Dermal Bones Research Articles

Development of the cranial lateral line system of Brook Trout, Salvelinus fontinalis (Teleostei: Salmonidae): Evolutionary and ecological implications.

The mechanosensory lateral line (LL) system of salmonid fishes has been the focus of comparative morphological studies and behavioral and physiologicalanalyses of flow sensing capabilities, but its morphology and development have not been studied in detail in any one species. Here, we describe the post-embryonic development of the cranial LL system in Brook Trout, Salvelinus fontinalis, using vital fluorescent staining (4-Di-2-ASP), scanning electron microscopy, µCT, and clearing and staining to visualize neuromasts and the process of cranial LL canal morphogenesis. We examined the relationship between the timing of LL development, the prolonged life history of salmonids, and potential ecological implications. The LL system is composed of seven canals containing canal neuromasts (CNs) and four lines of superficial neuromasts (SNs) on the skin. CNs and SNs increase in number and size during the alevin (larval) stage. CN number stabilizes as canal morphogenesis commences, but SN number increases well into the parr (juvenile) stage. CNs become larger and more elongated than SNs, but the relative area occupied by sensory hair cells decreases during ontogeny in both types of neuromasts. Neuromast-centered canal morphogenesis starts in alevins (yolk sac larvae), as they swim up into the water column from their gravel nests (~4 months post-fertilization), after which yolk sac absorption is completed and exogenous feeding begins. Canal morphogenesis proceeds asynchronously within and among canal series and is not complete until ~8 months post-fertilization (the parr stage). Three characters in the LL system and associated dermal bones were used to identify their homologs in other actinopterygians and to consider the evolution of LL canal reduction, thus demonstrating the value of salmonidsfor the study of LL evolution. The prolonged life history of Brook Trout and the onset of canal morphogenesis at swim-up are predicted to have implications for neuromast function at these critical behavioral and ecological transitions.

Development of Skin and Dermal Bone of the Shell in <i>Pelodiscus maackii</i> (Testudines: Trionychidae)

The article is devoted to the study of the ontogenetic development of the skin and the structure of the skin-bone shell of the trionychid turtle Pelodiscus maackii (Brandt, 1858). The process of formation of the epidermis and dermis, as well as their derivatives (epidermal scales and dermal ossifications), is described. The development of epidermal scales in Pelodiscus maackii has been shown to be similar to the development of asymmetric scales in other reptiles. Five stages of formation of the definitive structure of the dermal bones have been identified. Structural differences between the internal and external parts of the bony plates (rod-shaped elements and bony callosities) are due to the previous stratification of the dermis into histologically distinct strata: hypodermal, fibrous and papillary. In particular, the fibrous stratum, consisting of several layers of orthogonally oriented collagen bundles, is a precursor to the plywood-like structure of the trionychid bony plates. The growth of bone tissue around the vascular network in the papillary layer is the reason for the formation of surface ornamentation of the trionychid callosities. Some questions of the evolutionary significance of the unique features of the trionychid shell are discussed.

ФЛУКТУИРУЮЩАЯ АСИММЕТРИЯ СЕЙСМОСЕНСОРНЫХ КАНАЛОВ ГОЛОВЫ У ВЕРХОВКИ LEUCASPIUS DELINEATUS (HECKEL, 1843) ИЗ МАЛЫХ ПРУДОВ С РАЗНЫМИ ВЕЛИЧИНАМИ МИНЕРАЛИЗАЦИИ ВОДЫ И СОДЕРЖАНИЯ КАТИОНОВ ЖЕСТКОСТИ

Background. The change in external conditions is accompanied by various responses of organisms existing in an unstable and heterogeneous environment. Relevance. Under the influence of physico-chemical factors of the aquatic environment, the frequency and extent of asymmetric manifestations of bilateral morphological features in fish individuals may increase, which is a consequence of an imbalance in their development and reflects the level of environmental stress. The aim of this work was to examine the fluctuating asymmetry (FA) in the number of pores in the head seismosensory canals of belica Leucaspius delineatus (sunbleak) from the water bodies that differ in the content of total dissolved solids (water mineralization) and hardness cations. Methods. Ichthyological material was collected in nine small ponds belonging to the Kama River basin in the Udmurt Republic in 2018-2019. In the captured individuals of belica, the number of pores in the seismosensory canals in four paired head dermal bones has been identified, on the basis of which two FA indices have been calculated. Results. In several water bodies, a considerable proportion of belica mature individuals with incompletely ossified sections of the head seismosensory canals, often on one side only, or on the both sides but to different extents, has been recorded. This proportion determined the FA indices of the studied seismosensory features and had a positive correlation with the content of magnesium ions in the water bodies. In the belica individuals with fully ossified head seismosensory canals, the FA variance of the total number of pores in these canals had a positive correlation with the total dissolved solids in the water of the investigated small ponds; however, it had not depended on the content of hardness cations in them. Conclusion. The possible mechanisms of the influence of the investigated hydrochemical factors on the morphogenetic processes sequentially occurring in the belica early development - anlage of the raws of primary head neuromasts in its larvae and the subsequent formation of the ossified sections of the head seismosensory canals in its fry - are discussed.

