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The liver architecture of vertebrates can be classified into two types, the portal triad type (having periportal bile ducts) and the non-portal triad type (having non-periportal bile ducts). The former is detectable in the tetrapod liver whereas the lungfish liver has the latter. It remains to be revealed which type of hepatic architecture the coelacanth, which together with the lungfish belongs to the Sarcopterygii, possesses. The present study was undertaken to determine the histological characteristics of the coelacanth liver, and to compare with those of other vertebrates. The coelacanth liver had periportal bile ducts and ductules as detected in mammalian livers. The hepatic artery was found around large portal veins. Hagfish, shark, bichir, sturgeon, bowfin and frog livers had periportal bile ducts and bile ductules, whereas most intrahepatic bile ducts of the lungfish were independent of the distribution of the portal veins as seen in the Otocephala and Euteleostei. The lungfish liver developed duct and ductule structures in the parenchyma. These data indicate that the coelacanth liver had a mammalian-type hepatic architecture with a portal triad, and that the ancestors of tetrapods may have had a portal triad-type liver architecture.

Nitric oxide (NO) is an ancestral key signaling molecule essential for life and has enormous versatility in biological systems, including cardiovascular homeostasis, neurotransmission, and immunity. Although our knowledge of nitric oxide synthases (Nos), the enzymes that synthesize NO in vivo, is substantial, the origin of a large and diversified repertoire of nos gene orthologs in fish with respect to tetrapods remains a puzzle. The recent identification of nos3 in the ray-finned fish spotted gar, which was considered lost in the ray-finned fish lineage, changed this perspective. This prompted us to explore nos gene evolution and expression in depth, surveying vertebrate species representing key evolutionary nodes. This study provides noteworthy findings: first, nos2 experienced several lineage-specific gene duplications and losses. Second, nos3 was found to be lost independently in two different teleost lineages, Elopomorpha and Clupeocephala. Third, the expression of at least one nos paralog in the gills of developing shark, bichir, sturgeon, and gar but not in arctic lamprey, suggest that nos expression in this organ likely arose in the last common ancestor of gnathostomes. These results provide a framework for continuing research on nos genes’ roles, highlighting subfunctionalization and reciprocal loss of function that occurred in different lineages during vertebrate genome duplications.

The present paper is the first comparative study on the astroglia of several actinopterygian species at different phylogenetical positions, teleosts (16 species), and non-teleosts (3 species), based on the immunohistochemical staining of GFAP (glial fibrillary acidic protein), the characteristic cytoskeletal intermediary filament protein, and immunohistochemical marker of astroglia. The question was, how the astroglial architecture reflexes the high diversity of this largest vertebrate group. The actinopterygian telencephalon has a so-called ‘eversive’ development in contrast to the ‘evagination’ found in sarcopterygii (including tetrapods). Several brain parts either have no equivalents in tetrapod vertebrates (e.g., torus longitudinalis, lobus inferior, lobus nervi vagi), or have rather different shapes (e.g., the cerebellum). GFAP was visualized applying DAKO polyclonal anti-GFAP serum. The study was focused mainly on the telencephalon (eversion), tectum (visual orientation), and cerebellum (motor coordination) where the evolutionary changes were most expected, but the other areas were also investigated. The predominant astroglial elements were tanycytes (long, thin, fiber-like cells). In the teleost telencephala a ‘fan-shape’ re-arrangement of radial glia reflects the eversion. In bichir, starlet, and gar, in which the eversion is less pronounced, the ‘fan-shape’ re-arrangement did not form. In the tectum the radial glial processes were immunostained, but in Ostariophysi and Euteleostei it did not extend into their deep segments. In the cerebellum Bergmann-like glia was found in each group, including non-teleosts, except for Cyprinidae. The vagal lobe was uniquely enlarged and layered in Cyprininae, and had a corresponding layered astroglial system, which left almost free of GFAP the zones of sensory and motor neurons. In conclusion, despite the diversity and evolutionary alterations of Actinopterygii brains, the diversity of the astroglial architecture is moderate. In contrast to Chondrichthyes and Amniotes; in Actinopterygii true astrocytes (stellate-shaped extraependymal cells) did not appear during evolution, and the expansion of GFAP-free areas was limited.

