Development of branchial ionocytes in embryonic and larval stages of cloudy catshark, Scyliorhinus torazame.

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.

Zebrafish Klf4 maintains the ionocyte progenitor population by regulating epidermal stem cell proliferation and lateral inhibition.

In the skin and gill epidermis of fish, ionocytes develop alongside keratinocytes and maintain body fluid ionic homeostasis that is essential for adaptation to environmental fluctuations. It is known that ionocyte progenitors in zebrafish embryos are specified from p63+ epidermal stem cells through a patterning process involving DeltaC (Dlc)-Notch-mediated lateral inhibition, which selects scattered dlc+ cells into the ionocyte progenitor fate. However, mechanisms by which the ionocyte progenitor population is modulated remain unclear. Krüppel-like factor 4 (Klf4) transcription factor was previously implicated in the terminal differentiation of mammalian skin epidermis and is known for its bifunctional regulation of cell proliferation in a tissue context-dependent manner. Here, we report novel roles for zebrafish Klf4 in the ventral ectoderm during embryonic skin development. We found that Klf4 was expressed in p63+ epidermal stem cells of the ventral ectoderm from 90% epiboly onward. Knockdown or knockout of klf4 expression reduced the proliferation rate of p63+ stem cells, resulting in decreased numbers of p63+ stem cells, dlc-p63+ keratinocyte progenitors and dlc+ p63+ ionocyte progenitor cells. These reductions subsequently led to diminished keratinocyte and ionocyte densities and resulted from upregulation of the well-known cell cycle regulators, p53 and cdkn1a/p21. Moreover, mutation analyses of the KLF motif in the dlc promoter, combined with VP16-klf4 or engrailed-klf4 mRNA overexpression analyses, showed that Klf4 can bind the dlc promoter and modulate lateral inhibition by directly repressing dlc expression. This idea was further supported by observing the lateral inhibition outcomes in klf4-overexpressing or knockdown embryos. Overall, our experiments delineate novel roles for zebrafish Klf4 in regulating the ionocyte progenitor population throughout early stem cell stage to initiation of terminal differentiation, which is dependent on Dlc-Notch-mediated lateral inhibition.

The deuterostome context of chordate origins.

Our understanding of vertebrate origins is powerfully informed by comparative morphology, embryology and genomics of chordates, hemichordates and echinoderms, which together make up the deuterostome clade. Striking body-plan differences among these phyla have historically hindered the identification of ancestral morphological features, but recent progress in molecular genetics and embryology has revealed deep similarities in body-axis formation and organization across deuterostomes, at stages before morphological differences develop. These developmental genetic features, along with robust support of pharyngeal gill slits as a shared deuterostome character, provide the foundation for the emergence of chordates.

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.

Genetic lineage labeling in zebrafish uncovers novel neural crest contributions to the head, including gill pillar cells.

At the protochordate-vertebrate transition, a new predatory lifestyle and increased body size coincided with the appearance of a true head. Characteristic innovations of this head are a skull protecting and accommodating a centralized nervous system, a jaw for prey capture and gills as respiratory organs. The neural crest (NC) is a major ontogenetic source for the 'new head' of vertebrates and its contribution to the cranial skeleton has been intensively studied in different model organisms. However, the role of NC in the expansion of the respiratory surface of the gills has been neglected. Here, we use genetic lineage labeling to address the contribution of NC to specific head structures, in particular to the gills of adult zebrafish. We generated a sox10:ER(T2)-Cre line and labeled NC cells by inducing Cre/loxP recombination with tamoxifen at embryonic stages. In juvenile and adult fish, we identified numerous established NC derivatives and, in the cranium, we precisely defined the crest/mesoderm interface of the skull roof. We show the NC origin of the opercular bones and of multiple cell types contributing to the barbels, chemosensory organs located in the mouth region. In the gills, we observed labeled primary and secondary lamellae. Clonal analysis reveals that pillar cells, a craniate innovation that mechanically supports the filaments and forms gill-specific capillaries, have a NC origin. Our data point to a crucial role for the NC in enabling more efficient gas exchange, thus uncovering a novel, direct involvement of this embryonic tissue in the evolution of respiratory systems at the protochordate-vertebrate transition.