Embryonic cranial cartilage defects in the Fgfr3Y367C /+ mouse model of achondroplasia.

Achondroplasia, the most common chondrodysplasia in humans, is caused by one of two gain of function mutations localized in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) leading to constitutive activation of FGFR3 and subsequent growth plate cartilage and bone defects. Phenotypic features of achondroplasia include macrocephaly with frontal bossing, midface hypoplasia, disproportionate shortening of the extremities, brachydactyly with trident configuration of the hand, and bowed legs. The condition is defined primarily on postnatal effects on bone and cartilage, and embryonic development of tissues in affected individuals is not well studied. Using the Fgfr3Y367C/+ mouse model of achondroplasia, we investigated the developing chondrocranium and Meckel's cartilage (MC) at embryonic days (E)14.5 and E16.5. Sparse hand annotations of chondrocranial and MC cartilages visualized in phosphotungstic acid enhanced three-dimensional (3D) micro-computed tomography (microCT) images were used to train our automatic deep learning-based 3D segmentation model and produce 3D isosurfaces of the chondrocranium and MC. Using 3D coordinates of landmarks measured on the 3D isosurfaces, we quantified differences in the chondrocranium and MC of Fgfr3Y367C/+ mice relative to those of their unaffected littermates. Statistically significant differences in morphology and growth of the chondrocranium and MC were found, indicating direct effects of this Fgfr3 mutation on embryonic cranial and pharyngeal cartilages, which in turn can secondarily affect cranial dermal bone development. Our results support the suggestion that early therapeutic intervention during cartilage formation may lessen the effects of this condition.

Quantitative Live Imaging of Zebrafish Scale Regeneration: From Adult Fish to Signaling Patterns and Tissue Flows.

In regeneration, a damaged body part grows back to its original form. Understanding the mechanisms and physical principles underlying this process has been limited by the difficulties of visualizing cell signals and behaviors in regeneration. Zebrafish scales are emerging as a model system to investigate morphogenesis during vertebrate regeneration using quantitative live imaging. Scales are millimeter-sized dermal bone disks forming a skeletal armor on the body of the fish. The scale bone is deposited by an adjacent monolayer of osteoblasts that, after scale loss, regenerates in about 2weeks. This intriguing regenerative process is accessible to live confocal microscopy, quantifications, and mathematical modeling. Here, I describe methods to image scale regeneration live, tissue-wide and at sub-cellular resolution. Furthermore, I describe methods to process the resulting images and quantify cell, tissue, and signal dynamics.

Come together over me: Cells that form the dermatocranium and chondrocranium in mice.

Most bone develops either by intramembranous ossification where bone forms within a soft connective tissue, or by endochondral ossification by way of a cartilage anlagen or model. Bones of the skull can form endochondrally or intramembranously or represent a combination of the two types of ossification. Contrary to the classical definition of intramembranous ossification, we have previously described a tight temporo-spatial relationship between cranial cartilages and dermal bone formation and proposed a mechanistic relationship between chondrocranial cartilage and dermal bone. Here, we further investigate this relationship through an analysis of how cells organize to form cranial cartilages and dermal bone. Using Wnt1-Cre2 and Mesp1-Cre transgenic mice, we determine the derivation of cells that comprise cranial cartilages from either cranial neural crest (CNC) or paraxial mesoderm (PM). We confirm a previously determined CNC-PM boundary that runs through the hypophyseal fenestra in the cartilaginous braincase floor and identify four additional CNC-PM boundaries in the chondrocranial lateral wall, including a boundary that runs along the basal and apical ends of the hypochiasmatic cartilage. Based on the knowledge that as osteoblasts differentiate from CNC- and PM-derived mesenchyme, the differentiating cells express the transcription factor genes RUNX2 and osterix (OSX), we created a new transgenic mouse line called R2Tom. R2Tom mice carry a tdTomato reporter gene joined with an evolutionarily well-conserved enhancer sequence of RUNX2. R2Tom mice crossed with Osx-GFP mice yield R2Tom;Osx-GFP double transgenic mice in which various stages of osteoblasts and their precursors are detected with different fluorescent reporters. We use the R2Tom;Osx-GFP mice, new data on the cell derivation of cranial cartilages, histology, immunohistochemistry, and detailed morphological observations combined with data from other investigators to summarize the differentiation of cranial mesenchyme as it forms condensations that become chondrocranial cartilages and associated dermal bones of the lateral cranial wall. These data advance our previous findings of a tendency of cranial cartilage and dermal bone development to vary jointly in a coordinated manner, promoting a role for cranial cartilages in intramembranous bone formation.