The members of the globin superfamily are a classical model system to investigate gene evolution and their fates as well as the diversity of protein function. One of the best-known globins is myoglobin (Mb), which is mainly expressed in heart muscle and transports oxygen from the sarcolemma to the mitochondria. Most vertebrates harbor a single copy of the myoglobin gene, but some fish species have multiple myoglobin genes. Phylogenetic analyses indicate an independent emergence of multiple myoglobin genes, whereby the origin is mostly the last common ancestor of each order. By analyzing different transcriptome data sets, we found at least 15 multiple myoglobin genes in the polypterid gray bichir (Polypterus senegalus) and reedfish (Erpetoichthys calabaricus). In reedfish, the myoglobin genes are expressed in a broad range of tissues but show very different expression values. In contrast, the Mb genes of the gray bichir show a rather scattered expression pattern; only a few Mb genes were found expressed in the analyzed tissues. Both, gray bichir and reedfish possess lungs which enable them to inhabit shallow and swampy waters throughout tropical Africa with frequently fluctuating and low oxygen concentrations. The myoglobin repertoire probably reflects the molecular adaptation to these conditions. The sequence divergence, the substitution rate, and the different expression pattern of multiple myoglobin genes in gray bichir and reedfish imply different functions, probably through sub- and neofunctionalization during evolution.

BackgroundVertebrate teeth exhibit a wide range of regenerative systems. Many species, including most mammals, reptiles, and amphibians, form replacement teeth at a histologically distinct location called the successional dental lamina, while other species do not employ such a system. Notably, a ‘lamina-less’ tooth replacement condition is found in a paraphyletic array of ray-finned fishes, such as stickleback, trout, cod, medaka, and bichir. Furthermore, the position, renewal potential, and latency times appear to vary drastically across different vertebrate tooth regeneration systems. The progenitor cells underlying tooth regeneration thus present highly divergent arrangements and potentials. Given the spectrum of regeneration systems present in vertebrates, it is unclear if morphologically divergent tooth regeneration systems deploy an overlapping battery of genes in their naïve dental tissues.ResultsIn the present work, we aimed to determine whether or not tooth progenitor epithelia could be composed of a conserved cell type between vertebrate dentitions with divergent regeneration systems. To address this question, we compared the pharyngeal tooth regeneration processes in two ray-finned fishes: zebrafish (Danio rerio) and threespine stickleback (Gasterosteus aculeatus). These two teleost species diverged approximately 250 million years ago and demonstrate some stark differences in dental morphology and regeneration. Here, we find that the naïve successional dental lamina in zebrafish expresses a battery of nine genes (bmpr1aa, bmp6, cd34, gli1, igfbp5a, lgr4, lgr6, nfatc1,andpitx2), while active Wnt signaling andLef1expression occur during early morphogenesis stages of tooth development. We also find that, despite the absence of a histologically distinct successional dental lamina in stickleback tooth fields, the same battery of nine genes (Bmpr1a,Bmp6,CD34,Gli1,Igfbp5a,Lgr4,Lgr6,Nfatc1, andPitx2) are expressed in the basalmost endodermal cell layer, which is the region most closely associated with replacement tooth germs. Like zebrafish, stickleback replacement tooth germs additionally expressLef1and exhibit active Wnt signaling. Thus, two fish systems that either have an organized successional dental lamina (zebrafish) or lack a morphologically distinct successional dental lamina (sticklebacks) deploy similar genetic programs during tooth regeneration.ConclusionsWe propose that the expression domains described here delineate a highly conserved “successional dental epithelium” (SDE). Furthermore, a set of orthologous genes is known to mark hair follicle epithelial stem cells in mice, suggesting that regenerative systems in other epithelial appendages may utilize a related epithelial progenitor cell type, despite the highly derived nature of the resulting functional organs.

Ventricular natriuretic peptide (VNP) is a cardiac hormone found in chondrostei and primitive teleostei, such as sturgeon, bichir, eel, and trout. The VNP gene does not exist in mammals, reptiles, amphibians, and advanced teleost species (medaka and pufferfish). VNP has a long C-terminal tail sequence extending from an intramolecular ring, which is distinct in molecular structure from other NPs. VNP shares both A-type and B-type natriuretic peptide receptors (NPR-A and NPR-B) with ANP/BNP and CNP, respectively. A major stimulus of VNP release is acute salt-load in eels but chronic volume-load in trout. Like ANP, VNP potently decreases plasma Na+ concentration by inhibiting drinking and subsequent intestinal NaCl absorption in seawater-adapted eels. VNP also has a strong hypotensive effect in eel and trout. In summary, VNP is important for osmoregulation and blood pressure control in teleost fish.

The association between lateral‐line canals and skull bones in fishes has been the subject of several studies and raised a series of controversies, particularly with regard to the hypothesized role of lateral‐line organs (i.e. neuromasts) in osteogenesis and the consequences for hypotheses of homology of the bones associated with lateral‐line canals. Polypteridae, a group of freshwater fishes that occupies a key phylogenetic position as the most basal extant lineage of ray‐finned fishes (Actinopterygii), provides an interesting model for the study of the relationships between lateral‐line canals and skull bones. We describe the development of bones associated with lateral‐line canals in the Senegal Bichir, Polypterus senegalus, and use these data to re‐address previous hypotheses of homology of skull bones of polypterids. We demonstrate that the lateral‐line canals constitute a separate component of the dermatocranium that may interact with a membranodermal component, thereby forming compound bones in the adult. Differences in the interactions between these components determine the characteristics of the development of each independent bone in the skull of adult P. senegalus. Our results shed light on long‐standing controversies about the identity of skull bones such as the rostral, preopercle, and sphenotic in Polypteridae, and suggest the presence of an ancestral two‐component pattern of formation of bones associated with lateral‐line canals in bony fishes. These findings reveal the need to re‐address previous hypotheses of homology of bones associated with lateral‐line canals in different groups of bony fishes, especially fossil taxa.

