Sophie Sanchez

The enantiornithes, a group of Mesozoic birds, have morphological features suggesting a precocial mode of development; however, their detailed physiological characteristics, particularly hatchling metabolic rates, have yet to be explored. Here, we retrodicted metabolic rates for four Lower Cretaceous enantiornithines: one hatchling, one adult from Las Hoyas, one early post-hatching juvenile and one post-juvenile individual from the Jehol Biota, using phylogenetic comparative analyses and a comprehensive dataset of extant birds spanning the altricial–precocial spectrum. By examining vascular density (related to resting metabolic rate (RMR)) and nutrient foraminal size (proportional to blood flow rate) as proxies, we inferred the metabolic scope of the hatchling enantiornithine and determined that it fell toward the precocial end of the altricial–precocial spectrum. The RMR values of the two enantiornithine chicks were retrodicted as 0.67 mlO2 h–1 g–0.93. The peak metabolic rate (PMR) retrodicted is twice the resting metabolism for the Las Hoyas hatchling. These results are consistent with a semi-precocial/precocial development characterized by independent locomotion by climbing using the wing claws soon after hatching. However, flapping flight may have been delayed, owing to incomplete ossification of the sternum, as observed in one hatchling specimen.

Tessellated calcified cartilage (TCC) is a hallmark of the chondrichthyan skeleton, yet its development early in ontogeny across the four major groups (batoids, galeomorphs, squalomorphs, and holocephalans) remains poorly understood. Specialised traits of TCC, such as multi-layered TCC and internal mineralised trabeculae, typically develop in response to feeding mechanics. In this study, we evaluated TCC morphology in the jaws of 12 representative taxa to observe its structure at an early ontogenetic stage to determine whether these specialised features had yet developed. Batoids consistently exhibited well-developed, homogeneous, polygonal tesserae early in ontogeny regardless of jaw morphology or feeding habit. In contrast, galeomorphs displayed high morphological heterogeneity. Notably, we document the first report of an extensive internal trabecular network in a non-batoid elasmobranch, observed in Ginglymostoma cirratum , which may serve to resist the mechanical pressures of specialised suction feeding. Furthermore, we identified voussoir tesserae in galeomorphs for the first time, extending their documented presence across all elasmobranch groups, where they display an inverted aspect ratio (wider than tall) compared to mature forms. The durophagous Mustelus mustelus exhibited surprisingly poor TCC development despite being a durophagous feeder, pointing to a pronounced ontogenetic lag. In Squatina oculata , TCC was characterised by large and thick tesserae and extensive fused tesseral regions which may relate to its explosive ambush predation mode, whereas the holocephalan Chimaera exhibited a poorly mineralized, mesh-like structure without resolvable discrete tesserae or trabeculae-matching findings from previous studies. Across all specimens, multi-layered TCC was absent, confirming that multi-layering develops later in ontogeny. These results demonstrate that generalised models of TCC development based on one group or a few taxa fail to capture the broader diversity of TCC morphology. It also opens up many exciting avenues for further study, and forms the basis for comparisons with fossil chondrichthyans, to investigate the evolution of TCC.

Lungfishes are commonly considered to be the sister group to tetrapods. They appeared around 415 million years ago, then rapidly evolved, diversified and spread. Although many morphological characteristics have remained unchanged over the course of their evolution, their skeleton has evolved considerably towards reduced mineralisation. In particular, their fin skeleton has lost ossification in modern forms. In order to study this process of mineralisation loss, we examined the histology of the pectoral fin mesomeres of Glyptolepis groenlandica . This taxon, as a member of the Porolepiformes – i.e. the most rootward clade in the stem group of lungfish – is important for shedding light on the early evolution of the lungfish postcranial skeleton. Here we show that the central core of each mesomere of the pectoral fin of G. groenlandica remains cartilaginous. We hypothesise that the formation of a primary ossification centre is prevented in the skeletal elements of the pectoral fin of G. groenlandica . Instead, the mineralisation process displays a remarkable combination of ossification patterns. Endochondral ossification results from two processes: (1) in the diaphysis of each mesomere, the formation of connective vascular buds from the perichondrium induces endochondral ossification, as seen only in long bones of modern anurans and in the pectoral and pelvic girdles of extant coelacanths; and (2) in the metaphyses, the endochondral ossification replaces the cartilaginous matrix surrounding chondrocyte columns, as seen in early tetrapods and modern amniotes. Because of the basal position of G. groenlandica , our study likely illustrates the primitive state of fin ossification in lungfish and provides a reference point for understanding the steps that led to mineralisation loss in modern lungfishes.

