Xenopus tropicalis osteoblast-specific open chromatin regions reveal promoters and enhancers involved in human skeletal phenotypes and shed light on early vertebrate evolution.

While understanding the genetic underpinnings of osteogenesis has far-reaching implications for skeletal diseases and evolution, a comprehensive characterization of the osteoblastic regulatory landscape in non-mammalian vertebrates is still lacking. Here, we compared the ATAC-Seq profile of Xenopus tropicalis (Xt) osteoblasts to a variety of non mineralizing control tissues, and identified osteoblast-specific nucleosome free regions (NFRs) at 527 promoters and 6747 distal regions. Sequence analyses, Gene Ontology, RNA-Seq and ChIP-Seq against four key histone marks confirmed that the distal regions correspond to bona fide osteogenic transcriptional enhancers exhibiting a shared regulatory logic with mammals. We report 425 regulatory regions conserved with human and globally associated to skeletogenic genes. Of these, 35 regions have been shown to impact human skeletal phenotypes by GWAS, including one trps1 enhancer and the runx2 promoter, two genes which are respectively involved in trichorhinophalangeal syndrome type I and cleidocranial dysplasia. Intriguingly, 60 osteoblastic NFRs also align to the genome of the elephant shark, a species lacking osteoblasts and bone tissue. To tackle this paradox, we chose to focus on dlx5 because its conserved promoter, known to integrate regulatory inputs during mammalian osteogenesis, harbours an osteoblast-specific NFR in both frog and human. Hence, we show that dlx5 is expressed in Xt and elephant shark odontoblasts, supporting a common cellular and genetic origin of bone and dentine. Taken together, our work (i) unravels the Xt osteogenic regulatory landscape, (ii) illustrates how cross-species comparisons harvest data relevant to human biology and (iii) reveals that a set of genes including bnc2, dlx5, ebf3, mir199a, nfia, runx2 and zfhx4 drove the development of a primitive form of mineralized skeletal tissue deep in the vertebrate lineage.

Response to comment on "Exceptional preservation of organs in Devonian placoderms from the Gogo largerstätte".

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.

The Development of the Chimaeroid Pelvic Skeleton and the Evolution of Chondrichthyan Pelvic Fins.

Pelvic girdles, fins and claspers are evolutionary novelties first recorded in jawed vertebrates. Over the course of the evolution of chondrichthyans (cartilaginous fish) two trends in the morphology of the pelvic skeleton have been suggested to have occurred. These evolutionary shifts involved both an enlargement of the metapterygium (basipterygium) and a transition of fin radial articulation from the pelvic girdle to the metapterygium. To determine how these changes in morphology have occurred it is essential to understand the development of extant taxa as this can indicate potential developmental mechanisms that may have been responsible for these changes. The study of the morphology of the appendicular skeleton across development in chondrichthyans is almost entirely restricted to the historical literature with little contemporary research. Here, we have examined the morphology and development of the pelvic skeleton of a holocephalan chondrichthyan, the elephant shark (Callorhinchus milii), through a combination of dissections, histology, and nanoCT imaging and redescribed the pelvic skeleton of Cladoselache kepleri (NHMUK PV P 9269), a stem holocephalan. To put our findings in their evolutionary context we compare them with the fossil record of chondrichthyans and the literature on pelvic development in elasmobranchs from the late 19th century. Our findings demonstrate that the pelvic skeleton of C. milii initially forms as a single mesenchymal condensation, consisting of the pelvic girdle and a series of fin rays, which fuse to form the basipterygium. The girdle and fin skeleton subsequently segment into distinct components whilst chondrifying. This confirms descriptions of the early pelvic development in Scyliorhinid sharks from the historical literature and suggests that chimaeras and elasmobranchs share common developmental patterns in their pelvic anatomy. Alterations in the location and degree of radial fusion during early development may be the mechanism responsible for changes in pelvic fin morphology over the course of the evolution of both elasmobranchs and holocephalans, which appears to be an example of parallel evolution.

Exceptional preservation of organs in Devonian placoderms from the Gogo lagerstätte.