Despite the wide variety of adaptive modifications in the oral and facial regions of vertebrates, their early oropharyngeal development is considered strictly uniform. It involves sequential formation of the mouth and pharyngeal pouches, with ectoderm outlining the outer surface and endoderm the inner surface, as a rule. At the extreme anterior domain of vertebrate embryos, the ectoderm and endoderm directly juxtapose and initial development of this earliest ecto-endoderm interface, the primary mouth, typically involves ectodermal stomodeal invagination that limits the anterior expansion of the foregut endoderm. Here we present evidence that in embryos of extant non-teleost fishes, oral (stomodeal) formation is preceded by the development of prominent pre-oral gut diverticula (POGD) between the forebrain and roof of the forming mouth. Micro-computed tomography (micro-CT) imaging of bichir, sturgeon and gar embryos revealed that foregut outpocketing at the pre-oral domain begins even before the sequential formation of pharyngeal pouches. The presence of foregut-derived cells in the front of the mouth was further confirmed by in vivo experiments that allowed specific tracing of the early endodermal lining. We show that POGD in sturgeons contribute to the orofacial surface of their larvae, comprising oral teeth, lips, and sensory barbels. To our knowledge, this is the first thorough evidence for endodermal origin of external craniofacial structures in any vertebrate. In bichir and gar embryos, POGD form prominent cranial adhesive organs that are characteristic of the ancient bauplan of free-living chordate larvae. POGD hence seem arguably to be ancestral for all ray-finned fishes, and their topology, pharyngeal-like morphogenesis and gene expression suggest that they are evolutionarily related to the foregut-derived diverticula of early chordate and hemichordate embryos. The formation of POGD might thus represent an ancestral developmental module with deep deuterostome origins.

Polypterus senegalus Cuvier, 1829 is one of the most basal living actinopterygian fish and a member of the Actinopterygii. We analyzed the spatial and temporal pattern of cranial muscle development of P. senegalus using whole-mount immunostaining and serial sectioning. We described the detailed structure of the external gill muscles which divided into dorsal and ventral parts after yolk exhaustion. The pattern of the division is similar to that of urodeles. We suggest that, the external gill muscles of P. senegalus are involved in spreading and folding of the external gill stem and the branches. The fibers of the external gill muscles appear postero-lateral to the auditory capsule. In addition, the facial nerve passes through the external gills. Therefore, the external gill muscles are probably derived from the m. constrictor hyoideus dorsalis. In contrast to previous studies, we described the mm. interhyoideus and hyohyoideus fibers as independent components in the yolk-sac larvae. The m. hyohyoideus fibers appear lateral to the edge of the ventral portion of the external gill muscles, which are probably derived from the m. constrictor hyoideus dorsalis. These findings suggest that the m. hyohyoidues is derived from the m. constrictor hyoideus dorsalis in P. senegalus. In other actinopterygians, the m. hyohyoideus is derived from the m. constrictor hyoideus ventralis; therefore, the homology of the m. hyohyoidues of P. senegalus and other actinopterygians remains unclear. J. Morphol. 278:450-463, 2017. © 2017 Wiley Periodicals, Inc.

Recent progress in whole genome sequencing has revealed that animals have various kinds of opsin genes for photoreception. Among them, most opsin genes have introns in their coding regions. However, it has been known for a long time that teleost retinas express intron-less rhodopsin genes, which are presumed to have been formed by retroduplication from an ancestral intron-containing rhodopsin gene. In addition, teleosts have an intron-containing rhodopsin gene (exo-rhodopsin) exclusively for pineal photoreception. In this study, to unravel the evolutionary origin of the two teleost rhodopsin genes, we analyzed the rhodopsin genes of non-teleost fishes in the Actinopterygii. The phylogenetic analysis of full-length sequences of bichir, sturgeon and gar rhodopsins revealed that retroduplication of the rhodopsin gene occurred after branching of the bichir lineage. In addition, analysis of the tissue distribution and the molecular properties of bichir, sturgeon and gar rhodopsins showed that the abundant and exclusive expression of intron-containing rhodopsin in the pineal gland and the short lifetime of its meta II intermediate, which leads to optimization for pineal photoreception, were achieved after branching of the gar lineage. Based on these results, we propose a stepwise evolutionary model of teleost intron-containing and intron-less rhodopsin genes.