Developmental cardiorespiratory physiology of the air-breathing tropical gar, Atractosteus tropicus.

The physiological transition to aerial breathing in larval air-breathing fishes is poorly understood. We investigated gill ventilation frequency (fG), heart rate (fH), and air breathing frequency (fAB) as a function of development, activity, hypoxia, and temperature in embryos/larvae from day (D) 2.5 to D30 posthatch of the tropical gar, Atractosteus tropicus, an obligate air breather. Gill ventilation at 28°C began at approximately D2, peaking at ∼75 beats/min on D5, before declining to ∼55 beats/min at D30. Heart beat began ∼36-48 h postfertilization and ∼1 day before hatching. fH peaked between D3 and D10 at ∼140 beats/min, remaining at this level through D30. Air breathing started very early at D2.5 to D3.5 at 1-2 breaths/h, increasing to ∼30 breaths/h at D15 and D30. Forced activity at all stages resulted in a rapid but brief increase in both fG and fH, (but not fAB), indicating that even in these early larval stages, reflex control existed over both ventilation and circulation prior to its increasing importance in older fishes. Acute progressive hypoxia increased fG in D2.5-D10 larvae, but decreased fG in older larvae (≥D15), possibly to prevent branchial O2 loss into surrounding water. Temperature sensitivity of fG and fH measured at 20°C, 25°C, 28°C and 38°C was largely independent of development, with a Q10 between 20°C and 38°C of ∼2.4 and ∼1.5 for fG and fH, respectively. The rapid onset of air breathing, coupled with both respiratory and cardiovascular reflexes as early as D2.5, indicates that larval A. tropicus develops "in the fast lane."

Physiological and molecular ontogeny of branchial and extra-branchial urea excretion in posthatch rainbow trout (Oncorhynchus mykiss).

All teleost fish produce ammonia as a metabolic waste product. In embryos, ammonia excretion is limited by the chorion, and fish must detoxify ammonia by synthesizing urea via the ornithine urea cycle (OUC). Although urea is produced by embryos and larvae, urea excretion (J(urea)) is typically low until yolk sac absorption, increasing thereafter. The aim of this study was to determine the physiological and molecular characteristics of J(urea) by posthatch rainbow trout (Oncorhynchus mykiss). Following hatch, whole body urea concentration decreased over time, while J(urea) increased following yolk sac absorption. From 12 to 40 days posthatch (dph), extra-branchial routes of excretion accounted for the majority of J(urea), while the gills became the dominant site for J(urea) only after 55 dph. This represents the most delayed branchial ontogeny of any process studied to date. Urea transporter (UT) gene expression in the gills and skin increased over development, consistent with increases in branchial and extra-branchial J(urea). Following exposure to 25 mmol/l urea, the accumulation and subsequent elimination of exogenous urea was much greater at 55 dph than 12 dph, consistent with increased UT expression. Notably, UT gene expression in the gills of 55 dph larvae increased in response to high urea. In summary, there is a clear increase in urea transport capacity over posthatch development, despite a decrease in OUC activity.

Irradiation of rainbow trout at early life stages results in trans-generational effects including the induction of a bystander effect in non-irradiated fish.

The bystander effect, a non-targeted effect (NTE) of radiation, which describes the response by non-irradiated organisms to signals emitted by irradiated organisms, has been documented in a number of fish species. However transgenerational effects of radiation (including NTE) have yet to be studied in fish. Therefore rainbow trout, which were irradiated as eggs at 48h after fertilisation, eyed eggs, yolk sac larvae or first feeders, were bred to generate a F1 generation and these F1 fish were bred to generate a F2 generation. F1 and F2 fish were swam with non-irradiated bystander fish. Media from explants of F1 eyed eggs, F1 one year old fish gill and F1 two year old fish gill and spleen samples, and F2 two year old gill and spleen samples, as well as from bystander eggs/fish, was used to treat a reporter cell line, which was then assayed for changes in cellular survival/growth. The results were complex and dependent on irradiation history, age (in the case of the F1 generation), and were tissue specific. For example, irradiation of one parent often resulted in effects not seen with irradiation of both parents. This suggests that, unlike mammals, in certain circumstances maternal and paternal irradiation may be equally important. This study also showed that trout can induce a bystander effect 2 generations after irradiation, which further emphasises the importance of the bystander effect in aquatic radiobiology. Given the complex community structure in aquatic ecosystems, these results may have significant implications for environmental radiological protection.