The origin and early diversification of jawed vertebrates involved major changes to skeletal and soft anatomy. Skeletal transformations can be examined directly by studying fossil stem gnathostomes; however, preservation of soft anatomy is rare. We describe the only known example of a three-dimensionally mineralized heart, thick-walled stomach, and bilobed liver from arthrodire placoderms, stem gnathostomes from the Late Devonian Gogo Formation in Western Australia. The application of synchrotron and neutron microtomography to this material shows evidence of a flat S-shaped heart, which is well separated from the liver and other abdominal organs, and the absence of lungs. Arthrodires thus show the earliest phylogenetic evidence for repositioning of the gnathostome heart associated with the evolution of the complex neck region in jawed vertebrates.

Ontogeny and caudal autotomy fracture planes in a large scincid lizard, Egernia kingii.

Many lizard species use caudal autotomy, the ability to self-amputate a portion of the tail, as an effective but costly survival strategy. However, as a lizard grows, its increased size may reduce predation risk allowing for less costly strategies (e.g., biting and clawing) to be used as the primary defence. The King's skink (Egernia kingii) is a large scincid up to approximately 244 mm snout to vent length (SVL) in size when adult. Adults rely less on caudal autotomy than do juveniles due to their size and strength increase during maturation. It has been hypothesised that lower behavioural reliance on autotomy in adults is reflected in loss or restriction of caudal vertebrae fracture planes through ossification as caudal intra-vertebral fracture planes in some species ossify during ontogenetic growth. To test this, we used micro-CT to image the tails of a growth series of seven individuals of E. kingii. We show that fracture planes are not lost or restricted ontogenetically within E. kingii, with adults retaining between 39-44 autotomisable vertebrae following 5-6 non-autotomisable vertebrae. Even though mature E. kingii rely less on caudal autotomy than do juveniles, this research shows that they retain the maximum ability to autotomise their tails, providing a last resort option to avoid threats. The potential costs associated with retaining caudal autotomy are most likely mitigated through neurological control of autotomy and E. kingii's longevity.

At What Cost? Trade-Offs and Influences on Energetic Investment in Tail Regeneration in Lizards Following Autotomy.

Caudal autotomy, the ability to shed a portion of the tail, is a widespread defence strategy among lizards. Following caudal autotomy, and during regeneration, lizards face both short- and long-term costs associated with the physical loss of the tail and the energy required for regeneration. As such, the speed at which the individual regenerates its tail (regeneration rate) should reflect the fitness priorities of the individual. However, multiple factors influence the regeneration rate in lizards, making inter-specific comparisons difficult and hindering broader scale investigations. We review regeneration rates for lizards and tuatara from the published literature, discuss how species' fitness priorities and regeneration rates are influenced by specific, life history and environmental factors, and provide recommendations for future research. Regeneration rates varied extensively (0-4.3 mm/day) across the 56 species from 14 family groups. Species-specific factors, influencing regeneration rates, varied based on the type of fracture plane, age, sex, reproductive season, and longevity. Environmental factors including temperature, photoperiod, nutrition, and stress also affected regeneration rates, as did the method of autotomy induction, and the position of the tail also influenced regeneration rates for lizards. Additionally, regeneration could alter an individual's behaviour, growth, and reproductive output, but this varied depending on the species.

Mineralization of the Callorhinchus Vertebral Column (Holocephali; Chondrichthyes).

Members of the Chondrichthyes (Elasmobranchii and Holocephali) are distinguished by their largely cartilaginous endoskeletons, which comprise an uncalcified core overlain by a mineralized layer; in the Elasmobranchii (sharks, skates, rays) most of this mineralization takes the form of calcified polygonal tiles known as tesserae. In recent years, these skeletal tissues have been described in ever increasing detail in sharks and rays, but those of Holocephali (chimaeroids) have been less well-studied, with conflicting accounts as to whether or not tesserae are present. During embryonic ontogeny in holocephalans, cervical vertebrae fuse to form a structure called the synarcual. The synarcual mineralizes early and progressively, anteroposteriorly and dorsoventrally, and therefore presents a good skeletal structure in which to observe mineralized tissues in this group. Here, we describe the development and mineralization of the synarcual in an adult and stage 36 elephant shark embryo (Callorhinchus milii). Small, discrete, but irregular blocks of cortical mineralization are present in stage 36, similar to what has been described recently in embryos of other chimaeroid taxa such as Hydrolagus, while in Callorhinchus adults, the blocks of mineralization are more irregular, but remain small. This differs from fossil members of the holocephalan crown group (Edaphodon), as well as from stem group holocephalans (e.g., Symmorida, Helodus, Iniopterygiformes), where tesserae are notably larger than in Callorhinchus and show similarities to elasmobranch tesserae, for example with respect to polygonal shape.