In most vertebrates, pharyngeal arches form in a stereotypic anterior-to-posterior progression. To gain insight into the mechanisms underlying evolutionary changes in pharyngeal arch development, here we investigate embryos and larvae of bichirs. Bichirs represent the earliest diverged living group of ray-finned fishes, and possess intriguing traits otherwise typical for lobe-finned fishes such as ventral paired lungs and larval external gills. In bichir embryos, we find that the anteroposterior way of formation of cranial segments is modified by the unique acceleration of the entire hyoid arch segment, with earlier and orchestrated development of the endodermal, mesodermal, and neural crest tissues. This major heterochronic shift in the anteroposterior developmental sequence enables early appearance of the external gills that represent key breathing organs of bichir free-living embryos and early larvae. Bichirs thus stay as unique models for understanding developmental mechanisms facilitating increased breathing capacity.

Rich fossil evidence suggests that many traits and functions related to terrestrial evolution were present long before the ancestor of lobe- and ray-finned fishes. Here, we present genome sequences of the bichir, paddlefish, bowfin, and alligator gar, covering all major early divergent lineages of ray-finned fishes. Our analyses show that these species exhibit many mosaic genomic features of lobe- and ray-finned fishes. In particular, many regulatory elements for limb development are present in these fishes, supporting the hypothesis that the relevant ancestral regulation networks emerged before the origin of tetrapods. Transcriptome analyses confirm the homology between the lung and swim bladder and reveal the presence of functional lung-related genes in early ray-finned fishes. Furthermore, we functionally validate the essential role of a jawed vertebrate highly conserved element for cardiovascular development. Our results imply the ancestors of jawed vertebrates already had the potential gene networks for cardio-respiratory systems supporting air breathing.

In vertebrates, estrogens play fundamental roles in regulating reproductive activities through estrogen receptors (ESRs), and disruption of estrogen signaling is now of global concern for both wildlife and human health. To date, ESRs of only a limited number of species have been characterized. We investigated the functional diversity and molecular basis or ligand sensitivity of ESRs among ray-finned fish species (Actinopterygii), the most variable group within vertebrates. We cloned and characterized ESRs from several key species in the evolution of ray-finned fish including bichir (Polypteriformes, ESR1 and ESR2) at the basal lineage of ray-finned fish, and arowana (Osteoglossiformes, ESR1 and ESR2b) and eel (Anguilliformes, ESR1, ESR2a and ESR2b) both belonging to ancient early-branching lineages of teleosts, and suggest that ESR2a and ESR2b emerged through teleost-specific whole genome duplication, but an ESR1 paralogue has been lost in the early lineage of euteleost fish species. All cloned ESR isoforms showed similar responses to endogenous and synthetic steroidal estrogens, but they responded differently to non-steroidal estrogenic endocrine disrupting chemicals (EDCs) (e.g., ESR2a exhibits a weaker reporter activity compared with ESR2b). We show that variation in ligand sensitivity of ESRs can be attributed to phylogeny among species of different taxonomic groups in ray-finned fish. The molecular information provided contributes both to understanding of the comparative role of ESRs in the reproductive biology of fish and their comparative responses to EDCs.

Pattern recognition receptors (PRRs) play important roles in innate immunity system and trigger the specific pathogen recognition by detecting the pathogen-associated molecular patterns. The main four PRRs components including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs) and C-type lectin receptors (CLRs) were surveyed in the five genomes of non-teleost ray-finned fishes (NTR) including bichir (Polypterus senegalus), American paddlefish (Polyodon spathula), alligator gar (Atractosteus spatula), spotted gar (Lepisosteus oculatus) and bowfin (Amia calva), representing all the four major basal groups of ray-finned fishes. The result indicates that all the four PRRs components have been well established in these NTR fishes. In the RLR-MAVS signal pathway, which detects intracellular RNA ligands to induce production of type I interferons (IFNs), the MAVS was lost in bichir particularly. Also, the essential genes of recognition of Lipopolysaccharide (LPS) commonly in mammals like MD2, LY96 and LBP could not be identified in NTR fishes. It is speculated that TLR4 in NTR fishes may act as a cooperator with other PRRs and has a different pathway of recognizing LPS compared with that in mammals. In addition, we provide a survey of NLR and CLR in NTR fishes. The CLRs results suggest that Group V receptors are absent in fishes and Group II and VI receptors are well established in the early vertebrate evolution. Our comprehensive research of PRRs involving NTR fishes provides a new insight into PRR evolution in primitive vertebrate.

Bichirs are a sister group to sarcopterygian and tetrapods that can fully regenerate their endochondral-skeleton-fins. Histological and transcriptomic comparison approaches have been used to investigate the morphology and genetic basis of bichir lobe-fin regeneration, with strong down-regulation of muscle-related genes and up-regulation of ECM-related genes and developmental genes being observed. Bichir limb regeneration involves similar cellular processes to those employed by lungfish and salamander, with MARCKS-like protein (MLP) that is known to be a putative regeneration-initiating molecule in salamander, also up-regulated in the early stages of bichir lobe-fin regeneration. These gene expression results suggest that limb regeneration pathways in these amphibians have a common ancestral inheritance, consistent with evolution from endochondral-skeleton-fin structures to endochondral-skeleton-limb structures of vertebrates.