Teeth are a key innovation that underpinned the adaptive radiation of jawed vertebrates; however, their evolutionary origin must lie with the diverse tooth‐like structures of living and fossil jawless vertebrates. Most previous studies have focussed on the extinct stem‐gnathostomes that phylogenetically intercalate the living jawed and jawless vertebrates. The only two extant jawless cyclostome lineages, the lampreys and hagfish bearing keratinous toothlets, have long been overlooked, though they possess complex (but unmineralised) toothlets that some have interpreted as precursors to the teeth of jawed vertebrates. Regardless of whether the toothlets of cyclostomes are homologous or convergent on the teeth of jawed vertebrates, they have the potential to offer unparalleled molecular developmental insights into the evolutionary origin of teeth. To that end, we provide a synthesis of classical literature on cyclostome toothlet structure and development, as a basis for informing future molecular studies, to which we add new insights from X‐ray microtomography of three parasitic lamprey species spanning the breadth of the lamprey crown group. Based on detailed morphological analysis we describe their toothlet replacement mechanism at tissue level and uncover a relationship between toothlet size and the number of replacement cones. All examined species reveal the presence of replacement toothlets, suggesting this replacement mode is a conserved feature of the lamprey crown group. We discuss these results in comparison to hagfish, and conclude that toothlet replacement is a symplesiomorphy of cyclostomes. By describing lamprey toothlet development and replacement and comparing it with gnathostome teeth, this study lays the foundation for research into the development and evolution of teeth and tooth‐like structures across vertebrate lineages.

The endostyle is an endodermal organ unique to nonvertebrate chordates except for lamprey larvae, where it serves as forerunner to the adult thyroid. Here, we examine whether the acquisition of neural crest in the vertebrate lineage played a role in the elaboration of the endostyle. CM-DiI lineage tracing reveals a neural crest contribution to the endostyle, and CRISPR-Cas9 mutagenesis of key neural crest genes causes endostyle defects including formation of a single rather than bilobed structure. RNA sequencing reveals gene profiles characteristic of embryonic neural crest cells and Schwann cell precursors in the developing endostyle. Contrasting with the prevailing view that the endostyle is an endoderm-derived organ, we propose that the acquisition of the neural crest played a critical step in promoting thyroid evolution from chordate endostyle.

Cartilaginous fishes (chondrichthyans: chimeras and elasmobranchs -sharks, skates, and rays) hold a key phylogenetic position to explore the origin and diversifications of jawed vertebrates. Here, we report and integrate reference genomic, transcriptomic, and morphological data in the small-spotted catshark Scyliorhinus canicula to shed light on the evolution of sensory organs. We first characterize general aspects of the catshark genome, confirming the high conservation of genome organization across cartilaginous fishes, and investigate population genomic signatures. Taking advantage of a dense sampling of transcriptomic data, we also identify gene signatures for all major organs, including chondrichthyan specializations, and evaluate expression diversifications between paralogs within major gene families involved in sensory functions. Finally, we combine these data with 3D synchrotron imaging and in situ gene expression analyses to explore chondrichthyan-specific traits and more general evolutionary trends of sensory systems. This approach brings to light, among others, novel markers of the ampullae of Lorenzini electrosensory cells, a duplication hotspot for crystallin genes conserved in jawed vertebrates, and a new metazoan clade of the transient-receptor potential (TRP) family. These resources and results, obtained in an experimentally tractable chondrichthyan model, open new avenues to integrate multiomics analyses for the study of elasmobranchs and jawed vertebrates.