Evidence of artefacts made of giant sloth bones in central Brazil around the last glacial maximum.

The peopling of the Americas and human interaction with the Pleistocene megafauna in South America remain hotly debated. The Santa Elina rock shelter in Central Brazil shows evidence of successive human settlements from around the last glacial maximum (LGM) to the Early Holocene. Two Pleistocene archaeological layers include rich lithic industry associated with remains of the extinct giant ground sloth Glossotherium phoenesis. The remains include thousands of osteoderms (i.e. dermal bones), three of which were human-modified. In this study, we perform a traceological analysis of these artefacts by optical microscopy, non-destructive scanning electron microscopy, UV/visible photoluminescence and synchrotron-based microtomography. We also describe the spatial association between the giant sloth bone remains and stone tools and provide a Bayesian age model that confirms the timing of this association in two time horizons of the Pleistocene in Santa Elina. The conclusion from our traceological study is that the three giant sloth osteoderms were intentionally modified into artefacts before fossilization of the bones. This provides additional evidence for the contemporaneity of humans and megafauna, and for the human manufacturing of personal artefacts on bone remains of ground sloths, around the LGM in Central Brazil.

A fish for Phoebe: a new actinopterygian from the Upper Carboniferous Coal Measures of Saddleworth, Greater Manchester, UK, and a revision of Kansasiella eatoni

Abstract The origin of crown Actinopterygii has been dated to near the Devonian-Carboniferous boundary, but poor phylogenetic signal occludes the origin of modern actinopterygian biodiversity. Scarcity of comparative endoskeletal anatomy from this period likely contributes to the uncertainty. For example, only a handful of neurocrania have been described from the Carboniferous period despite an abundance of fossil taxa. Here we present a new actinopterygian, Phoebeannaia mossae gen. et sp. nov., from the Bashkirian Coal Measures of Saddleworth, Greater Manchester, UK, represented by an exceptionally preserved neurocranium and associated dermal bones. Morphological similarities to the Virgilian Kansasiella eatoni prompted comparative study and construction of a new neurocranial character matrix. Subsequent systematic analysis enabled identification of a cluster of similar Late Palaeozoic neurocrania, exploration of trends in braincase evolution across time, and investigation of potential causes of cladistic instability. Our neurocranial dataset reliably places this new specimen on the neopterygian stem near Kansasiella, but it struggles to incorporate extant taxa such as Polypteriformes, leading to uncertainty in branching pattern and inferred neurocranial transformation series. This work triples the number of complete Carboniferous neurocrania described with modern microcomputed tomography and provides a framework for future testing of large-scale hypotheses regarding the diversification and origin of ray-finned fishes.

Mef2ca and mef2cb Double Mutant Zebrafish Show Altered Craniofacial Phenotype and Motor Behaviour.

The transcription factor MEF2C is crucial in neuronal, cardiac, bone and cartilage molecular processes, as well as for craniofacial development. MEF2C was associated with the human disease MRD20, whose patients show abnormal neuronal and craniofacial development. Zebrafish mef2ca;mef2cb double mutants were analysed for abnormalities in craniofacial and behaviour development through phenotypic analysis. Quantitative PCR was performed to investigate the expression levels of neuronal marker genes in mutant larvae. The motor behaviour was analysed by the swimming activity of 6 dpf larvae. We found that mef2ca;mef2cb double mutants display several abnormal phenotypes during early development, including those already described in zebrafish carrying mutations in each paralog, but also (i) a severe craniofacial phenotype (comprising both cartilaginous and dermal bone structures), (ii) developmental arrest due to the disruption of cardiac oedema and (iii) clear alterations in behaviour. We demonstrate that the defects observed in zebrafish mef2ca;mef2cb double mutants are similar to those previously described in MEF2C-null mice and MRD20 patients, confirming the usefulness of these mutant lines as a model for studies concerning MRD20 disease, the identification of new therapeutic targets and screening for possible rescue strategies.