Cladistians are a group of basal nonteleost actinopterygian fishes that represent an interesting group for the study of primitive brain features, most likely present in the ancestral Osteichthyes. We have investigated the catecholaminergic (CA) systems in the brain of two representative cladistian species, the bichir Polypterus senegalus and the reedfish Erpetoichthys calabaricus, by means of antibodies against tyrosine hydroxylase (TH; the first enzyme in the synthesis of catecholamines) and dopamine (DA). Double immunohistofluorescence was performed for simultaneous detection of TH with nitric oxide synthase, choline acetyltransferase, calbindin, calretinin, and serotonin, aiming to accurately establish the localization of the CA neurons and to assess possible interactions between these neuroactive substances. All forebrain CA groups of cladistians are dopaminergic, whereas noradrenergic cells are located within the rhombencephalon. Distinct groups of DA immunoreactive (DA-ir) cells were observed in the olfactory bulb, subpallium, and preoptic area of the telencephalon. Hypothalamic groups were detected in the suprachiasmatic nucleus, retrotuberal and retromamillary areas and, in particular, the paraventricular organ showed immunoreactivity to dopamine but not to TH. Diencephalic DA-ir groups were detected in the prethalamus, posterior tubercle, and pretectum. A small DA-ir cell population was observed in the midbrain tegmentum only in Polypterus. CA cell groups were also located in the locus coeruleus, solitary tract nucleus, and area postrema within the rhombencephalon, the spinal cord, and the retina. The comparison of these results with other vertebrates, using a neuromeric analysis, shows highly conserved traits in all vertebrates studied but also evidences particular characteristics of actinopterygian fishes.

Cladistians are a group of basal actinopterygian fishes that constitute a good model for studying primitive brain features, most likely present in the ancestral bony fishes. The analysis of the nitrergic neurons (with the enzyme nitric oxide synthase; NOS) has helped in understanding important aspects of brain organization in all vertebrates studied. We investigated the nitrergic system of two cladistian species by means of specific antibodies against NOS and NADPH-diaphorase (NADPH-d) histochemistry, which, with the exception of the primary olfactory and terminal nerve fibers, labeled only for NADPH-d, yielded identical results. Double immunohistochemistry was conducted for simultaneous detection of NOS with tyrosine hydroxylase, choline acetyltransferase, calbindin, calretinin, and serotonin, to establish accurately the localization of the nitrergic neurons and fibers and to assess possible interactions between these neuroactive substances. The pattern of distribution in both species showed only subtle differences in the density of labeled cells. Distinct groups of NOS-immunoreactive cells were observed in pallial and subpallial areas, paraventricular region, tuberal and retromammillary hypothalamic areas, posterior tubercle, prethalamic and thalamic areas, optic tectum, torus semicircularis, mesencephalic tegmentum, interpeduncular nucleus, superior and middle reticular nuclei, magnocellular vestibular nucleus, solitary tract nucleus, nucleus medianus magnocellularis, the spinal cord and amacrine cells in the retina. Large neurons in cranial nerve sensory ganglia were also labeled. The comparison of these results with those from other vertebrates, using a neuromeric analysis, reveals a conserved pattern of organization of the nitrergic system from this primitive fish group to amniotes, including mammals.

The coelacanth, Latimeria chalumnae Smith, 1939 [1] (Sarcopterygii: Actinistia), together with the closely related L. menadoensis Pouyaud et al ., 1999 [2], remains the only living representative of one of the most basally-branching primary radiations of lobe-finned fishes (Sarcopterygii). Even though extant species cannot be considered ‘primitive’ due to the inherent logic of phylogenetic theory, the coelacanth nonetheless is invaluable for understanding evolutionary transformations in basal sarcopterygians as it can help in the determination of character polarity. The appearance of one novelty during early vertebrate evolution that had major implications for the success of a huge number of species is the origin of lungs. The conventional interpretation is that lungs evolved in basal bony fishes (Osteichthyes or Osteognathostomata), were maintained in the lobe-finned fishes, and eventually were transformed into a swimbladder among the ray-finned fishes (Actinopterygii) (e.g. [3]). However, the currently available data do not rule out separate origins of lungs and swimbladders from a common ‘respiratory pharynx’, even though this would require a slightly less parsimonious course of evolution [4,5]. The coelacanth is a key species in addressing this question and for this reason the data recently provided by Cupello and colleagues [6] are a very welcome addition to the discussion. Here, I would like to add a few points pertinent to lung evolution that appear to be a consequence of these exciting data. One of the most interesting aspects of the coelacanth is that it apparently exhibits an unpaired structure of putative homology with lungs [6–8]. In the Polypteriformes (bichir and reed fish), the lungs are paired [5,9,10], as are those of the lungfishes (Dipnoi) [11], except the Australian lungfish, Neoceratodus forsteri (Krefft, 1870) …

