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Jawed vertebrates have inner ears with three semicircular canals, the presence of which has been used as a key to understanding evolutionary relationships. Ostracoderms, the jawless stem gnathostomes, had only two canals and lacked the lateral canal 1 – 3 . Lampreys, which are modern cyclostomes, are generally thought to possess two semicircular canals whereas the hagfishes—which are also cyclostomes—have only a single canal, which used to be regarded as a more primitive trait 1 , 4 . However, recent molecular and developmental analyses have strongly supported the monophyly of cyclostomes 5 – 7 , which has left the evolutionary trajectory of the vertebrate inner ear unclear 8 . Here we show the differentiation of the otic vesicle of the lamprey Lethenteron camtschaticum and inshore hagfish Eptatretus burgeri . This is the first time, to our knowledge, that the development of the hagfish inner ear is reported. We found that canal development in the lamprey starts with two depressions—which is reminiscent of the early developmental pattern of the inner ear in modern gnathostomes. These cyclostome otic vesicles show a pattern of expression of regulatory genes, including OTX genes, that is comparable to that of gnathosomes. Although two depressions appear in the lamprey vesicle, they subsequently fuse to form a single canal that is similar to that of hagfishes. Complete separation of the depressions results in anterior and posterior canals in gnathostomes. The single depression of the vesicle in hagfishes thus appears to be a secondarily derived trait. Furthermore, the lateral canal in crown gnathostomes was acquired secondarily—not by de novo acquisition of an OTX expression domain, but by the evolution of a developmental program downstream of the OTX genes. The differentiation of the inner ear in the lamprey Lethenteron camtschaticum and hagfish Eptatretus burgeri sheds light on the evolution of the semicircular canals of jawed vertebrates.

Sharks are one of the most threatened groups of marine animals worldwide, mostly owing to overfishing and habitat degradation/loss. Although these cartilaginous fish have evolved to fill many ecological niches across a wide range of habitats, they have limited capability to rapidly adapt to human-induced changes in their environments. Contrary to global warming, ocean acidification was not considered as a direct climate-related threat to sharks. Here we show, for the first time, that an early ontogenetic acclimation process of a tropical shark (Chiloscyllium punctatum) to the projected scenarios of ocean acidification (ΔpH = 0.5) and warming (+4°C; 30°C) for 2100 elicited significant impairments on juvenile shark condition and survival. The mortality of shark embryos at the present-day thermal scenarios was 0% both at normocapnic and hypercapnic conditions. Yet routine metabolic rates (RMRs) were significantly affected by temperature, pH and embryonic stage. Immediately after hatching, the Fulton condition of juvenile bamboo sharks was significantly different in individuals that experienced future warming and hypercapnia; 30 days after hatching, survival rapidly declined in individuals experiencing both ocean warming and acidification (up to 44%). The RMR of juvenile sharks was also significantly affected by temperature and pH. The impact of low pH on ventilation rates was significant only under the higher thermal scenario. This study highlights the need of experimental-based risk assessments of sharks to climate change. In other words, it is critical to directly assess risk and vulnerability of sharks to ocean acidification and warming, and such effort can ultimately help managers and policy-makers to take proactive measures targeting most endangered species.

The amniote middle ear is a classical example of the evolutionary novelty. Although paleontological evidence supports the view that mammals and diapsids (modern reptiles and birds) independently acquired the middle ear after divergence from their common ancestor, the developmental bases of these transformations remain unknown. Here we show that lower-to-upper jaw transformation induced by inactivation of the Endothelin1-Dlx5/6 cascade involving Goosecoid results in loss of the tympanic membrane in mouse, but causes duplication of the tympanic membrane in chicken. Detailed anatomical analysis indicates that the relative positions of the primary jaw joint and first pharyngeal pouch led to the coupling of tympanic membrane formation with the lower jaw in mammals, but with the upper jaw in diapsids. We propose that differences in connection and release by various pharyngeal skeletal elements resulted in structural diversity, leading to the acquisition of the tympanic membrane in two distinct manners during amniote evolution. The evolution of the amniote middle ear remains unclear. Here, the authors show that inactivation of the Edn1-Dlx5/6 cascade during development results in loss of the tympanic membrane in mouse and duplication in chicken, which suggests independent evolution of the tympanic membrane in different amniotes.