Evaluating Dermal Bone as a Novel Source of Endocrine Information in Ninespine and Threespine Stickleback Fish.

Monitoring the physiology of small aquatic and marine teleost fish presents challenges. Blood samples, often the first choice for endocrinologists, can be difficult or even impossible to obtain and alternative matrices currently used for hormone analyses do not occur in fishes (e.g., hair, feathers etc.) or are not easily collected from small aquatic organisms (e.g., urine and feces). Some teleosts, however, have enlarged bony dermal elements that possibly accumulate and store steroid hormones in physiological relevant concentrations. Both threespine stickleback (Gasterosteus aculeatus) and ninespine stickleback (Pungitius pungitius) have a series of external, lateral bony plates, dorsal spines, and a pair of pelvic spines attached to the pelvic girdle. We investigated if cortisol, the primary circulating glucocorticoid in teleosts, could be extracted from stickleback dermal bone and quantified using a commercially available enzyme immunoassay (EIA). We successfully validated a cortisol EIA for dermal bone extracts, determined that cortisol was detectable in both species, and found that dermal bone cortisol levels significantly correlated with cortisol levels in whole body homogenate. Ninespine stickleback had significantly higher dermal bone cortisol concentrations than threespine stickleback and female threespine stickleback tended to have over twice the mean dermal bone cortisol concentration than males. Because both stickleback species are widely used for ecotoxicological studies, using dermal bone as a source of endocrine information, while leaving the body for contaminant, genomic, histological, and stable isotope analyses, could be a powerful and parsimonious tool. Further investigation and physiological validations are necessary to fully understand the utility of this new sample matrix.

Osteohistological characterization of notosuchian osteoderms: Evidence for an overlying thick leathery layer of skin.

Osteoderms are mineralized structures embedded in the dermis, known for nonavian archosaurs, squamates, xenarthrans, and amphibians. Herein, we compared the osteoderm histology of Brazilian Notosuchia of Cretaceous age using three neosuchians for comparative purposes. Microanatomical analyses showed that most of them present a diploe structure similar to those of other pseudosuchians, lizards, and turtles. This structure contains two cortices (the external cortex composed of an outer and an inner layers, and the basal cortex) and a core in-between them. Notosuchian osteoderms show high bone compactness (>0.85) with varying degrees of cancellous bone in the core. The neosuchian Guarinisuchus shows the lowest bone compactness with a well-developed cancellous layer. From an ontogenetic perspective, most tissues are formed through periosteal ossification, although the mineralized tissues observed in baurusuchid LPRP/USP 0634 suggest a late metaplastic development. Histology suggests that the ossification center of notosuchian osteoderm is located at the keel. Interestingly, we identified Sharpey's fibers running perpendicularly to the outer layer of the external cortex in Armadillosuchus arrudai, Itasuchus jesuinoi, and Baurusuchidae (LPRP/USP 0642). This feature indicates a tight attachment within the dermis, and it is evidence for the presence of an overlying thick leathery layer of skin over these osteoderms. These data allow a better understanding of the osteohistological structure of crocodylomorph dermal bones, and highlight their structural diversity. We suggest that the vascular canals present in some sampled osteoderms connecting the inner layer of the external cortex and the core with the external surface may increase osteoderm surface and the capacity of heat transfer in terrestrial notosuchians.

Reinterpreting the work of Charles Breder: Sensory neuromasts and orbital skeleton variation in eyeless Astyanax cavefish