This study describes the distribution and the coexpression of specific neurochemical markers in both neuroendocrine-like cells (NEC-like) and polymorphous granular cells (PGCs) that populate the mucociliated epithelium of the lung in the air-breathing fish Polypterus senegalus, using confocal immunohistochemistry. Using confocal immunohistochemistry, we determined the coexpression of specific neurochemical markers. Colocalization studies showed that 5HT is coexpressed with calbindin and nNOS and choline acetyltransferase (ChAT) is coexpressed with nNOS in the PGCs. This study also shows for the first time the simultaneous occurrence of piscidin 1 and 5HT in the PGCs. The function of these cells being equivalent to ones found in fish gill subepithelial parenchyma is still not known. Due to the importance of piscidin 1 in local immune defence, more research is to be useful to understand a possible interaction of PGCs with immune response in the bichir lung. In fact, the capacity of PGCs to produce NO and other neuroactive substances found in immune cells of fish may represent a primitive form of immunoregulation of innate immunity and specifically antimicrobial function as NO induction and respiratory burst activity are correlated with immune response.

Mitotic chromosome sets in four fish species representing four different orders (Polypteriformes, Osteoglossiformes, Cypriniformes and Characiformes) occurring in the White Nile, Blue Nile and Omo-Turkana basins in south-western and northern Ethiopia were studied with the air-dried method of Kligerman and Bloom (1977). The following chromosome (2n) and arm (NF) numbers were found in these fishes: 2n = 36, NF = 72 in the gray bichir Polypterus senegalus (Polypteridae), 2n = 40, NF = 42 in the stonebasher Pollimyrus cf. nigricans (Mormyridae), 2n = 50, NF = 66 in the Abyssinian loach Afronemacheilus abyssinicus (Balitoridae) and 2n = 54, NF = 78 in the big-scale tetra Brycinus macrolepidotus (Alestiidae). Comparisons with the previously published data (reviewed by Arai, 2011) show general similarity in the gross karyotype features at the level of conspecific populations and congeneric species for the gray bichir, at the level of phylogenetically close genera and families for the Abyssinian loach and big-scale tetra, while substantial karyological differences were revealed between the stonebasher and two other mormyrid genera (Gnathonemus and Marcusenius) studied by Uyeno (1973).

It has been reported that in chondrichthyans the cardiac outflow tract is composed of the myocardial conus arteriosus, while in most teleosteans it consists of the nonmyocardial bulbus arteriosus. Classical studies already indicated that a conus and a bulbus coexist in several ancient actinopterygian and teleost groups. Recent work has shown that a cardiac outflow tract consisting of a conus and a bulbus is common to both cartilaginous and bony fishes. Nonetheless and despite their position at the base of the actinopterygian phylogenetic lineage, the anatomical arrangement of the cardiac outflow tract of the Polypteriformes remained uncertain. The present study of hearts from gray bichirs was intended to fill this gap. The cardiac outflow tract of the bichir consists of two main components, namely a very long conus arteriosus, furnished with valves, and a short, intrapericardial, arterial-like bulbus arteriosus, which differs from the ventral aorta because it is covered by epicardium, shows a slightly different spatial arrangement of the histological elements and is crossed by coronary arteries. Histomorphologically, the outflow tract consists of three longitudinal regions, distal, middle and proximal, an arrangement which has been suggested to be common to all vertebrates. The distal region corresponds to the bulbus, while the conus comprises the middle and proximal regions. The present findings reinforce the notion that the bulbus arteriosus of fish has played an essential role in vertebrate heart evolution as it is the precursor of the intrapericardial trunks of the aorta and pulmonary artery of birds and mammals.

Summary The occurrence of pigment cells in the heart is well documented in amphibians, birds and mammals. By contrast, information on heart pigmentation in fish is extremely sparse. The aim is to report the presence of pigment cells over the entire surface of the heart in the gray bichir, Polypterus senegalus. The sample consisted of 12 hearts, which, after gross anatomical examination, were studied using histochemical and immunohistochemical techniques for light microscopy, and transmission electron microscopy. The pigment cells were located in the subepicardium, showing a regular distribution pattern across the whole heart, except for the anterior end of the outflow tract, where the pigmentation was much more intense. The cells contained dark, ovoid-shaped organelles which was consistent with a melanosome cell identity. As in other vertebrates, the physiological role of the pigment cells in the heart of the gray bichir is unknown. The absence of such cells in hearts of other polypteriforms suggests that cells containing melanin are not essential for normal fish heart function. Basing on literature data concerning tetrapods, it can be inferred that the pigment cells of the heart of the gray bichir derive from the neural crest. If this were true, our findings would provide the first evidence for the presence of neural crest-derived cells in the subepicardium of adult hearts of early actinopterygians.