Re-regeneration to reduce negative effects associated with tail loss in lizards.

Many species of lizard use caudal autotomy, the ability to self-amputate a portion of their tail, regenerated over time, as an effective anti-predation mechanism. The importance of this tactic for survival depends on the degree of predation risk. There are, however, negative trade-offs to losing a tail, such as loss of further autotomy opportunities with the regenerated tail vertebrae being replaced by a continuous cartilaginous rod. The common consensus has been that once a tail has been autotomised and regenerated it can only be autotomised proximal to the last vertebral autotomy point, as the cartilage rod lacks autotomy planes. However, anecdotal evidence suggests that although the regenerated portion of the tail is unable to autotomise, it can re-regenerate following a physical shearing event. We assessed re-regeneration in three populations of the King's skink (Egernia kingii), a large lizard endemic to south-west Western Australia and surrounding islands. We show that re-regeneration is present at an average of 17.2% across the three populations, and re-regenerated tissue can comprise up to 23.3% of an individual's total tail length. The ability to re-regenerate may minimise the costs to an individual's fitness associated with tail loss, efficiently restoring ecological functions of the tail.

The Ancient Origins of Neural Substrates for Land Walking.

Walking is the predominant locomotor behavior expressed by land-dwelling vertebrates, but it is unknown when the neural circuits that are essential for limb control first appeared. Certain fish species display walking-like behaviors, raising the possibility that the underlying circuitry originated in primitive marine vertebrates. We show that the neural substrates of bipedalism are present in the little skate Leucoraja erinacea, whose common ancestor with tetrapods existed ∼420 million years ago. Leucoraja exhibits core features of tetrapod locomotor gaits, including left-right alternation and reciprocal extension-flexion of the pelvic fins. Leucoraja also deploys a remarkably conserved Hox transcription factor-dependent program that is essential for selective innervation of fin/limb muscle. This network encodes peripheral connectivity modules that are distinct from those used in axial muscle-based swimming and has apparently been diminished in most modern fish. These findings indicate that the circuits that are essential for walking evolved through adaptation of a genetic regulatory network shared by all vertebrates with paired appendages. VIDEO ABSTRACT.

Development of the Synarcual in the Elephant Sharks (Holocephali; Chondrichthyes): Implications for Vertebral Formation and Fusion.

The synarcual is a structure incorporating multiple elements of two or more anterior vertebrae of the axial skeleton, forming immediately posterior to the cranium. It has been convergently acquired in the fossil group 'Placodermi', in Chondrichthyes (Holocephali, Batoidea), within the teleost group Syngnathiformes, and to varying degrees in a range of mammalian taxa. In addition, cervical vertebral fusion presents as an abnormal pathology in a variety of human disorders. Vertebrae develop from axially arranged somites, so that fusion could result from a failure of somite segmentation early in development, or from later heterotopic development of intervertebral bone or cartilage. Examination of early developmental stages indicates that in the Batoidea and the 'Placodermi', individual vertebrae developed normally and only later become incorporated into the synarcual, implying regular somite segmentation and vertebral development. Here we show that in the holocephalan Callorhinchus milii, uniform and regular vertebral segmentation also occurs, with anterior individual vertebra developing separately with subsequent fusion into a synarcual. Vertebral elements forming directly behind the synarcual continue to be incorporated into the synarcual through growth. This appears to be a common pattern through the Vertebrata. Research into human disorders, presenting as cervical fusion at birth, focuses on gene misexpression studies in humans and other mammals such as the mouse. However, in chondrichthyans, vertebral fusion represents the normal morphology, moreover, taxa such Leucoraja (Batoidea) and Callorhinchus (Holocephali) are increasingly used as laboratory animals, and the Callorhinchus genome has been sequenced and is available for study. Our observations on synarcual development in three major groups of early jawed vertebrates indicate that fusion involves heterotopic cartilage and perichondral bone/mineralised cartilage developing outside the regular skeleton. We suggest that chondrichthyans have potential as ideal extant models for identifying the genes involved in these processes, for application to human skeletal heterotopic disorders.