Shark and ray (elasmobranch) dentitions are well known for their multiple generations of teeth, with isolated teeth being common in the fossil record. However, how the diverse dentitions characteristic of elasmobranchs form is still poorly understood. Data on the development and maintenance of the dental patterning in this major vertebrate group will allow comparisons to other morphologically diverse taxa, including the bony fishes, in order to identify shared pattern characters for the vertebrate dentition as a whole. Data is especially lacking from the Batoidea (skates and rays), hence our objective is to compile data on embryonic and adult batoid tooth development contributing to ordering of the dentition, from cleared and stained specimens and micro-CT scans, with 3D rendered models. We selected species (adult and embryonic) spanning phylogenetically significant batoid clades, such that our observations may raise questions about relationships within the batoids, particularly with respect to current molecular-based analyses. We include developmental data from embryos of recent model organisms Leucoraja erinacea and Raja clavata to evaluate the earliest establishment of the dentition. Characters of the batoid dentition investigated include alternate addition of teeth as offset successional tooth rows (versus single separate files), presence of a symphyseal initiator region (symphyseal tooth present, or absent, but with two parasymphyseal teeth) and a restriction to tooth addition along each jaw reducing the number of tooth families, relative to addition of successor teeth within each family. Our ultimate aim is to understand the shared characters of the batoids, and whether or not these dental characters are shared more broadly within elasmobranchs, by comparing these to dentitions in shark outgroups. These developmental morphological analyses will provide a solid basis to better understand dental evolution in these important vertebrate groups as well as the general plesiomorphic vertebrate dental condition.

How genetic changes are linked to morphological novelties and developmental constraints remains elusive. Here, we investigate genetic apparatuses that distinguish fish fins from tetrapod limbs by analyzing transcriptomes and open-chromatin regions (OCRs). Specifically, we compared mouse forelimb buds with the pectoral fin buds of an elasmobranch, the brown-banded bamboo shark ( Chiloscyllium punctatum ). A transcriptomic comparison with an accurate orthology map revealed both a mass heterochrony and hourglass-shaped conservation of gene expression between fins and limbs. Furthermore, open-chromatin analysis suggested that access to conserved regulatory sequences is transiently increased during mid-stage limb development. During this stage, stage-specific and tissue-specific OCRs were also enriched. Together, early and late stages of fin/limb development are more permissive to mutations than middle stages, which may have contributed to major morphological changes during the fin-to-limb evolution. We hypothesize that the middle stages are constrained by regulatory complexity that results from dynamic and tissue-specific transcriptional controls. Animals come in all shapes and sizes. This diversity arose through genetic mutations during evolution, but it is unclear exactly how these variations led to the formation of new shapes. There is increasing evidence to suggest that not all shapes are possible and that variability between animals is limited by a phenomenon known as “developmental constraint”. These limitations direct parts of the body towards a specific shape as they develop in the embryo. Therefore, understanding the mechanisms underlying these developmental constraints could help explain how different body shapes evolved. The limbs of humans and other mammals evolved from the fins of fish, and this transition is often used to study the role developmental constraints play in evolution. This is an ideal model as there is already a detailed fossil record mapping this evolutionary event, and data pinpointing some of the genes involved in the development of limbs and fins. But this data is incomplete, and a full comparison between the genes activated in the fin and the limb during embryonic development had not been achieved. This is because most fish used for research have undergone recent genetic changes, making it hard to spot which genetic differences are linked to the evolution of the limb. To overcome this barrier, Onimaru et al. compared genetic data from the developing limbs of mice to the developing fins of the brown-banded bamboo shark, which evolves much slower than other fish. This revealed that although many genes commonly played a role in the development of the fin and the limb in the embryo, the activity of these shared genes was not the same. For example, genes that switched on in the late stages of limb development, switched off in the late stages of fin development. But in the middle of development, those differences were relatively small and both species activated very similar sets of genes. Many of these genes were pleiotropic, which means they have important roles in other tissues and therefore mutate less often. This suggests that the mid-stage of limb development is under the strongest level of constraint. Darwin’s theory of natural selection explains that mutations drive evolution. But the theory cannot predict what kinds of new body shapes new mutations will produce. Understanding how the activity levels of different genes affect development could help to fill this knowledge gap. This has potential medical applications, for example, understanding why some genetic changes cause more serious problems than others. This work suggests that mutations in genes that are active during the mid-stage of limb development may have the most serious impact.