Most actinopterygians replace their teeth continuously throughout life. To address the question of where and how replacement teeth form in actinopterygians, it is advisable to investigate well-chosen representatives within the lineage. The African bichir, Polypterus senegalus, belongs to the earliest diverged group of the actinopterygian lineage with currently living representatives. Its well characterized dentition, together with its phylogenetic position, make this species an attractive model to answer following questions: (1) when and where does the replacement tooth form and how is it connected with the dental organ of the predecessor, and (2) is there any evidence for the presence of epithelial stem cells, hypothesized to play a role in replacement? Serial sections show that one tooth family can contain up to three members, which are all interconnected by dental epithelium. Replacement teeth develop without the presence of a successional dental lamina. We propose that this is the plesiomorphic condition for tooth replacement in actinopterygians. BrdU pulse-chase experiments reveal cells in the outer and middle dental epithelium, proliferating at the time of initiation of a new replacement tooth. It is tempting to assume that these cell layers provide a stem cell niche. The observed absence of label-retaining cells after long chase times (up to 8 weeks) is held against the light of divergent views on cell cycling properties of stem cells. At present, our data do not support, neither reject, the hypothesis on involvement of epithelial stem cells within the process of continuous tooth replacement. J. Exp. Zool. (Mol. Dev. Evol.) 322B: 281–293, 2014. © 2014 Wiley Periodicals, Inc.

Cladistians (Polypteriformes) are currently considered basal to other living ray-finned fishes (actinopterygians), and their brain organization is therefore critical to providing information about the primitive neural characters that existed in the earliest ray-finned fishes. The organization of the serotonergic system in the brain has been carefully analyzed in most vertebrate groups, and in the present study we provide the first detailed information on the distribution of serotonergic cell bodies and fibers in the central nervous system of representative species of the two extant genera of cladistians, i.e. Polypterus senegalus and Erpetoichthys calabaricus, by means of immunohistochemistry against serotonin (5-HT). Distinct groups of immunoreactive cells were detected in the preoptic area, the hypothalamic paraventricular organ, the pineal organ, the pretectal region, the long column of the raphe in the rhombencephalic midline, the spinal cord, and amacrine cells in the inner nuclear layer of the retina. Fiber labeling was widely distributed in all main brain subdivisions but was more abundant in distinct pallial and subpallial areas, the preoptic area, the thalamus, the optic tectum, the tori semicircularis and lateralis, the rhombencephalic reticular formation, the nucleus of the solitary tract, and the dorsal aspect of the spinal cord. Our analysis makes it possible to establish which serotonergic structures characterized the earliest ray-finned fishes, and a comparison of these results with those from other classes of vertebrates, including a segmental analysis to correlate cell populations, reveals that most characteristics, such as the presence of serotonergic cells in the preoptic area and the basal hypothalamus, are preserved in all anamniotes. However, this system seems to be reduced in amniotes, mainly mammals, although important features are shared, such as the presence of serotonergic cells in the pineal organ, the retina, and the raphe nuclei.

Polypteriform fishes are believed to be basal to other living ray-finned bony fishes, and they may be useful for providing information of the neural organization that existed in the brain of the earliest ray-finned fishes. The calcium-binding proteins calretinin (CR) and calbindin-D28k (CB) have been widely used to characterize neuronal populations in vertebrate brains. Here, the distribution of the immunoreactivity against CR and CB was investigated in the olfactory organ and brain of Polypterus senegalus and compared to the distribution of these molecules in other ray-finned fishes. In general, CB-immunoreactive (ir) neurons were less abundant than CR-ir cells. CR immunohistochemistry revealed segregation of CR-ir olfactory receptor neurons in the olfactory mucosa and their bulbar projections. Our results confirmed important differences between pallial regions in terms of CR immunoreactivity of cell populations and afferent fibers. In the habenula, these calcium-binding proteins revealed right-left asymmetry of habenular subpopulations and segregation of their interpeduncular projections. CR immunohistochemistry distinguished among some thalamic, pretectal, and posterior tubercle-derived populations. Abundant CR-ir populations were observed in the midbrain, including the tectum. CR immunoreactivity was also useful for characterizing a putative secondary gustatory/visceral nucleus in the isthmus, and for distinguishing territories in the primary viscerosensory column and octavolateral region. Comparison of the data obtained within a segmental neuromeric context indicates that some CB-ir and CR-ir populations in polypteriform fishes are shared with other ray-finned fishes, but other positive structures appear to have evolved following the separation between polypterids and other ray-finned fishes.