The role of the Pax1/9 gene in the early development of amphioxus pharyngeal gill slits.

The pharynx is a major characteristic of chordates. Compared with vertebrates, amphioxus has an advantage for the study of pharynx development, as embryos lack neural crest, and the pharynx is mainly derived from endoderm cells. The Pax1/9 subfamily genes have essential roles in vertebrate pharyngeal patterning, but it is not known if the Pax1/9 gene has similar functions in amphioxus pharynx development. To answer this question, we examined the Pax1/9 gene expression pattern in amphioxus embryos at different developmental stages, and observed morphological changes following Pax1/9 knockdown. RT-qPCR analysis indicated that Pax1/9 expression was initiated during early neurula stage and rapidly peaked during mid-neurula stage. Furthermore, in situ hybridization analysis showed that Pax1/9 transcripts were localized exclusively in the most endodermal region of the developing pharynx in early neurula stage embryos; however, Pax1/9 expression was strikingly down-regulated in the region where gill slits would form from the fusion of endoderm and ectoderm in subsequent developmental stages and was maintained in the border regions between adjacent gill slits. Knockdown of Pax1/9 function using both morpholino and siRNA approaches led to embryonic defects in the first three gill slits, and fusion of the first two gill slits. Moreover, the expression levels of the pharyngeal marker genes Six1/2 and Tbx1/10 were reduced in Pax1/9 knockdown embryos. From these observations, we concluded that the Pax1/9 gene has an important role in the initial differentiation of amphioxus pharyngeal endoderm and in the formation of gill slits, most likely via modulation of Six1/2 and Tbx1/10 expression.

Two developmentally temporal quantitative trait loci underlie convergent evolution of increased branchial bone length in sticklebacks.

In convergent evolution, similar phenotypes evolve repeatedly in independent populations, often reflecting adaptation to similar environments. Understanding whether convergent evolution proceeds via similar or different genetic and developmental mechanisms offers insight towards the repeatability and predictability of evolution. Oceanic populations of threespine stickleback fish, Gasterosteus aculeatus, have repeatedly colonized countless freshwater lakes and streams, where new diets lead to morphological adaptations related to feeding. Here, we show that heritable increases in branchial bone length have convergently evolved in two independently derived freshwater stickleback populations. In both populations, an increased bone growth rate in juveniles underlies the convergent adult phenotype, and one population also has a longer cartilage template. Using F2 crosses from these two freshwater populations, we show that two quantitative trait loci (QTL) control branchial bone length at distinct points in development. In both populations, a QTL on chromosome 21 controls bone length throughout juvenile development, and a QTL on chromosome 4 controls bone length only in adults. In addition to these similar developmental profiles, these QTL show similar chromosomal locations in both populations. Our results suggest that sticklebacks have convergently evolved longer branchial bones using similar genetic and developmental programmes in two independently derived populations.

α-ENaC in bullfrog embryo: expression in cement gland, gills and skin.

The epithelial sodium channel (ENaC) is involved in Na(+) responses such as Na(+) absorption and salt taste. The alpha ENaC subunit (α-ENaC) is expressed in the skin of both the adult and larval (tadpole) bullfrog. α-ENaC expression in the developing bullfrog embryo has not been previously investigated. In this study, the expression of α-ENaC at various stages (Sts.) of bullfrog embryonic development is assessed by western blot and immunofluorescence analysis. Bullfrog α-ENaC (α-fENaC) protein was detected by western blot in embryos at Sts. (Gosner/Shumway) 19, 21 and 25. Immunofluorescence studies indicate that α-fENaC was localized to the embryonic cement glands at St. 18 (muscular response), St. 19 (heart beat) and St. 21 (mouth open and/or cornea transparent), to the external gills at St. 21 and to the outermost cell-layer of the skin at St. 25 (operculum complete). The function(s) of ENaC in these embryonic structures remain to be elucidated.

