hox gene expression predicts tetrapod-like axial regionalization in the skate, Leucoraja erinacea.

The axial skeleton of tetrapods is organized into distinct anteroposterior regions of the vertebral column (cervical, trunk, sacral, and caudal), and transitions between these regions are determined by colinear anterior expression boundaries of Hox5/6, -9, -10, and -11 paralogy group genes within embryonic paraxial mesoderm. Fishes, conversely, exhibit little in the way of discrete axial regionalization, and this has led to scenarios of an origin of Hox-mediated axial skeletal complexity with the evolutionary transition to land in tetrapods. Here, combining geometric morphometric analysis of vertebral column morphology with cell lineage tracing of hox gene expression boundaries in developing embryos, we recover evidence of at least five distinct regions in the vertebral skeleton of a cartilaginous fish, the little skate (Leucoraja erinacea). We find that skate embryos exhibit tetrapod-like anteroposterior nesting of hox gene expression in their paraxial mesoderm, and we show that anterior expression boundaries of hox5/6, hox9, hox10, and hox11 paralogy group genes predict regional transitions in the differentiated skate axial skeleton. Our findings suggest that hox-based axial skeletal regionalization did not originate with tetrapods but rather has a much deeper evolutionary history than was previously appreciated.

Embryonic origin and serial homology of gill arches and paired fins in the skate, Leucoraja erinacea.

Paired fins are a defining feature of the jawed vertebrate body plan, but their evolutionary origin remains unresolved. Gegenbaur proposed that paired fins evolved as gill arch serial homologues, but this hypothesis is now widely discounted, owing largely to the presumed distinct embryonic origins of these structures from mesoderm and neural crest, respectively. Here, we use cell lineage tracing to test the embryonic origin of the pharyngeal and paired fin skeleton in the skate (Leucoraja erinacea). We find that while the jaw and hyoid arch skeleton derive from neural crest, and the pectoral fin skeleton from mesoderm, the gill arches are of dual origin, receiving contributions from both germ layers. We propose that gill arches and paired fins are serially homologous as derivatives of a continuous, dual-origin mesenchyme with common skeletogenic competence, and that this serial homology accounts for their parallel anatomical organization and shared responses to axial patterning signals.

A common way to evolve new body parts is to copy existing ones and to remodel them. In insects for example, the antennae, mouth parts and legs all follow the same basic body plan, with modifications that adapt them for different uses. In the late 19th century, anatomist Karl Gegenbaur noticed a similar pattern in fish. He saw similarities between pairs of fins and pairs of gills, suggesting that one evolved from the other. But there is currently no fossil evidence documenting such a transformation. Modern research has shown that the development of both gill and fin skeletons shares common genetic pathways. But the cells that form the two structures do not come from the same place. Gill skeletons develop from a part of the embryo called the neural crest, while fin skeletons come from a region called the mesoderm. One way to test Gegenbaur's idea is to look more closely at the cells that form gill and fin skeletons as fish embryos develop. Here, Sleight and Gillis examined the gills and fins of a cartilaginous fish called Leucoraja erinacea, also known as the little skate. Sleight and Gillis labelled the cells from the neural crest and mesoderm of little skate embryos with a fluorescent dye and then tracked the cells over several weeks. While the fins did form from mesoderm cells, the gills did not develop as expected. The first gill contained only neural crest cells, but the rest were a mixture of both cell types. This suggests that fins and gills develop from a common pool of cells that consists of both neural crest and mesoderm cells, which have the potential to develop into either body part. This previously unrecognised embryonic continuity between gills and fins explains why these structures respond in the same way to the same genetic cues, regardless of what cell type they develop from. Based on this new evidence, Sleight and Gillis believe that Gegenbaur was right, and that fins and gills do indeed share an evolutionary history. While firm evidence for the transformation of gills into fins remains elusive, this work suggests it is possible. A deeper understanding of the process could shed light on the development of other repeated structures in nature. Research shows that animals use a relatively small number of genetic cues to set out their body plans. This can make it hard to use genetics alone to study their evolutionary history. But, looking at how different cell types respond to those cues to build anatomical features, like fins and gills, could help to fill in the gaps.