Charles Breder, a pioneering researcher of blind Mexican cavefish was the first to note extreme variation in the facial skeleton of this intriguing subterranean-dwelling organism. Using a system of polar coordinate plots, he identified substantial dysmorphic changes affecting bones of the orbital skeleton. A complication of his landmark publication from 1944 was an error in the number of orbital bones depicted for this species. Intriguingly, however, he proposed an unknown “organizing force” likely influences final bone position and associated dysmorphia. At the time this was merely hypothetical. Roughly eight decades since its publication, however, insights into sensory influences on facial bone development may explain dysmorphia and variation in bone numbers for Astyanax cavefish. A morphological association between mechano-sensory neuromasts of the lateral line and dermal bones of the facial skeleton had been appreciated in the classical literature, but the polarity of this interaction has long remained unclear. Here, we propose that sensory-skeletal integration between sensory neuromasts and bones explain the incomplete numbers of bones, and dysmorphic features such as fusion between neighboring elements. We propose that in closely-related surface fish (and most teleost fish) this developmental coupling enables the sensory and skeletal systems to become integrated into a functional unit over the course of life history. In this opinion article, we discuss the relevance of this (poorly understood) phenomenon as a potential evolutionary source of variation in the facial bone structures of taxa across deep geologic time. We provide three potential explanations for the error in Breder's drawings, that may be explained by natural developmental variation documented in other related species. Moreover, we argue that the natural variation in this “evolutionary” model system is useful for explaining diverse cranial features by uniting aberrations occurring during embryogenesis with long-term adult dysmorphia.

To be or not to be heavier: The role of dermal bones in the buoyancy of the Late Triassic temnospondyl amphibian Metoposaurus krasiejowensis.

Stereospondyli are a clade of large aquatic temnospondyls known to have evolved a large dermal pectoral girdle. Among the Stereospondyli, metoposaurids in particular possess large interclavicles and clavicles relative to the rest of the postcranial skeleton. Because of the large size of these dermal bones, it was first proposed that they served as ballast during hydrostatic buoyancy control which assisted metoposaurids to live a bottom-dwelling mode of life. However, a large bone need not necessarily be heavy, for which determining the bone compactness becomes crucial for understanding any such adaptation in these dermal bones. Previous studies on the evolution of bone adaptations to aquatic lifestyles such as osteosclerosis, pachyostosis, osteoporotic-like pattern and pachyosteosclerosis have been observed in the long bones of secondarily aquatic amniotes. However, there are no known studies on the analysis of bone compactness in the dermal pectoral girdle of non-amniote taxa including Temnospondyli. This study looks at evidence of changes in bone mass adaptations in the dermal bones of the pectoral girdle of two stereospondyls occurring in the Late Triassic Krasiejόw locality (Southwestern Poland), namely: Metoposaurus krasiejowensis and Cyclotosaurus intermedius. However, because of lack of research on bone compactness of temnospondyls in general, there is no existent frame of reference to infer bone mass increase (BMI) in the M. krasiejowensis samples, and thus the bone compactness results of this taxon are compared with that of the samples of C. intermedius. Results of this study indicate that the interclavicles of M. krasiejowensis partially evolved BMI-like condition rendering these bones to be heavy enough to get selected as ballast during hydrostatic buoyancy control. Additionally, M. krasiejowensis shared its habitat with C. intermedius, however, the dermal pectoral girdle sample of the latter taxon does not display signs of BMI-like condition. Furthermore, the absence of variation in hydrostatic buoyancy control in the ontogenetic series of interclavicles of M. krasiejowensis could imply lack of ontogenetic niche shift along the water column.

Developmental osteology of Ictalurus punctatus and Noturus gyrinus (Siluriformes: Ictaluridae) with a discussion of siluriform bone homologies

Abstract The skeleton of Siluriformes is characterized by several autapomorphies, including secondary absence, extreme modification, and purported fusion of several ossifications. Although well documented in adults, information on skeletal development in catfishes is relatively sparse and typically focused on particular regions of the skeleton (e.g., Weberian apparatus). To further our understanding of the siluriform skeleton, I document the development of the entire skeleton in two ictalurid species, Ictalurus punctatus (channel catfish) and Noturus gyrinus (tadpole madtom) from five days pre-hatch to adult. I reexamine the homologies of bones previously hypothesized to represent compound elements in catfishes as well as an additional element only known to occur in some ictalurids. Development of the skeleton is complete in I. punctatus at 22.4 mm SL and almost complete in N. gyrinus (except dorsal- and anal-fin distal radials) at 14.1 mm SL. No signs of ontogenetic fusion were observed in any of the purported compound elements. Previous hypotheses of the homology of these elements and of additional ossifications are reviewed in light of developmental information obtained herein. No dermal parietal component is present at any stage in the so-called parieto-supraoccipital. The bone is the supraoccipital which ossifies from two lateral centers of ossification which later fuse, rather than from a median center. The ‘posttemporo-supracleithrum’ originates from a single center of ossification and represents the supracleithrum. The posttemporal is present in ictalurids and many other catfishes as a canal-bearing bone between the supracleithrum and the pterotic, a bone sometimes identified as the extrascapular. The extrascapular is missing in catfishes. Ictalurids have an additional dermal bone above the posttemporal, which is either an independently ossifying fragment of the posttemporal or a neoformation restricted to some members of this family. The single chondral bone of the pectoral girdle originates from a single center of ossification that represents the coracoid. The scapula is missing in catfishes. Dorsal-fin distal radial 2 is absent in catfishes and the foramen of dorsal-fin spine 2 is formed from modifications to the base of the fin-ray itself. Unlike loricarioid catfishes, the urohyal of ictalurids originates solely as an ossification of the sternohyoideus tendons. The anteriormost infraorbital element ossifies from a single center of ossification around the infraorbital sensory canal and represents the lacrimal. The antorbital is missing in catfishes. Finally, skeletal development of I. punctatus is compared to that available for other otophysans, including the cypriniforms Danio rerio and Enteromius holotaenia and the characiform Salminus brasiliensis.