The cholinergic system in the brain has been widely studied in most vertebrate groups, but there is no information available about this neurotransmission system in the brains of holostean fishes, a primitive and poorly understood group of actinopterygian fishes. The present study provides the first detailed information on the distribution of cholinergic cell bodies and fibers in the central nervous system in two holostean species, the Florida gar, Lepisosteus platyrhincus, and the bowfin, Amia calva. Immmunohistochemistry against the enzyme choline acetyltransferase (ChAT) revealed distinct groups of ChAT-immunoreactive (ChAT-ir) cells in the habenula, isthmic nucleus, laterodorsal tegmental nucleus, octavolateral area, reticular formation, cranial nerve motor nuclei and the motor column of the spinal cord, all of which seem to be highly conserved among vertebrates. Some ChAT-ir cells were detected in the basal telencephalon that appear in actinopterygians for the first time in the evolution of this neurotransmission system, whereas the remarkable cholinergic population in the optic tectum is a peculiar characteristic, the presence of which varies throughout evolution, although it is present in all teleosts studied. Abundant cholinergic fibers were found in the pretectal region and optic tectum, where they probably modulate vision, and in the hypothalamus and the interpeduncular neuropil. Some interspecific differences were also observed, such as the presence of ChAT-ir cells in the supraoptoparaventricular band only in Lepisosteus and in in the nucleus subglomerulosus only in Amia. In addition, ChAT-ir fibers in the olfactory bulb were detected only in Amia. Comparison of these results with those from other classes of vertebrates, and a segmental analysis to correlate cell populations, reveal that the pattern of the cholinergic system in holosteans is very close to that in ancestral actinopterygian fishes, as recently described in the bichir (Cladistia), although an important evolutionary novelty in holosteans is the presence of cholinergic cells in the basal telencephalon.

As a member of the most basal clade of extant ray-finned fishes (actinopterygians) and of one of the most basal clades of osteichthyans (bony fishes + tetrapods), Polypterus can provide insights into the ancestral anatomy of both ray-finned and lobe-finned fishes, including those that gave rise to tetrapods. The pectoral fin of Polypterus has been well described but, surprisingly, neither the bones nor the muscles of the pelvic fin are well known. We stained and dissected the pelvic fin of Polypterus senegalus and Polypterus delhezi to offer a detailed description of its musculoskeletal anatomy. In addition to the previously described adductor and abductor muscles, we found preaxial and postaxial muscles similar to those in the pectoral fin of members of this genus. The presence of pre- and postaxial muscles in both the pectoral and pelvic fins of Polypterus, combined with recent descriptions of similar muscles in the lobe-finned fishes Latimeria and Neoceratodus, suggests that they were present in the most recent common ancestor of bony fishes. These results have crucial implications for the evolution of appendicular muscles in both fish and tetrapods.

The lung is an important organ for air breathing in tetrapods and originated well before the terrestrialization of vertebrates. Therefore, to better understand lung evolution, we investigated lung development in the extant basal actinopterygian fish Senegal bichir (Polypterus senegalus). First, we histologically confirmed that lung development in this species is very similar to that of tetrapods. We also found that the mesenchymal expression patterns of three genes that are known to play important roles in early lung development in tetrapods (Fgf10, Tbx4 and Tbx5) were quite similar to those of tetrapods. Moreover, we found a Tbx4 core lung mesenchyme-specific enhancer (C-LME) in the genomes of bichir and coelacanth (Latimeria chalumnae) and experimentally confirmed that these were functional in tetrapods. These findings provide the first molecular evidence that the developmental program for lung was already established in the common ancestor of actinopterygians and sarcopterygians.

Hatching gland cells (HGCs) originate from different germ layers between frogs and teleosts, although the hatching enzyme genes are orthologous. Teleostei HGCs differentiate in the mesoendodermal cells at the anterior end of the involved hypoblast layer (known as the polster) in late gastrula embryos. Conversely, frog HGCs differentiate in the epidermal cells at the neural plate border in early neurula embryos. To infer the transition in the developmental origin of HGCs, we studied two basal ray-finned fishes, bichir (Polypterus) and sturgeon. We observed expression patterns of their hatching enzyme (HE) and that of three transcription factors that are critical for HGC differentiation: KLF17 is common to both teleosts and frogs; whereas FoxA3 and Pax3 are specific to teleosts and frogs, respectively. We then inferred the transition in the developmental origin of HGCs. In sturgeon, the KLF17, FoxA3, and HE genes were expressed during the tailbud stage in the cell mass at the anterior region of the body axis, a region corresponding to the polster in teleost embryos. In contrast, the bichir was suggested to possess both teleost- and amphibian-type HGCs, i.e. the KLF17 and FoxA3 genes were expressed in the anterior cell mass corresponding to the polster, and the KLF17, Pax3 and HE genes were expressed in dorsal epidermal layer of the head. The change in developmental origin is thought to have occurred during the evolution of basal ray-finned fish, because bichir has two HGCs, while sturgeon only has the teleost-type.