Background The gene regulatory network involved in tooth morphogenesis has been extremely well described in mammals and its modeling has allowed predictions of variations in regulatory pathway that may have led to evolution of tooth shapes. However, very little is known outside of mammals to understand how this regulatory framework may also account for tooth shape evolution at the level of gnathostomes. In this work, we describe expression patterns and proliferation/apoptosis assays to uncover homologous regulatory pathways in the catshark Scyliorhinus canicula . Results Because of their similar structural and developmental features, gene expression patterns were described over the four developmental stages of both tooth and scale buds in the catshark. These gene expression patterns differ from mouse tooth development, and discrepancies are also observed between tooth and scale development within the catshark. However, a similar nested expression of Shh and Fgf suggests similar signaling involved in morphogenesis of all structures, although apoptosis assays do not support a strictly equivalent enamel knot system in sharks. Similarities in the topology of gene expression pattern, including Bmp signaling pathway, suggest that mouse molar development is more similar to scale bud development in the catshark. Conclusions These results support the fact that no enamel knot, as described in mammalian teeth, can be described in the morphogenesis of shark teeth or scales. However, homologous signaling pathways are involved in growth and morphogenesis with variations in their respective expression patterns. We speculate that variations in this topology of expression are also a substrate for tooth shape evolution, notably in regulating the growth axis and symmetry of the developing structure.

Background Skeletal mineralisation, achieved through the controlled precipitation of calcium phosphate, represents a major evolutionary innovation in early vertebrates. While this process has been extensively studied in bony species, it remains poorly understood in cartilaginous fishes, which lack endochondral bone. In this study, the small-spotted catshark ( Scyliorhinus canicula ) was used as a model to investigate the genetic basis of cartilage differentiation and subsequent mineralisation. Results Two stages in late embryogenesis were examined: one preceding and one following the onset of vertebral mineralisation. Using a bulk RNA sequencing approach, we identified a set of genes characterising the early differentiation of chondrocytes and cartilage synthesis, shared among jawed vertebrates. Strikingly, only a very limited number of genes previously identified in mammalian skeletogenesis showed upregulated expression in the catshark mineralising chondrocytes. Among these, particular attention was given to the spp2 (secreted phosphoprotein 2) gene family. Unlike in bony vertebrates, where spp2 exists as a single and rather little-studied gene, cartilaginous fishes possess multiple gene duplicates with contrasted sites of expression. Through phylogenetic analyses and gene expression studies in the catshark, we showed how, despite the presence of shared molecular components likely inherited from their last jawed vertebrate ancestor, cartilaginous and bony fishes have independently evolved distinct cartilage mineralisation strategies. Conclusions This work highlights the diversity of skeletal development mechanisms and underscores the importance of cartilaginous fishes in broadening our understanding of how vertebrate mineralisation evolved. By contrasting conserved and lineage-specific features, the study provides new insights into the independent trajectories that shaped the skeletal structures of modern vertebrates.