A dysmorphic mouse model reveals developmental interactions of chondrocranium and dermatocranium.

The cranial endo and dermal skeletons, which comprise the vertebrate skull, evolved independently over 470 million years ago and form separately during embryogenesis. In mammals, much of the cartilaginous chondrocranium is transient, undergoing endochondral ossification or disappearing, so its role in skull morphogenesis is not well studied and it remains an enigmatic structure. We provide complete 3D reconstructions of the laboratory mouse chondrocranium from embryonic day (E) 13.5 through E17.5 using a novel methodology of uncertainty-guided segmentation of phosphotungstic enhanced 3D micro-computed tomography images with sparse annotation. We evaluate the embryonic mouse chondrocranium and dermatocranium in 3D, and delineate the effects of a Fgfr2 variant on embryonic chondrocranial cartilages and on their association with forming dermal bones using the Fgfr2cC342Y/+ Crouzon syndrome mouse. We show that the dermatocranium develops outside of and in shapes that conform to the chondrocranium. Results reveal direct effects of the Fgfr2 variant on embryonic cartilage, on chondrocranium morphology, and on the association between chondrocranium and dermatocranium development. Histologically, we observe a trend of relatively more chondrocytes, larger chondrocytes, and/or more matrix in the Fgfr2cC342Y/+ embryos at all timepoints before the chondrocranium begins to disintegrate at E16.5. The chondrocrania and forming dermatocrania of Fgfr2cC342Y/+ embryos are relatively large, but a contrasting trend begins at E16.5 and continues into early postnatal (P0 and P2) timepoints, with the skulls of older Fgfr2cC342Y/+ mice reduced in most dimensions compared to Fgfr2c+/+ littermates. Our findings have implications for the study and treatment of human craniofacial disease, for understanding the impact of chondrocranial morphology on skull growth, and potentially on the evolution of skull morphology.

Morphology of Palaeospondylus shows affinity to tetrapod ancestors.

Palaeospondylus gunni, from the Middle Devonian period, is one of the most enigmatic fossil vertebrates, and its phylogenetic position has remained unclear since its discovery in Scotland in 1890(ref.1). The fossil's strange set of morphological features has made comparisons with known vertebrate morphotype diversity difficult. Here we use synchrotron radiation X-ray micro-computed tomography to show that Palaeospondylus was a sarcopterygian, and most probably a stem-tetrapod. The skeleton of Palaeospondylus consisted solely of endoskeletal elements in which hypertrophied chondrocyte cell lacunae, osteoids and a small fraction of perichondral bones developed. Despite the complete lack of teeth and dermal bones, the neurocranium of Palaeospondylus resembles those of stem-tetrapod Eusthenopteron2 and Panderichthys3, and phylogenetic analyses place Palaeospondylus in between them. Because the unique features of Palaeospondylus, such as the cartilaginous skeleton and the absence of paired appendages, are present in the larva of crown tetrapods, our study highlights an unanticipated heterochronic evolution at the root of tetrapods.

Mechanisms of dermal bone repair after predatory attack in the giant stem-group teleost Leedsichthys problematicus Woodward, 1889a (Pachycormiformes).