Development and regeneration of the crushing dentition in skates (Rajidae).

Sharks and rays (elasmobranchs) have the remarkable capacity to continuously regenerate their teeth. The polyphyodont system is considered the ancestral condition of the gnathostome dentition. Despite this shared regenerative ability, sharks and rays exhibit dramatic interspecific variation in their tooth morphology. Ray (batoidea) teeth typically constitute crushing pads of flattened teeth, whereas shark teeth are pointed, multi-cuspid units. Although recent research has addressed the molecular development of the shark dentition, little is known about that of the ray. Furthermore, how dental diversity within the elasmobranch lineage is achieved remains unknown. Here, we examine dental development and regeneration in two Batoid species: the thornback skate (Raja clavata) and the little skate (Leucoraja erinacea). Using in situ hybridization and immunohistochemistry, we examine the expression of a core gnathostome dental gene set during early development of the skate dentition and compare it to development in the shark. Elasmobranch tooth development is highly conserved, with sox2 likely playing an important role in the initiation and regeneration of teeth. Alterations to conserved genes expressed in an enamel knot-like signalling centre may explain the morphological diversity of elasmobranch teeth, thereby enabling sharks and rays to occupy diverse dietary and ecological niches.

Resegmentation is an ancestral feature of the gnathostome vertebral skeleton.

The vertebral skeleton is a defining feature of vertebrate animals. However, the mode of vertebral segmentation varies considerably between major lineages. In tetrapods, adjacent somite halves recombine to form a single vertebra through the process of 'resegmentation'. In teleost fishes, there is considerable mixing between cells of the anterior and posterior somite halves, without clear resegmentation. To determine whether resegmentation is a tetrapod novelty, or an ancestral feature of jawed vertebrates, we tested the relationship between somites and vertebrae in a cartilaginous fish, the skate (Leucoraja erinacea). Using cell lineage tracing, we show that skate trunk vertebrae arise through tetrapod-like resegmentation, with anterior and posterior halves of each vertebra deriving from adjacent somites. We further show that tail vertebrae also arise through resegmentation, though with a duplication of the number of vertebrae per body segment. These findings resolve axial resegmentation as an ancestral feature of the jawed vertebrate body plan.

Reproductive characteristics and reproductive tract anatomy of the California butterfly ray Gymnura marmorata (Myliobatiformes: Gymnuridae).

The California butterfly ray Gymnura marmorata is frequently caught by artisanal fisheries in north-western Mexico (Baja California Sur; BCS). The aim of this study was to estimate reproductive characteristics and describe the micro and macroscopic reproductive structures of female and male G. marmorata. Sampling was carried out at two fisheries landing sites located on the west coast BCS, from 2008 to 2012. The largest disc width (WD ) ever recorded for this species was 131.0 cm and females were larger than males. The male sample was composed exclusively of adult specimens, with a median size at 50% maturity (WD50 ) estimated at 44.9 cm WD . For females, size at onset of maturity was estimated at 63.0 cm WD ; WD50 , at 64.7 cm WD ; and size at pregnancy at 80.1 cm WD . Gonadal asymmetry was observed for the first time in this species. Indeed, in males only the left testis was functional (testicular asymmetry), while in females, only the left ovary was functional (ovarian asymmetry).

Mother-embryo isotope fractionation in the pygmy devilray Mobula kuhlii cf. eregoodootenkee.

