acquisitions., Competing Interests: JE, PT, AC, JK, GN, CB, SS No competing interests declared, (© 2021, Estefa et al.)

The ontogeny of the paired appendages has been extensively studied in lungfishes and tetrapods, but remains poorly known in coelacanths. Recent work has shed light on the anatomy and development of the pectoral fin in Latimeria chalumnae. Yet, information on the development of the pelvic fin and girdle is still lacking. Here, we described the development of the pelvic fin and girdle in Latimeria chalumnae based on 3D reconstructions generated from conventional and X-ray synchrotron microtomography, as well as MRI acquisitions. As in other jawed vertebrates, the development of the pelvic fin occurs later than that of the pectoral fin in Latimeria. Many elements of the endoskeleton are not yet formed at the earliest stage sampled. The four mesomeres are already formed in the fetus, but only the most proximal radial elements (preaxial radial 0-1) are formed and individualized at this stage. We suggest that all the preaxial radial elements in the pelvic and pectoral fin of Latimeria are formed through the fragmentation of the mesomeres. We document the progressive ossification of the pelvic girdle, and the presence of a trabecular system in the adult. This trabecular system likely reinforces the cartilaginous girdle to resist the muscle forces exerted during locomotion. Finally, the presence of a preaxial element in contact with the pelvic girdle from the earliest stage of development onward questions the mono-basal condition of the pelvic fin in Latimeria. However, the particular shape of the mesomeres may explain the presence of this element in contact with the girdle., (© 2020 American Association for Anatomy.)

The ontogenetic trajectory of a marginal jawbone of Lophosteus superbus (Late Silurian, 422 Million years old), the phylogenetically most basal stem osteichthyan, visualized by synchrotron microtomography, reveals a developmental relationship between teeth and dermal odontodes that is not evident from the adult morphology. The earliest odontodes are two longitudinal founder ridges formed at the ossification center. Subsequent odontodes that are added lingually to the ridges turn into conical teeth and undergo cyclic replacement, while those added labially achieve a stellate appearance. Stellate odontodes deposited directly on the bony plate are aligned with the alternate files of teeth, whereas new tooth positions are inserted into the files of sequential addition when a gap appears. Successive teeth and overgrowing odontodes show hybrid morphologies around the oral-dermal boundary, suggesting signal cross-communication. We propose that teeth and dermal odontodes are modifications of a single system, regulated and differentiated by the oral and dermal epithelia., Competing Interests: DC, HB, SS, PT, TM, PA No competing interests declared, (© 2020, Chen et al.)

The monobasal pectoral fins of living coelacanths and lungfishes are homologous to the forelimbs of tetrapods and are thus critical to investigate the origin thereof. However, it remains unclear whether the similarity in the asymmetrical endoskeletal arrangement of the pectoral fins of coelacanths reflects the evolution of the pectoral appendages in sarcopterygians. Here, we describe for the first time the development of the pectoral fin and shoulder girdle in the extant coelacanth Latimeria chalumnae, based on the tomographic acquisition of a growth series. The pectoral girdle and pectoral fin endoskeleton are formed early in development with a radially outward growth of the endoskeletal elements. The visualization of the pectoral girdle during development shows a reorientation of the girdle between the fetus and pup 1 stages, creating a contact between the scapulocoracoids and the clavicles in the ventro-medial region. Moreover, we observed a splitting of the pre- and post-axial cartilaginous plates in respectively pre-axial radials and accessory elements on one hand, and in post-axial accessory elements on the other hand. However, the mechanisms involved in the splitting of the cartilaginous plates appear different from those involved in the formation of radials in actinopterygians. Our results show a proportional reduction of the proximal pre-axial radial of the fin, rendering the external morphology of the fin more lobe-shaped, and a spatial reorganization of elements resulting from the fragmentation of the two cartilaginous plates. Latimeria development hence supports previous interpretations of the asymmetrical pectoral fin skeleton as being plesiomorphic for coelacanths and sarcopterygians., (© 2019 Anatomical Society.)

The neurocranium of sarcopterygian fishes was originally divided into an anterior (ethmosphenoid) and posterior (otoccipital) portion by an intracranial joint, and underwent major changes in its overall geometry before fusing into a single unit in lungfishes and early tetrapods 1 . Although the pattern of these changes is well-documented, the developmental mechanisms that underpin variation in the form of the neurocranium and its associated soft tissues during the evolution of sarcopterygian fishes remain poorly understood. The coelacanth Latimeria is the only known living vertebrate that retains an intracranial joint 2,3 . Despite its importance for understanding neurocranial evolution, the development of the neurocranium of this ovoviviparous fish remains unknown. Here we investigate the ontogeny of the neurocranium and brain in Latimeria chalumnae using conventional and synchrotron X-ray micro-computed tomography as well as magnetic resonance imaging, performed on an extensive growth series for this species. We describe the neurocranium at the earliest developmental stage known for Latimeria, as well as the major changes that the neurocranium undergoes during ontogeny. Changes in the neurocranium are associated with an extreme reduction in the relative size of the brain along with an enlargement of the notochord. The development of the notochord appears to have a major effect on the surrounding cranial components, and might underpin the formation of the intracranial joint. Our results shed light on the interplay between the neurocranium and its adjacent soft tissues during development in Latimeria, and provide insights into the developmental mechanisms that are likely to have underpinned the evolution of neurocranial diversity in sarcopterygian fishes.