Oldest pathology in a tetrapod bone illuminates the origin of terrestrial vertebrates.

The origin of terrestrial tetrapods was a key event in vertebrate evolution, yet how and when it occurred remains obscure, due to scarce fossil evidence. Here, we show that the study of palaeopathologies, such as broken and healed bones, can help elucidate poorly understood behavioural transitions such as this. Using high-resolution finite element analysis, we demonstrate that the oldest known broken tetrapod bone, a radius of the primitive stem tetrapod Ossinodus pueri from the mid-Viséan (333 million years ago) of Australia, fractured under a high-force, impact-type loading scenario. The nature of the fracture suggests that it most plausibly occurred during a fall on land. Augmenting this are new osteological observations, including a preferred directionality to the trabecular architecture of cancellous bone. Together, these results suggest that Ossinodus, one of the first large (>2m length) tetrapods, spent a significant proportion of its life on land. Our findings have important implications for understanding the temporal, biogeographical and physiological contexts under which terrestriality in vertebrates evolved. They push the date for the origin of terrestrial tetrapods further back into the Carboniferous by at least two million years. Moreover, they raise the possibility that terrestriality in vertebrates first evolved in large tetrapods in Gondwana rather than in small European forms, warranting a re-evaluation of this important evolutionary event.

Capture, transport, and husbandry of elephant sharks (Callorhinchus milii) adults, eggs, and hatchlings for research and display.

Elephant sharks (Callorhinchus milii) have the slowest evolving genome of all vertebrates and are an interesting model species for evolution research and a prized display animal. However, their deep water habitat, short breeding season, fragility, and susceptibility to stress-induced mortality have made them difficult animals to capture, keep in captivity, and obtain fertilized eggs from. Gravid females were captured by rod and reel from Western Port Bay, Australia and transferred to a 40 000 L closed aquaculture system to lay their eggs before being released. The water quality parameters, averaged over three seasons of 4-6 weeks (mean ± standard deviation) were: 16.8°C ± 2.31, salinity 37.1 ± 2.9 g/L, ammonia 0.137 ± 0.2 mg/L, nitrite levels 0.89 ± 0.9 mg/L, nitrate 66.8 ± 45.6 mg/L, pH 7.8 ± 0.18, dissolved oxygen levels 93.6 ± 5.3%, ORP 307 ± 63.3 mV. Eggs were incubated in purpose built egg cages and embryos hatched after 143.6 days ± 1.3 at 16.9 ± 0.9°C of incubation. These procedures led to no adult mortality in the last 2 years and 620 eggs with known deposition date were collected over 4 years, of which 81.5% (±4.8) were viable. Collection of abundant embryological material with known deposition date is of paramount importance for evolutionary developmental research. We attribute this success to excellent water quality, maximum reduction of stress during capture, transport, handling, and captive care.

Pelvic and reproductive structures in placoderms (stem gnathostomes).

Newly discovered pelvic and reproductive structures within placoderms, representing some of the most crownward members of the gnathostome stem group and the most basal jawed vertebrates, challenge established ideas on the origin of the pelvic girdle and reproductive complexity. Here we critically review previous descriptions of the pelvic structures in placoderms and reinterpret the morphology of the pelvic region within the arthrodires and ptyctodonts, in particular the position of the pelvic fin and the relationship of the male clasper to the pelvic girdle. Absence of clear articular surfaces on the clasper and girdle in the Arthrodira, along with evidence from the Ptyctodontida, suggest that these are separate structures along the body. We describe similarities between the pectoral and pelvic girdles and claspers, for example, all these have both dermal and perichondral (cartilaginous) components. Claspers in placoderms and chondrichthyans develop in very different ways; in sharks, claspers develop from the pelvic fin while the claspers in placoderms develop separately, suggesting that their independent development involved a posterior extension of the 'competent stripes' for fin development previously limited to the region between the paired pectoral and pelvic fins. Within this expanded zone, we suggest that clasper position relative to the pelvic fins was determined by genes responsible for limb position. Information on early gnathostome reproductive processes is preserved in both the Ptyctodontida and Arthrodira, including the presence of multiple embryos in pregnant females, embryos of differing sizes and of different sexes (e.g. male claspers preserved in some embyros). By comparison with chondrichthyans, these observations suggest more complex reproductive strategies in placoderms than previously appreciated.