We determined stable-isotope ratios for replicate muscle tissues in 13 gravid Mobula kuhlii cf. eregoodootenkee (110.4-120.4 cm disc width; WD ) and their embryos (7.0-42.3 cm WD ) and also yolks and histrotroph, to assess the potential implications for juvenile nutrition and habitat use. Irrespective of their development in the uterus, embryos had similar δ13 C values in their muscle tissue as the mothers and both had greater values than in the histotroph. During gestation, δ13 C values increased across all sample types. However, while embryo muscle tissue and the histotroph were associated with increasing 15 N levels during embryonic development, this was depleted in the mothers' muscle tissue and yolk. Although speculative, the observed variation in stable-isotope ratios might imply a dietary shift among gravid females during their early gestation. Irrespective of the underlying mechanisms, the results indicate neonates will have relatively greater δ15 N values than post-partum females, which would probably confound juvenile foraging-ecology estimates.

Steroid concentrations in maternal serum and uterine histotroph in round stingrays (Urobatis halleri).

Despite a wide range of elasmobranch (sharks, skates and rays) matrotrophic strategies, and thus potentially diverse pathways for maternal-fetal hormone exchange, little attention has been given to uterine steroids during development. Round Stingrays (Urobatis halleri) with matrotrophic histotrophy were captured during every month of their annual reproductive season from post-ovulation to near parturition, and paired samples of plasma and histotroph were analyzed for a suite of steroid hormones using LC-ESI/MRM. Hormone concentrations within and between maternal and uterine compartments were compared using two markers of embryo development. Histotroph had consistently higher detection rates and concentrations of hormones than maternal plasma, especially during early pregnancy when embryos are yolk sac-dependent for nutrition. Peaks in histotroph testosterone concentrations preceded maternal plasma, suggesting that hormones were locally produced within the uterine compartment. Embryonic sexual differentiation based on the presence of visible claspers (male copulatory organs) coincided with peaks in histotroph progesterone, testosterone, 17-hydroxyprogesterone and estradiol, suggesting that, like mammalian pregnancy, elasmobranch embryonic steroids also contribute to their own developmental environment.

Embryonic transcriptome sequencing of the ocellate spot skate Okamejei kenojei.

Chondrichthyans (cartilaginous fishes) exhibit highly variable reproductive styles, categorized as viviparity and oviparity. Among these, species with oviparity provide an enormous potential of molecular experimentation with stable sample supply which does not demand the sacrifices of live mothers. Cartilaginous fishes are divided into two subclasses, chimaeras (Holocephali) and elasmobranchs (Elasmobranchii), and the latter consists of two monophyletic groups, Batoidea (rays, skates and torpedoes) and Selachimorpha (sharks). Here we report transcriptome assemblies of the ocellate spot skate Okamejei kenojei, produced by strand-specific RNA-seq of its embryonic tissues. We obtained a total of 325 million illumina short reads from libraries prepared using four different tissue domains and assembled them all together. Our assembly result confirmed the species authenticity and high continuity of contig sequences. Also, assessment of its coverage of pre-selected one-to-one orthologs supported high diversity of transcripts in the assemblies. Our products are expected to provide a basis of comparative molecular studies encompassing other chondrichthyan species with emerging genomic and transcriptomic sequence information.

Legacy polychlorinated biphenyl contamination impairs male embryonic development in an elasmobranch with matrotrophic histotrophy, the round stingray (Urobatis halleri).

Anthropogenic chemical exposure can result in overall reductions in reproductive success. Using the round stingray (Urobatis halleri) as an elasmobranch model with internal gestation, we measured female fecundity and embryo growth from postovulation to near parturition to test the hypothesis that environmental polychlorinated biphenyl (PCB) contamination would impair reproductive success. Two sites were sampled from southern California, USA: the mainland site was exposed to legacy PCB contamination (with low exposure to other anthropogenic contaminants), and the offshore reference site at Catalina Island was a separate population with low anthropogenic influence. Contaminant-exposed embryos weighed less at each stage of development than reference embryos, while accumulating proportionately more liver mass over development. Furthermore, environmental contamination negatively affected male embryos more than female embryos. The present study is the first study to demonstrate a negative effect of contaminant exposure on elasmobranch embryo growth, with probable fitness costs later in life. Environ Toxicol Chem 2018;37:2904-2911. © 2018 SETAC.