Abstract The mode of evolution of left-right asymmetries in the vertebrate habenulae remains largely unknown. Using a transcriptomic approach, we show that in a cartilaginous fish, the catshark Scyliorhinus canicula , habenulae exhibit marked asymmetries, in both their medial and lateral components. Comparisons across vertebrates suggest that those identified in lateral habenulae reflect an ancestral gnathostome trait, partially conserved in lampreys, and independently lost in tetrapods and neopterygians. Asymmetry formation involves distinct mechanisms in the catshark lateral and medial habenulae. Medial habenulae are submitted to a marked, asymmetric temporal regulation of neurogenesis, undetectable in their lateral counterparts. Conversely, asymmetry formation in lateral habenulae results from asymmetric choices of neuronal identity in post-mitotic progenitors, a regulation dependent on the repression of Wnt signaling by Nodal on the left. Based on comparisons with the mouse and the zebrafish, we propose that habenular asymmetry formation involves a recurrent developmental logic across vertebrates, which relies on conserved, temporally regulated genetic programs sequentially shaping choices of neuronal identity on both sides and asymmetrically modified by Wnt activity.

Non-lethal methods for semen collection from elasmobranchs to better understand species reproduction has accompanied the development of artificial insemination. Ejaculates (n = 82) collected from whitespotted bamboo sharks Chiloscyllium plagiosum (n = 19) were assessed and cold-stored raw or extended at 4 °C. Females (n = 20) were inseminated with fresh or 24–48 h cold-stored raw or extended semen and paternity of offspring determined with microsatellite markers. Insemination of females with fresh semen (n = 10) resulted in 80 hatchlings and 27.6% fertility. Insemination of females with semen cold-stored 24 h (n = 4) and 48 h (n = 1) semen resulted in 17 hatchlings and fertilization rates of 28.1% and 7.1% respectively. Two females inseminated with fresh or cold-stored semen laid eggs that hatched from fertilization and parthenogenesis within the same clutch. Parthenogenesis rate for inseminated females was 0.71%. Results demonstrate artificial insemination with cold-stored semen can provide a strategy for transport of male genetics nationally and internationally, precluding the need to transport sharks. Production of parthenotes in the same clutch as sexually fertilized eggs highlights the prevalence of parthenogenesis in whitespotted bamboo sharks and poses important considerations for population management.

In teleost fish, branchial ionocytes are important sites for osmoregulation and acid-base regulation by maintaining ionic balance in the body fluid. During the early developmental stages before the formation of the gills, teleost ionocytes are localized in the yolk-sac membrane and body skin. By comparing with teleost fish, much less is known about ionocytes in developing embryos of elasmobranch fish. The present study investigated the development of ionocytes in the embryo and larva of cloudy catshark, Scyliorhinus torazame. We first observed ionocyte distribution by immunohistochemical staining with anti-Na+/K+-ATPase (NKA) and anti-vacuolar-type H+-ATPase (V-ATPase) antibodies. The NKA- and V-ATPase-rich ionocytes appeared as single cells in the gill filaments from stage 31, the stage of pre-hatching, while the ionocytes on the body skin and yolk-sac membrane were also observed. From stage 32, in addition to single ionocytes on the gill filaments, some outstanding follicular structures of NKA-immunoreactive cells were developed to fill the inter-filament region of the gill septa. The follicular ionocytes possess NKA in the basolateral membrane and Na+/H+ exchanger 3 in the apical membrane, indicating that they are involved in acid-base regulation like single NKA-rich ionocytes. Three-dimensional analysis and whole-mount immunohistochemistry revealed that the distribution of follicular ionocytes was limited to the rostral side of gill septum. The rostral sides of gill septum might be exposed to faster water flow than caudal side because the gills of sharks gently curved backward. This dissymmetric distribution of follicular ionocytes is considered to facilitate efficient body-fluid homeostasis of catshark embryo.