The teeth of gnathostomes (jawed vertebrates) show rigidly patterned, unidirectional replacement that may or may not be associated with a shedding mechanism. These mechanisms, which are critical for the maintenance of the dentition, are incongruently distributed among extant gnathostomes. Although a permanent tooth-generating dental lamina is present in all chondrichthyans, many tetrapods and some teleosts, it is absent in the non-teleost actinopterygians. Tooth-shedding by basal hard tissue resorption occurs in most osteichthyans (including tetrapods) but not in chondrichthyans. Here we report a three-dimensional virtual dissection of the dentition of a 424-million-year-old stem osteichthyan, Andreolepis hedei, using propagation phase-contrast synchrotron microtomography, with a reconstruction of its growth history. Andreolepis, close to the common ancestor of all extant osteichthyans, shed its teeth by basal resorption but probably lacked a permanent dental lamina. This is the earliest documented instance of resorptive tooth shedding and may represent the primitive osteichthyan mode of tooth replacement.

Extant vertebrates form two clades, the jawless Cyclostomata (lampreys and hagfishes) and the jawed Gnathostomata (all other vertebrates), with contrasting facial architectures. These arise during development from just a few key differences in the growth patterns of the cranial primordia: notably, the nasal sacs and hypophysis originate from a single placode in cyclostomes but from separate placodes in gnathostomes, and infraoptic ectomesenchyme migrates forward either side of the single placode in cyclostomes but between the placodes in gnathostomes. Fossil stem gnathostomes preserve cranial anatomies rich in landmarks that provide proxies for developmental processes and allow the transition from jawless to jawed vertebrates to be broken down into evolutionary steps. Here we use propagation phase contrast synchrotron microtomography to image the cranial anatomy of the primitive placoderm (jawed stem gnathostome) Romundina, and show that it combines jawed vertebrate architecture with cranial and cerebral proportions resembling those of cyclostomes and the galeaspid (jawless stem gnathostome) Shuyu. This combination seems to be primitive for jawed vertebrates, and suggests a decoupling between ectomesenchymal growth trajectory, ectomesenchymal proliferation, and cerebral shape change during the origin of gnathostomes.

Because of its close relationship to tetrapods, Eusthenopteron is an important taxon for understanding the establishment of the tetrapod body plan. Notably, it is one of the earliest sarcopterygians in which the humerus of the pectoral fin skeleton is preserved. The microanatomical and histological organization of this humerus provides important data for understanding the evolutionary steps that built up the distinctive architecture of tetrapod limb bones. Previous histological studies showed that Eusthenopteron's long-bone organization was established through typical tetrapod ossification modalities. Based on a three-dimensional reconstruction of the inner microstructure of Eusthenopteron's humerus, obtained from propagation phase-contrast X-ray synchrotron microtomography, we are now able to show that, despite ossification mechanisms and growth patterns similar to those of tetrapods, it also retains plesiomorphic characters such as a large medullary cavity, partly resulting from the perichondral ossification around a large cartilaginous bud as in actinopterygians. It also exhibits a distinctive tubular organization of bone-marrow processes. The connection between these processes and epiphyseal structures highlights their close functional relationship, suggesting that either bone marrow played a crucial role in the long-bone elongation processes or that trabecular bone resulting from the erosion of hypertrophied cartilage created a microenvironment for haematopoietic stem cell niches.

The debate about the origin of the vertebrate dentition has been given fresh fuel by new fossil discoveries and developmental studies of extant animals. Odontodes (teeth or tooth-like structures) can be found in two distinct regions, the 'internal' oropharyngeal cavity and the 'external' skin. A recent hypothesis argues that regularly patterned odontodes is a specific oropharyngeal feature, whereas odontodes in the external skeleton lack this organization. However, this argument relies on the skeletal system of modern chondrichthyans (sharks and their relatives), which differ from other gnathostome (jawed vertebrate) groups in not having dermal bones associated with the odontodes. Their external skeleton is also composed of monoodontode 'placoid scales', whereas the scales of most early fossil gnathostomes are polyodontode, i.e. constructed from several odontodes on a shared bony base. Propagation phase contrast X-ray Synchrotron microtomography (PPC-SRµCT) is used to study the polyodontode scales of the early bony fish Andreolepis hedei. The odontodes constructing a single scale are reconstructed in 3D, and a linear and regular growth mechanism similar to that in a gnathostome dentition is confirmed, together with a second, gap-filling growth mechanism. Acanthodian tooth whorls are described, which show that ossification of the whorl base preceded and probably patterned the development of the dental lamina, in contrast to the condition in sharks where the dental lamina develops early and patterns the dentition.The new findings reveal, for the first time, how polyodontode scales grow in 3D in an extinct bony fish. They show that dentition-like odontode patterning occurs on scales and that the primary patterning unit of a tooth whorl may be the bony base rather than the odontodes it carries. These results contradict the hypothesis that oropharyngeal and external odontode skeletons are fundamentally separate and suggest that the importance of dermal bone interactions to odontode patterning has been underestimated.