Development and evolution of tooth renewal in neoselachian sharks as a model for transformation in chondrichthyan dentitions.

A defining feature of dentitions in modern sharks and rays is the regulated pattern order that generates multiple replacement teeth. These are arranged in labio-lingual files of replacement teeth that form in sequential time order both along the jaw and within successively initiated teeth in a deep dental lamina. Two distinct adult dentitions have been described: alternate, in which timing of new teeth alternates between two adjacent files, each erupting separately, and the other arranged as single files, where teeth of each file are timed to erupt together, in some taxa facilitating similarly timed teeth to join to form a cutting blade. Both are dependent on spatiotemporally regulated formation of new teeth. The adult Angel shark Squatina (Squalomorphii) exemplifies a single file dentition, but we obtained new data on the developmental order of teeth in the files of Squatina embryos, showing alternate timing of tooth initiation. This was based on micro-CT scans revealing that the earliest mineralised teeth at the jaw margin and their replacements in file pairs (odd and even jaw positions) alternate in their initiation timing. Along with Squatina, new observations from other squalomorphs such as Hexanchus and Chlamydoselachus, together with representatives of the sister group Galeomorphii, have established that the alternate tooth pattern (initiation time and replacement order) characterises the embryonic dentition of extant sharks; however, this can change in adults. These character states were plotted onto a recent phylogeny, demonstrating that the Squalomorphii show considerable plasticity of dental development. We propose a developmental-evolutionary model to allow change from the alternate to a single file alignment of replacement teeth. This establishes new dental morphologies in adult sharks from inherited alternate order.

Embryonic origin of the gnathostome vertebral skeleton.

The vertebral column is a key component of the jawed vertebrate (gnathostome) body plan, but the primitive embryonic origin of this skeleton remains unclear. In tetrapods, all vertebral components (neural arches, haemal arches and centra) derive from paraxial mesoderm (somites). However, in teleost fishes, vertebrae have a dual embryonic origin, with arches derived from somites, but centra formed, in part, by secretion of bone matrix from the notochord. Here, we test the embryonic origin of the vertebral skeleton in a cartilaginous fish (the skate, Leucoraja erinacea) which serves as an outgroup to tetrapods and teleosts. We demonstrate, by cell lineage tracing, that both arches and centra are somite-derived. We find no evidence of cellular or matrix contribution from the notochord to the skate vertebral skeleton. These findings indicate that the earliest gnathostome vertebral skeleton was exclusively of somitic origin, with a notochord contribution arising secondarily in teleosts.

Biological notes on the reef stingray, Urobatis concentricus, an endemic species of Mexico.

This study found that the reef stingray Urobatis concentricus presents a matrotrophic aplacental viviparous reproduction with yolk sac and trophonemata formation. Growth of ovarian follicles occurs asynchronously and continuously with fecundity of three embryos per female. A maximum size of 58·4 cm total length and 37·6 cm disc width was recorded, corresponding to a mature female of 3 or 4 years of age.

Embryonic development of the axial column in the little skate, Leucoraja erinacea.