Holocephalan embryos provide evidence for gill arch appendage reduction and opercular evolution in cartilaginous fishes.

Chondrichthyans possess endoskeletal appendages called branchial rays that extend laterally from their hyoid and gill-bearing (branchial) arches. Branchial ray outgrowth, like tetrapod limb outgrowth, is maintained by Sonic hedgehog (Shh) signaling. In limbs, distal endoskeletal elements fail to form in the absence of normal Shh signaling, whereas shortened duration of Shh expression correlates with distal endoskeletal reduction in naturally variable populations. Chondrichthyans also exhibit natural variation with respect to branchial ray distribution--elasmobranchs (sharks and batoids) possess a series of ray-supported septa on their hyoid and gill arches, whereas holocephalans (chimaeras) possess a single hyoid arch ray-supported operculum. Here we show that the elongate hyoid rays of the holocephalan Callorhinchus milii grow in association with sustained Shh expression within an opercular epithelial fold, whereas Shh is only transiently expressed in the gill arches. Coincident with this transient Shh expression, branchial ray outgrowth is initiated in C. milii but is not maintained, yielding previously unrecognized vestigial gill arch branchial rays. This is in contrast to the condition seen in sharks, where sustained Shh expression corresponds to the presence of fully formed branchial rays on the hyoid and gill arches. Considered in light of current hypotheses of chondrichthyan phylogeny, our data suggest that the holocephalan operculum evolved in concert with gill arch appendage reduction by attenuation of Shh-mediated branchial ray outgrowth, and that chondrichthyan branchial rays and tetrapod limbs exhibit parallel developmental mechanisms of evolutionary reduction.

Adaptation to freshwater in the palaemonid shrimp Macrobrachium amazonicum: comparative ontogeny of osmoregulatory organs.

The ontogeny of osmoregulatory organs was studied in two geographically isolated populations of the palaemonid shrimp Macrobrachium amazonicum, one originating from the Amazon estuary (A) and the other from inland waters of the Pantanal (P) in northeastern and southwestern Brazil, respectively. A previous investigation had shown that the estuarine population is able to hypo-osmoregulate in seawater, whereas the hololimnetic inland population has lost this physiological function. In the present study, the structural development of the branchial chamber and excretory glands and the presence of Na(+)/K(+)-ATPase (NKA) were compared between populations and between larval and juvenile stages after exposure to two salinities representing hypo- and hypertonic environments. In the newly hatched zoea I stage of both populations, gills were absent and NKA was localized along the inner epithelium of the branchiostegite. In intermediate (zoea V) and late larval stages (decapodids), significant differences between the two populations were observed in gill development and NKA expression. In juveniles, NKA was detected in the gills and branchiostegite, with no differences between populations. At all developmental stages and in both populations, NKA was present in the antennal glands upon hatching. The strong hypo-osmoregulatory capacity of the early developmental stages in population A could be linked to ion transport along the inner side of the branchiostegite; this seemed to be absent or weak in population P. The presence of fully functional gills expressing NKA appears to be essential for efficient hyper-osmoregulation in late developmental stages during successful freshwater adaptation and colonization.

Serotonergic and cholinergic elements of the hypoxic ventilatory response in developing zebrafish.