Embryonic development of fin spines in Callorhinchus milii (Holocephali); implications for chondrichthyan fin spine evolution.

Fin spines are commonly known from fossil gnathostomes (jawed vertebrates) and are usually associated with paired and unpaired fins. They are less common among extant gnathostomes, being restricted to the median fins of certain chondrichthyans (cartilaginous fish), including chimaerids (elephant sharks) and neoselachians (sharks, skates, and rays). Fin spine growth is of great interest and relevance but few studies have considered their evolution and development. We investigated the development of the fin spine of the chimaerid Callorhinchus milii using stained histological sections from a series of larval, hatchling, and adult individuals. The lamellar trunk dentine of the Callorhinchus spine first condenses within the mesenchyme, rather than at the contact surface between mesenchyme and epithelium, in a manner more comparable to dermal bone formation than to normal odontode development. Trabecular dentine forms a small component of the spine under the keel; it is covered externally with a thin layer of lamellar trunk dentine, which is difficult to distinguish in sectioned adult spines. We suggest that the distinctive characteristics of the trunk dentine may reflect an origin through co-option of developmental processes involved in dermal bone formation. Comparison with extant Squalus and a range of fossil chondrichthyans shows that Callorhinchus is more representative than Squalus of generalized chondrichthyan fin-spine architecture, highlighting its value as a developmental model organism.

Fossil musculature of the most primitive jawed vertebrates.

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

Comparative pelvic development of the axolotl (Ambystoma mexicanum) and the Australian lungfish (Neoceratodus forsteri): conservation and innovation across the fish-tetrapod transition.

BACKGROUND: The fish-tetrapod transition was one of the major events in vertebrate evolution and was enabled by many morphological changes. Although the transformation of paired fish fins into tetrapod limbs has been a major topic of study in recent years, both from paleontological and comparative developmental perspectives, the interest has focused almost exclusively on the distal part of the appendage and in particular the origin of digits. Relatively little attention has been paid to the transformation of the pelvic girdle from a small unipartite structure to a large tripartite weight-bearing structure, allowing tetrapods to rely mostly on their hindlimbs for locomotion. In order to understand how the ischium and the ilium evolved and how the acetabulum was reoriented during this transition, growth series of the Australian lungfish Neoceratodus forsteri and the Mexican axolotl Ambystoma mexicanum were cleared and stained for cartilage and bone and immunostained for skeletal muscles. In order to understand the myological developmental data, hypotheses about the homologies of pelvic muscles in adults of Latimeria, Neoceratodus and Necturus were formulated based on descriptions from the literature of the coelacanth (Latimeria), the Australian Lungfish (Neoceratodus) and a salamander (Necturus). RESULTS: In the axolotl and the lungfish, the chondrification of the pelvic girdle starts at the acetabula and progresses anteriorly in the lungfish and anteriorly and posteriorly in the salamander. The ilium develops by extending dorsally to meet and connect to the sacral rib in the axolotl. Homologous muscles develop in the same order with the hypaxial musculature developing first, followed by the deep, then the superficial pelvic musculature. CONCLUSIONS: Development of the pelvic endoskeleton and musculature is very similar in Neoceratodus and Ambystoma. If the acetabulum is seen as being a fixed landmark, the evolution of the ischium only required pubic pre-chondrogenic cells to migrate posteriorly. It is hypothesized that the iliac process or ridge present in most tetrapodomorph fish is the precursor to the tetrapod ilium and that its evolution mimicked its development in modern salamanders.

Development and evolution of the muscles of the pelvic fin.