The morphological patterns and molecular mechanisms of vertebral column development are well understood in bony fishes (osteichthyans). However, vertebral column morphology in elasmobranch chondrichthyans (e.g., sharks and skates) differs from that of osteichthyans, and its development has not been extensively studied. Here, we characterize vertebral development in an elasmobranch fish, the little skate, Leucoraja erinacea, using microCT, paraffin histology, and whole-mount skeletal preparations. Vertebral development begins with the condensation of mesenchyme, first around the notochord, and subsequently around the neural tube and caudal artery and vein. Mesenchyme surrounding the notochord differentiates into a continuous sheath of spindle-shaped cells, which forms the precursor to the mineralized areolar calcification of the centrum. Mesenchyme around the neural tube and caudal artery/vein becomes united by a population of mesenchymal cells that condenses lateral to the sheath of spindle-shaped cells, with this mesenchymal complex eventually differentiating into the hyaline cartilage of the future neural arches, hemal arches, and outer centrum. The initially continuous layers of areolar tissue and outer hyaline cartilage eventually subdivide into discrete centra and arches, with the notochord constricted in the center of each vertebra by a late-forming "inner layer" of hyaline cartilage, and by a ring of areolar calcification located medial to the outer vertebral cartilage. The vertebrae of elasmobranchs are distinct among vertebrates, both in terms of their composition (i.e., with centra consisting of up to three tissues layers-an inner cartilage layer, a calcified areolar ring, and an outer layer of hyaline cartilage), and their mode of development (i.e., the subdivision of arch and outer centrum cartilage from an initially continuous layer of hyaline cartilage). Given the evident variation in patterns of vertebral construction, broad taxon sampling, and comparative developmental analyses are required to understand the diversity of mechanisms at work in the developing axial skeleton of vertebrates. J. Morphol. 278:300-320, 2017. © 2017 Wiley Periodicals, Inc.

Near-term embryos in a Pristis pristis (Elasmobranchii: Pristidae) from Brazil.

This paper describes the record of a gravid Pristis pristis from the north coast of Maranhão State (Brazil) in May of 2009 by an artisanal vessel. Size, mass and rostrum length of the adult female were estimated at 5000 mm, 750000 g and 1536 mm, respectively. Total length (LT ) of miscarried embryos ranged from 755 to 800 mm and total mass from 890 to 1120 g. Although this is a single record, it indicates that P. pristis births may be taking place during May or coming months and there may be population differences between Central and South America.

A shared role for sonic hedgehog signalling in patterning chondrichthyan gill arch appendages and tetrapod limbs.

Chondrichthyans (sharks, skates, rays and holocephalans) possess paired appendages that project laterally from their gill arches, known as branchial rays. This led Carl Gegenbaur to propose that paired fins (and hence tetrapod limbs) originally evolved via transformation of gill arches. Tetrapod limbs are patterned by asonic hedgehog(Shh)-expressing signalling centre known as the zone of polarising activity, which establishes the anteroposterior axis of the limb bud and maintains proliferative expansion of limb endoskeletal progenitors. Here, we use loss-of-function, label-retention and fate-mapping approaches in the little skate to demonstrate that Shh secretion from a signalling centre in the developing gill arches establishes gill arch anteroposterior polarity and maintains the proliferative expansion of branchial ray endoskeletal progenitor cells. These findings highlight striking parallels in the axial patterning mechanisms employed by chondrichthyan branchial rays and paired fins/limbs, and provide mechanistic insight into the anatomical foundation of Gegenbaur's gill arch hypothesis.

Molecular mechanisms underlying the exceptional adaptations of batoid fins.

Extreme novelties in the shape and size of paired fins are exemplified by extinct and extant cartilaginous and bony fishes. Pectoral fins of skates and rays, such as the little skate (Batoid, Leucoraja erinacea), show a strikingly unique morphology where the pectoral fin extends anteriorly to ultimately fuse with the head. This results in a morphology that essentially surrounds the body and is associated with the evolution of novel swimming mechanisms in the group. In an approach that extends from RNA sequencing to in situ hybridization to functional assays, we show that anterior and posterior portions of the pectoral fin have different genetic underpinnings: canonical genes of appendage development control posterior fin development via an apical ectodermal ridge (AER), whereas an alternative Homeobox (Hox)-Fibroblast growth factor (Fgf)-Wingless type MMTV integration site family (Wnt) genetic module in the anterior region creates an AER-like structure that drives anterior fin expansion. Finally, we show that GLI family zinc finger 3 (Gli3), which is an anterior repressor of tetrapod digits, is expressed in the posterior half of the pectoral fin of skate, shark, and zebrafish but in the anterior side of the pelvic fin. Taken together, these data point to both highly derived and deeply ancestral patterns of gene expression in skate pectoral fins, shedding light on the molecular mechanisms behind the evolution of novel fin morphologies.