The chemosensory roles of gill neuroepithelial cells (NECs) in mediating the hyperventilatory response to hypoxia are not clearly defined in fish. While serotonin (5-HT) is the predominant neurotransmitter in O(2)-sensitive gill NECs, acetylcholine (ACh) plays a more prominent role in O(2) sensing in terrestrial vertebrates. The present study characterized the developmental chronology of potential serotonergic and cholinergic chemosensory pathways of the gill in the model vertebrate, the zebrafish (Danio rerio). In immunolabelled whole gills from larvae, serotonergic NECs were observed in epithelia of the gill filaments and gill arches, while non-serotonergic NECs were found primarily in the gill arches. Acclimation of developing zebrafish to hypoxia (P(O2)=75 mmHg) reduced the number of serotonergic NECs observed at 7 days post-fertilization (d.p.f.), and this effect was absent at 10 d.p.f. In vivo administration of 5-HT mimicked hypoxia by increasing ventilation frequency (f(V)) in early stage (7-10 d.p.f.) and late stage larvae (14-21 d.p.f.), while ACh increased f(V) only in late stage larvae. In time course experiments, application of ketanserin inhibited the hyperventilatory response to acute hypoxia (P(O2)=25 mmHg) at 10 d.p.f., while hexamethonium did not have this effect until 12 d.p.f. Cells immunoreactive for the vesicular acetylcholine transporter (VAChT) began to appear in the gill filaments by 14 d.p.f. Characterization in adult gills revealed that VAChT-positive cells were a separate population of neurosecretory cells of the gill filaments. These studies suggest that serotonergic and cholinergic pathways in the zebrafish gill develop at different times and contribute to the hyperventilatory response to hypoxia.

The dynamic nitric oxide pattern in developing cuttlefish Sepia officinalis.

BACKGROUND: Nitric oxide (NO) is implied in many important biological processes in all metazoans from porifera to chordates. In the cuttlefish Sepia officinalis NO plays a key role in the defense system and neurotransmission. RESULTS: Here, we detected for the first time NO, NO synthase (NOS) and transcript levels during the development of S. officinalis. The spatial pattern of NO and NOS is very dynamic, it begins during organogenesis in ganglia and epithelial tissues, as well as in sensory cells. At later stages, NO and NOS appear in organs and/or structures, including Hoyle organ, gills and suckers. Temporal expression of NOS, followed by real-time PCR, changes during development reaching the maximum level of expression at stage 26. CONCLUSIONS: Overall these data suggest the involvement of NO during cuttlefish development in different fundamental processes, such as differentiation of neural and nonneural structures, ciliary beating, sensory cell maintaining, and organ functioning.

Ammonia excretion via Rhcg1 facilitates Na⁺ uptake in larval zebrafish, Danio rerio, in acidic water.

The involvement of a Na(+)/H(+) exchanger (NHE) in mediating Na(+) uptake by freshwater fish is currently debated. Although supported indirectly by empirical molecular and pharmacological data, theoretically its operation should be constrained thermodynamically, owing to unfavorable chemical gradients. Recently, there has been an increasing focus on ammonia channels (Rh proteins) as potentially contributing to Na(+) uptake across the freshwater fish gill. In this study, we tested the hypothesis that Rhcg1, a specific apical isoform of Rh protein, is critically important in facilitating Na(+) uptake in zebrafish larvae via its interaction with NHE. Treating larvae (4 days postfertilization) with 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of NHE, caused a significant reduction in Na(+) uptake in fish reared in acidic water (pH ∼ 4.0). A role for NHE in Na(+) uptake was further confirmed by translational knockdown of NHE3b, an isoform of NHE thought to be responsible for Na(+)/H(+) exchange in zebrafish larvae. Exposing the larvae reared in acidic water to 5 mM external ammonium sulfate or increasing the buffering capacity of the water with 10 mM HEPES caused concurrent reductions in ammonia excretion and Na(+) uptake. Furthermore, translational knockdown of Rhcg1 significantly reduced ammonia excretion and Na(+) uptake in larvae chronically (4 days) or acutely (24 h) exposed to acidic water. Unlike in sham-injected larvae, EIPA did not affect Na(+) uptake in fish experiencing Rhcg1 knockdown. Additionally, exposure of larvae to bafilomycin A1 (an inhibitor of H(+)-ATPase) significantly reduced Na(+) uptake in fish reared in acidic water. These observations suggest the existence of multiple mechanisms of Na(+) uptake in larval zebrafish in acidic water: one in which Na(+) uptake via NHE3b is linked to ammonia excretion via Rhcg1, and another facilitated by H(+)-ATPase.