Locomotor strategies in terrestrial tetrapods have evolved from the utilisation of sinusoidal contractions of axial musculature, evident in ancestral fish species, to the reliance on powerful and complex limb muscles to provide propulsive force. Within tetrapods, a hindlimb-dominant locomotor strategy predominates, and its evolution is considered critical for the evident success of the tetrapod transition onto land. Here, we determine the developmental mechanisms of pelvic fin muscle formation in living fish species at critical points within the vertebrate phylogeny and reveal a stepwise modification from a primitive to a more derived mode of pelvic fin muscle formation. A distinct process generates pelvic fin muscle in bony fishes that incorporates both primitive and derived characteristics of vertebrate appendicular muscle formation. We propose that the adoption of the fully derived mode of hindlimb muscle formation from this bimodal character state is an evolutionary innovation that was critical to the success of the tetrapod transition.

Vertebral development of modern salamanders provides insights into a unique event of their evolutionary history.

The origin of salamanders and their interrelationships to the two other modern amphibian orders (frogs and caecilians) are problematic owing to an 80-100 million year gap in the fossil record between the Carboniferous to the Lower Jurassic. This is compounded by a scarcity of adult skeletal characters linking the early representatives of the modern orders to their stem-group in the Paleozoic. The use of ontogenetic characters can be of great use in the resolution of these questions. Growth series of all ten modern salamander families (a 120 cleared and stained larvae) were examined for pattern and timing of vertebral elements chondrification and ossification. The primitive pattern is that of the neural arches developing before the centra, while the reverse represents the derived condition. Both the primitive and derived conditions are observed within the family Hynobiidae, whereas only the derived condition is observed in all other salamanders. This provides support to the claims that Hynobiidae is both the most basal of modern families and potentially polyphyletic (with Ranodon and Hybobius forming the most basal clade and Salamandrella being a part of the most derived clade). This provides insight into a unique event in salamander evolutionary history and suggests that the developmental pattern switch occurred between the Triassic and the mid-Jurassic before the last major radiation.

The pectoral fin of Panderichthys and the origin of digits.

One of the identifying characteristics of tetrapods (limbed vertebrates) is the presence of fingers and toes. Whereas the proximal part of the tetrapod limb skeleton can easily be homologized with the paired fin skeletons of sarcopterygian (lobe-finned) fish, there has been much debate about the origin of digits. Early hypotheses interpreted digits as derivatives of fin radials, but during the 1990s the idea gained acceptance that digits are evolutionary novelties without direct equivalents in fish fin skeletons. This was partly based on developmental genetic data, but also substantially on the pectoral fin skeleton of the elpistostegid (transitional fish/tetrapod) Panderichthys, which appeared to lack distal digit-like radials. Here we present a CT scan study of an undisturbed pectoral fin of Panderichthys demonstrating that the plate-like 'ulnare' of previous reconstructions is an artefact and that distal radials are in fact present. This distal portion is more tetrapod-like than that found in Tiktaalik and, in combination with new data about fin development in basal actinopterygians, sharks and lungfish, makes a strong case for fingers not being a novelty of tetrapods but derived from pre-existing distal radials present in all sarcopterygian fish.

Fish fingers: digit homologues in sarcopterygian fish fins.

A defining feature of tetrapod evolutionary origins is the transition from fish fins to tetrapod limbs. A major change during this transition is the appearance of the autopod (hands, feet), which comprises two distinct regions, the wrist/ankle and the digits. When the autopod first appeared in Late Devonian fossil tetrapods, it was incomplete: digits evolved before the full complement of wrist/ankle bones. Early tetrapod wrists/ankles, including those with a full complement of bones, also show a sharp pattern discontinuity between proximal elements and distal elements. This suggests the presence of a discontinuity in the proximal-distal sequence of development. Such a discontinuity occurs in living urodeles, where digits form before completion of the wrist/ankle, implying developmental independence of the digits from wrist/ankle elements. We have observed comparable independent development of pectoral fin radials in the lungfish Neoceratodus (Osteichthyes: Sarcopterygii), relative to homologues of the tetrapod limb and proximal wrist elements in the main fin axis. Moreover, in the Neoceratodus fin, expression of Hoxd13 closely matches late expression patterns observed in the tetrapod autopod. This evidence suggests that Neoceratodus fin radials and tetrapod digits may be patterned by shared mechanisms distinct from those patterning the proximal fin/limb elements, and in that sense are homologous. The presence of independently developing radials in the distal part of the pectoral (and pelvic) fin may be a general feature of the Sarcopterygii.