Leedsichthys problematicus is a suspension-feeding member of the Mesozoic clade Pachycormiformes (stem-group Teleostei), and the largest known ray-finned fish (Actinopterygii). As in some larger fish, the skeleton is poorly ossified, but the caudal fin (tail) is well-preserved. Bony calluses have been found here, on the dermal fin rays, and when sectioned, show evidence of bone repair in response to damage. As part of this repair, distinctive tissue changes are observed, including the deposition of woven bone onto broken bone fragments and the surface of the lepidotrichium, after resorption of the edges of these fragments and the lepidotrichial surface itself. Within the woven bone are many clear elongate spaces, consistent with their interpretation as bundles of unmineralized collagen (Sharpey's fibres). These normally provide attachment within dermal bones, and here attach new bone to old, particularly to resorbed surfaces, identified by scalloped reversal lines. Haversian systems are retained in the old bone, from which vasculature initially invaded the callus, hence bringing stem cells committed to forming bone onto the surfaces of the damaged area. These observations provide strong evidence of a vital response through survival of a predatory attack by a large marine reptile, coeval with Leedsichthys in the Jurassic seas.

The Mesozoic terminated in boreal spring

The Cretaceous–Palaeogene mass extinction around 66 million years ago was triggered by the Chicxulub asteroid impact on the present-day Yucatán Peninsula1,2. This event caused the highly selective extinction that eliminated about 76% of species3,4, including all non-avian dinosaurs, pterosaurs, ammonites, rudists and most marine reptiles. The timing of the impact and its aftermath have been studied mainly on millennial timescales, leaving the season of the impact unconstrained. Here, by studying fishes that died on the day the Mesozoic era ended, we demonstrate that the impact that caused the Cretaceous–Palaeogene mass extinction took place during boreal spring. Osteohistology together with stable isotope records of exceptionally preserved perichondral and dermal bones in acipenseriform fishes from the Tanis impact-induced seiche deposits5 reveal annual cyclicity across the final years of the Cretaceous period. Annual life cycles, including seasonal timing and duration of reproduction, feeding, hibernation and aestivation, vary strongly across latest Cretaceous biotic clades. We postulate that the timing of the Chicxulub impact in boreal spring and austral autumn was a major influence on selective biotic survival across the Cretaceous–Palaeogene boundary.

Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition.

The teeth of sharks famously form a series of transversely organized files with a conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here, we use tomographic methods to investigate mandibular structures in several early branching ‘acanthodian’-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckel's cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly.

Phenotypic Variation Through Ontogeny: Thyroid Axis Disruption During Larval Development in the Frog Pleurodema borellii

Studies of the effects of thyroid hormones on larval development in the frog Xenopus spp. have provided baseline information to identify developmental constraints and elucidate genetic and hormonal mechanisms driving development, growth, and life history transitions. However, this knowledge requires data based on other anurans to complete a comprehensive approach to the understanding of larval developmental diversity and phenotypic variation through ontogeny. Mesocosm experiments provide realistic data about environmental conditions and timing; this information is useful to describe anuran larval development and/or analyze endocrine disruption. In this study, mesocosm experiments of the larval development of the frog Pleurodema borellii were conducted to explore the consequences of thyroid axis disruption; the sensitivity of tadpoles to the methimazole (2.66 mg/l) and thyroxine (T4) (1.66 μg/l) was compared. These concentrations were selected based on previous studies in Pleurodema borellii. We test the effects of methimazole and thyroxine on development in early exposure (from beginning of larval development) and late exposure, 18 days after hatching, with doses administered every 48 h. Tadpoles were evaluated 31 days after hatching. Methimazole caused moderate hypertrophy of the thyroid gland, alteration in the growth rates, differentiation without inhibition of development, and an increase of developmental variability. Thyroxine produced slight atrophy of the thyroid gland, accelerated growth rates and differentiation, and minor developmental variability. In tadpoles at stages previous to metamorphose, skull development (differentiation of olfactory capsules, appearance of dermal bones, and cartilage remodeling) seemed to be unaltered by the disruptors. Moreover, similar abnormal morphogenesis converged in specimens under methimazole and thyroxine exposures. Abnormalities occurred in pelvic and pectoral girdles, and vent tube, and could have been originated at the time of differentiation of musculoskeletal tissues of girdles. Our results indicate that premetamorphic stages (Gosner Stages 25–35) are sensitive to minimal thyroid axis disruption, which produces changes in developmental rates; these stages would also be critical for appendicular musculoskeletal morphogenesis to achieve the optimal condition to start metamorphosis.