Embryonic ionocytes in the European sea bass (Dicentrarchus labrax): Structure and functionality.

Early ionocytes have been studied in the European sea bass (Dicentrarchus labrax) embryos. Structural and functional aspects were analyzed and compared with those observed in the same conditions (38 ppt) in post hatching stages. Immunolocalization of Na(+) /K(+) -ATPase (NKA) in embryos revealed the presence of ionocytes on the yolk sac membrane from a stage 12 pair of somites (S), and an original cluster around the first gill slits from stage 14S. Histological investigations suggested that from these cells, close to the future gill chambers, originate the ionocytes observed on gill arches and gill filaments after hatching. Triple immunocytochemical staining, including NKA, various Na(+) /K(+) /2Cl⁻ cotransporters (NKCCs) and the chloride channel "cystic fibrosis transmembrane regulator" (CFTR), point to the occurrence of immature and mature ionocytes in early and late embryonic stages at different sites. These observations were completed with transmission electronic microscopy. The degree of functionality of ionocytes is discussed according to these results. Yolk sac membrane ionocytes and enteric ionocytes seem to have an early role in embryonic osmoregulation, whereas gill slits tegumentary ionocytes are presumed to be fully efficient after hatching.

Stage-dependent toxicity of 2,4-dichlorophenoxyacetic on the embryonic development of a South American toad, Rhinella arenarum.

The acute and short term chronic toxicity of both the herbicide butyl ester of 2,4-Dichlorophenoxyacetic acid (2,4-D) and a commercial formulation (CF) were evaluated on Rhinella (= Bufo) arenarum embryos at different developmental stages. Adverse effects were analyzed by means of the isotoxicity curves for lethality, malformations, stage-dependent susceptibility, and ultrastructural features. For all experimental conditions, the CF was more toxic, up to 10 times, than the active ingredient, being the open mouth stage (S.21) the most susceptible to the herbicide. For continuous treatment conditions, the early embryonic development was the most susceptible to 2,4-D and the LC50s for 96 and 168 h were 9.06 and 7.76 mg L(-1) respectively. In addition, both the active ingredient and the CF were highly teratogenic, resulting in reduced body size, delayed development, microcephaly, agenesis of gills, and abnormal cellular proliferation processes as the main adverse effects. According to US EPA, 2,4-D in agricultural scenarios may be up to three times higher than the NOEC values for teratogenic effects reported in this study. Therefore, they might represent a risk for amphibians. This study also points out the relevance of reporting the susceptibility of embryos at different developmental stages to both the active ingredient and the CF of agrochemicals in order to protect nontarget organisms.

Embryonic ontogeny of three species of Horned Frogs, with a review of early development in Ceratophryidae.

Horned Frogs of the family Ceratophryidae are conspicuous anurans represented by three endemic South American genera. Most ceratophryids inhabit semiarid environments, but three species of Ceratophrys occupy tropical or temperate humid areas. Several morphological and behavioral characters of larvae and adults are conserved across the family. Based on examination of specimens and accounts in the literature, the embryonic development of C. ornata, C. cranwelli, and the monotypic genus Chacophrys are described and compared with that of species of Lepidobatrachus. Ceratophryid embryos share a suite of morphological features and heterochronic shifts during development. Most features, such as gill structure, ciliation, early hatching, and precocious differentiation of the gut and hind limbs, are shared by all the species regardless the differences in the habitats that occupy. This is consistent with previous observations of some adult characters, and likely supports the hypothesis of an early diversification of ceratophryids in semiarid environments. Other embryonic features, such as the morphology and ontogeny of the oral disc and digestive tract, are correlated with larval feeding habits and vary within the family. The evolutionary and ecological significance of some conserved characters (e.g., gastrulation pattern, Type-A adhesive glands) and other taxon-specific features (e.g., nasal appendix) remain to be explored in the group.

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.