Development and evolution of dentition pattern and tooth order in the skates and rays (batoidea; chondrichthyes).

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.

Maternal transfer of organohalogenated compounds in sharks and stingrays.

Elasmobranchs can bioaccumulate considerable amounts of persistent organic pollutants (POPs) and utilize several reproductive strategies thereby influencing maternal transfer of contaminants. This study provides preliminary data on the POP transfer from pregnant females to offspring of three species (Atlantic stingrays, bonnethead, blacktip sharks) with different reproduction modes (aplacental, placental viviparity). Polychlorinated biphenyl (PCB) levels were generally higher than any other POPs. Stingrays and blacktip shark embryos contained the lowest POP concentrations while bonnetheads and the blacktip adult female had the highest concentrations. Results suggest that POPs are more readily transferred from the mother to the embryo compared to what is transferred to ova in stingrays. Statistically significant differences in levels of selected POPs were found between embryos from the left and right uterus within the same litter as well as between female and male embryos within the same litter for bonnetheads, but not for the blacktip sharks.

The evolution of the vertebrate cerebellum: absence of a proliferative external granule layer in a non-teleost ray-finned fish.

The cerebellum represents one of the most morphologically variable structures in the vertebrate brain. To shed light on its evolutionary history, we have examined the molecular anatomy and proliferation of the developing cerebellum of the North American paddlefish, Polyodon spathula. Absence of an external proliferative cerebellar layer and the restriction of Atonal1 expression to the rhombic lip and valvular primordium demonstrate that transit amplification in a cerebellar external germinal layer, a prominent feature of amniote cerebellum development, is absent in paddlefish. Furthermore, expression of Sonic hedgehog, which drives secondary proliferation in the mouse cerebellum, is absent from the paddlefish cerebellum. These data are consistent with what has been observed in zebrafish and suggest that the transit amplification seen in the amniote cerebellum was either lost very early in the ray-finned fish lineage or evolved in the lobe-finned fish lineage. We also suggest that the Atoh1-positive proliferative valvular primordium may represent a synapomorphy (shared derived character) of ray-finned fishes. The topology of valvular primordium development in paddlefish differs significantly from that of zebrafish and correlates with the adult cerebellar form. The distribution of proliferative granule cell precursors in different vertebrate taxa is thus the likely determining factor in cerebellar morphological diversity.

Quantification of maternal offloading of organic contaminants in elasmobranchs using the histotrophic round stingray (Urobatis halleri) as a model.

Maternal offloading is one route by which young animals may accumulate persistent organic pollutants, such as dichlorodiphenyltrichloroethane (DDT) and polychlorinated biphenyls (PCBs), but has not been well documented in elasmobranchs despite their propensity to accumulate high concentrations of contaminants. Using the round stingray (Urobatis halleri) as a coastal elasmobranch model, we examined maternal offloading processes at two stages in the stingray's entire reproductive cycle. Post-ovulated and near-term pregnant female stingrays were sampled from southern California, and organic contaminants were measured in the ova and embryonic tissues and compared to concentrations measured in corresponding female livers to determine route and extent of transfer. Total organic contaminant loads measured in ovulated eggs were about two times lower than loads measured in embryos (p < 0.001) indicating mothers have the ability to transfer contaminants throughout pregnancy. Contaminant loads measured in pups showed a positive relationship with mother's contaminant concentrations (p < 0.001); however, mothers offloaded relatively low percentages (1.5 ± 1.7%) of their total contaminant load using contaminants measured in the liver as a proxy. However, histotrophy is only one form of supplemental provisioning utilized by elasmobranchs and variation in reproductive modes likely influences the extent to which female elasmobranchs may maternally offload contaminants.