Ankylosaurs were a group of heavily armored non‐avian dinosaurs (Dinosauria, Ankylosauria), represented by a relatively abundant fossil record from the Cretaceous of North and South America. Their dermal skeleton was characterized by large osteoderms whose development and functional role have been largely investigated. However, interstitial small ossicles, forming between these osteoderms, have been far more overlooked and it remains unknown whether they were formed through the ossification of a preexisting fibrous matrix of connective tissue (i.e., metaplasia) or by a cell‐induced differentiation of new fiber bundles followed by mineralization (i.e., neoplasia sensu (Zeitschrift für Wissenschaftliche Zoologie, 1858, 9, 147)). Here, we propose a hypothesis on the developmental origin of these small ossicles in the ankylosaurian Antarctopelta oliveroi using light microcopy, scanning electron microscopy and three‐dimensional virtual histology through propagation phase‐contrast synchrotron radiation micro‐computed tomography (PPC‐SRμCT). Ossicles are located in the dermis. They are composed of two layers: (1) a thin external layer, and (2) a thick basal plate, composed of collagen fiber bundles, which forms the main part of the ossicle. The external layer is made of a smooth, vitreous mineralized tissue that does not look like bone. The basal plate, however, is of osseous origin. In this basal plate, the collagen fiber bundles are organized in two orthogonal systems: one horizontal—observable in cross‐sections—and one vertical—observable in the primary plane of sections sensu (Journal of Vertebrate Paleontology, 2004, 24, 874). The horizontal system is itself composed of successive layers of collagen fiber bundles arranged into an orthogonal plywood‐like structure. The bundles of the vertical system radiate from the center of the ossicle at the level of the transition between the external layer and the basal plate and run towards the periphery of the basal plate. Their thickness increases from the center of the ossicle towards its periphery. Numerous bundles of the vertical system form thin threads that interweave and penetrate within the thick bundles of the horizontal system. Our new data suggest that the ossicles were at least partially formed by metaplasia, that is, through the ossification of a preexisting fibrous matrix of connective tissue. This process was probably supplemented by a cell‐induced differentiation of new fiber bundles laid down prior to their incorporation into the fibrous system and its mineralization. This process looks more akin to neoplasia sensu (Zeitschrift für Wissenschaftliche Zoologie, 1858, 9, 147) than to metaplasia. Consequently, metaplastic and neoplastic processes may coexist in these ossicles with a possible differential expression during ontogeny.

Our study shows that the von Mises stress, induced by external load on the humerus of Eusthenopteron, dissipates through the cortex, trabeculae and the muscles of the pectoral appendage involved in elevation and protraction. As Eusthenopteron’s microanatomy is similar to that of Devonian tetrapods, we expect them to share the same process of load dissipation and energy absorption through 1) cortical stress distribution; and 2) longitudinal trabecular conduction. Our FE simulations in hypothetical terrestrial conditions demonstrate that this type of microanatomical architecture could withstand the weight of Tiktaalik proportionally to the size of Eusthenopteron in standing posture. This tubular arrangement, including marrow processes originally involved in long-bone elongation, would have acquired a key secondary biomechanical function to increase the resistance and strength of the cancellous bone to external compressive load. As an exaptation, this specific trabecular architecture may have played a major role in the tetrapod land exploration about 400 million years ago.

Jensen et al . ( 1 ) question evidence presented of a chambered heart within placoderms, citing its small size and apparently ventral atrium. However, they fail to note the belly-up orientation of the placoderm within one nodule, and the variability of heart morphology within extant taxa. Thus, we remain confident in our interpretation of the mineralized organ as the heart.

Detailed histological analyses are desirable for zebrafish mutants that are models for human skeletal diseases, but traditional histological techniques are limited to two-dimensional thin sections with orientations highly dependent on careful sample preparation. On the other hand, techniques that provide three-dimensional (3D) datasets including µCT scanning are typically limited to visualizing the bony skeleton and lack histological resolution. We combined diffusible iodine-based contrast enhancement (DICE) and propagation phase-contrast synchrotron radiation micro-computed tomography (PPC-SRµCT) to image late larval and juvenile zebrafish, obtaining high-quality 3D virtual histology datasets of the mineralized skeleton and surrounding soft tissues. To demonstrate this technique, we used virtual histological thin sections and 3D segmentation to qualitatively and quantitatively compare wild-type zebrafish and nkx3.2^-/- mutants to characterize novel soft-tissue phenotypes in the muscles and tendons of the jaw and ligaments of the Weberian apparatus, as well as the sinus perilymphaticus associated with the inner ear. We could observe disrupted fiber organization and tendons of the adductor mandibulae and protractor hyoideus muscles associated with the jaws, and show that despite this, the overall muscle volumes appeared unaffected. Ligaments associated with the malformed Weberian ossicles were mostly absent in nkx3.2^-/- mutants, and the sinus perilymphaticus was severely constricted or absent as a result of the fused exoccipital and basioccipital elements. These soft-tissue phenotypes have implications for the physiology of nkx3.2^-/- zebrafish, and demonstrate the promise of DICE-PPC-SRµCT for histopathological investigations of bone-associated soft tissues in small-fish skeletal disease models and developmental studies more broadly.