The transition from jawless to jawed vertebrates (gnathostomes) resulted in the reconfiguration of the muscles and skeleton of the head, including the creation of a separate shoulder girdle with distinct neck muscles. We describe here the only known examples of preserved musculature from placoderms (extinct armored fishes), the phylogenetically most basal jawed vertebrates. Placoderms possess a regionalized muscular anatomy that differs radically from the musculature of extant sharks, which is often viewed as primitive for gnathostomes. The placoderm data suggest that neck musculature evolved together with a dermal joint between skull and shoulder girdle, not as part of a broadly flexible neck as in sharks, and that transverse abdominal muscles are an innovation of gnathostomes rather than of tetrapods.

The phylogenetic relationships between the different groups of Paleozoic gnathostomes are still debated, mainly because of incomplete datasets on Paleozoic jawed vertebrate fossils and ontogeny of some modern taxa. This issue is illustrated by the condition of the glossopharyngeal nerve relative to the parachordal plate, the otic capsules and the metotic fissure in gnathostomes. Two main conditions are observed in elasmobranchs (shark and rays) and osteichthyans (bony fishes and tetrapods). The condition in the other chondrichthyan taxon, the holocephalans, is still poorly known, and without any information on this taxon, it remains difficult to polarize the condition in gnathostomes. Based on the anatomical study of an embryo of the holocephalan Callorhinchus milii by means of propagation X-Ray Synchrotron phase contrast microtomography using both holotomography and single distance phase retrieval process, we show that, contrary to what was previously inferred for holocephalans (i.e. an osteichthyan-like condition), the arrangement of the glossopharyngeal nerve relative to the surrounding structure in holocephalans is more similar to that of elasmobranchs. Furthermore, the holocephalan condition represents a combination of plesiomorphic characters for gnathostomes (e.g., the glossopharyngeal nerve leaves the braincase via the metotic fissure) and homoplastic characters. By contrast, the crown osteichthyans are probably derived in having the glossopharyngeal nerve that enters the saccular chamber and in having the glossopharyngeal foramen separated from the metotic fissure.

Growth and patterning of craniofacial sutures is subjected to the effects of mechanical stress. Mechanotransduction processes occurring at the margins of the sutures are not precisely understood. Here, we propose a simple theoretical model based on the orientation of collagen fibres within the suture in response to local stress. We demonstrate that fibre alignment generates an instability leading to the emergence of interdigitations. We confirm the appearance of this instability both analytically and numerically. To support our model, we use histology and synchrotron X-ray microtomography and reveal the fine structure of fibres within the sutural mesenchyme and their insertion into the bone. Furthermore, using a mouse model with impaired mechanotransduction, we show that the architecture of sutures is disturbed when forces are not interpreted properly. Finally, by studying the structure of sutures in the mouse, the rat, an actinopterygian (Polypterus bichir) and a placoderm (Compagopiscis croucheri), we show that bone deposition patterns during dermal bone growth are conserved within jawed vertebrates. In total, these results support the role of mechanical constraints in the growth and patterning of craniofacial sutures, a process that was probably effective at the emergence of gnathostomes, and provide new directions for the understanding of normal and pathological suture fusion.

Living cartilaginous fishes, or chondrichthyans, include numerous elasmobranch (sharks and rays) species but only few chimaeroid (ratfish) species. The early history of chimaeroids, or holocephalans, and the modalities of their divergence from elasmobranchs are much debated. During Carboniferous times, 358-300 million years (Myr) ago, they underwent a remarkable evolutionary radiation, with some odd and poorly understood forms, including the enigmatic iniopterygians that were known until now from poorly informative flattened impressions. Here, we report iniopterygian skulls found preserved in 3 dimensions in approximately 300-Myr-old concretions from Oklahoma and Kansas. The study was performed by using conventional X-ray microtomography (muCT), as well as absorption-based synchrotron microtomography (SR-muCT) [Tafforeau P, et al. (2006) Applications of X-ray synchrotron microtomography for non-destructive 3D studies of paleontological specimens. Appl Phys A 83:95-202] and a new holotomographic approach [Guigay P, Langer M, Boistel R, Cloetens P (2007) Mixed transfer function and transport of intensity approach for phase retrieval in the Fresnel region. Opt Lett 32:1617-1619], which revealed their peculiar anatomy. Iniopterygians also share unique characters with living chimaeroids, suggesting that the key chimaeroid skull features were already established 300 Myr ago. Moreover, SR-muCT of an articulated skull revealed a strikingly brain-shaped structure inside the endocranial cavity, which seems to be an exceptional case of soft-tissue mineralization of the brain, presumably as a result of microbially induced postmortem phosphatization. This was imaged with exceptional accuracy by using holotomography, which demonstrates its great potential to image preserved soft parts in dense fossils.