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Teleost major histocompatibility complex (MHC) shows several unique features not shared by the shark and tetrapod MHC. (1) The MHC class I and class II genes are not linked but dispersed on several chromosomes. Because the linkage between the class I and II genes is conserved by basal ray-finned fish such as bichir and spotted gar, it is likely that this linkage was broken by the gross genome rearrangement postulated at the early stage of teleost evolution. (2) The alpha 1 domain of the teleost class I gene shows a long-term lineage conservation in a span of hundreds of millions of years. (3) The teleost lacks the DM gene, suggesting the presence of a unique class II antigen presentation pathway. (4) The basal teleost and bichir share the dimorphic proteasome subunit beta type-8 (PSMB8) alleles, providing an example of an exceptionally long-term trans-species polymorphism. All of these unique points make the teleost MHC one of the best model systems to analyze MHC evolution.

Epimorphic regeneration of fins was studied in different ray-finned fishes (Actinopterygii), but species representing the phylogenetically basal lineages of the taxon have remained outside the attention of researchers. Information on the regenerative abilities of these groups is important both for understanding the evolutionary origins of the epimorphic regeneration phenomenon and for assessing the universality of regenerative potencies in Actinopterygii. Addressing this problem, we studied for the first time fin regeneration in two members of the archaic family Polypteridae: the ropefish (Erpetoichthys calabaricus) and the Senegal bichir (Polypterus senegalus). Along with the ability to regenerate the bony rays of fins, widespread among Actinopterygii, polypterids show the ability to effectively regenerate the endoskeleton and musculature of their fins. This unusual feature allows us to suggest polypterids as new model organisms for the study of the mechanisms of vertebrate limb regeneration.

Vertebrates have experienced two rounds of whole-genome duplication (WGD) in the stem lineages of deep nodes within the group and a subsequent duplication event in the stem lineage of the teleosts—a highly diverse group of ray-finned fishes. Here, we present the first full Hox gene sequences for any member of the Acipenseriformes, the American paddlefish, and confirm that an independent WGD occurred in the paddlefish lineage, approximately 42 Ma based on sequences spanning the entire HoxA cluster and eight genes on the HoxD gene cluster. These clusters comprise different HOX loci and maintain conserved synteny relative to bichir, zebrafish, stickleback, and pufferfish, as well as human, mouse, and chick. We also provide a gene genealogy for the duplicated fzd8 gene in paddlefish and present evidence for the first Hox14 gene in any ray-finned fish. Taken together, these data demonstrate that the American paddlefish has an independently duplicated genome. Substitution patterns of the “alpha” paralogs on both the HoxA and HoxD gene clusters suggest transcriptional inactivation consistent with functional diploidization. Further, there are similarities in the pattern of sequence divergence among duplicated Hox genes in paddlefish and teleost lineages, even though they occurred independently approximately 200 Myr apart. We highlight implications on comparative analyses in the study of the “fin-limb transition” as well as gene and genome duplication in bony fishes, which includes all ray-finned fishes as well as the lobe-finned fishes and tetrapod vertebrates.

Because of their crucial phylogenetic positions, hagfishes, sharks, and bichirs are recognized as key taxa in our understanding of vertebrate evolution. The expression patterns of the regulatory genes involved in developmental patterning have been analyzed in the context of evolutionary developmental studies. However, in a survey of public sequence databases, we found that the large-scale sequence data for these taxa are still limited. To address this deficit, we used conventional Sanger DNA sequencing and a next-generation sequencing technology based on 454 GS FLX sequencing to obtain expressed sequence tags (ESTs) of the Japanese inshore hagfish (Eptatretus burgeri; 161,482 ESTs), cloudy catshark (Scyliorhinus torazame; 165,819 ESTs), and gray bichir (Polypterus senegalus; 34,336 ESTs). We deposited the ESTs in a newly constructed database, designated the “Vertebrate TimeCapsule.” The ESTs include sequences from genes that can be effectively used in evolutionary developmental studies; for instance, several encode cartilaginous extracellular matrix proteins, which are central to an understanding of the ways in which evolutionary processes affected the skeletal elements, whereas others encode regulatory genes involved in craniofacial development and early embryogenesis. Here, we discuss how hagfishes, sharks, and bichirs contribute to our understanding of vertebrate evolution, we review the current status of the publicly available sequence data for these three taxa, and we introduce our EST projects and newly developed database. J. Exp. Zool. (Mol. Dev. Evol.) 316:526–546, 2011. © 2011 Wiley Periodicals, Inc.

In vertebrates, the receptor neurons of the olfactory/vomeronasal systems express different receptor gene families and related G-protein types (in particular the G protein alpha subunit). There are no data in the literature about the molecular features of the olfactory/vomeronasal systems of Cladistia thus, in this work, the presence and distribution of different types of G protein alpha subunits were investigated in the olfactory organs of the bichir Polypterus senegalus, using immunohistochemistry. Gαo-like immunoreactivity was detected in the microvillous receptor neurons, with the cell body in the basal zone of the sensory epithelium, and in the crypt neurons. Gαo-like ir glomeruli were mainly localized in the anterior part of the olfactory bulb. Gαolf-like immunoreactivity in the sensory epithelium was detected in the ciliated receptor neurons, while the immunoreactive glomeruli in the olfactory bulb were mainly localized in the ventral-posterior part. No Gαq nor Gαi3 immunoreactivity was detected. These data are partially in agreement with studies that show the distribution of G protein alpha subunits in teleosts, allowing to hypothesize a common organization of the olfactory/vomeronasal systems in the group of Actinopterigians.

In tetrapods, vasopressin (VP) and vasotocin (VT) are involved in various aspects of physiology and behavior including osmoregulation, cardiovascular function, reproduction, stress response and social behavior. Pharmacological and molecular studies have identified three types of VP/VT receptors, V1a-type (V1aR), V1b-type (V1bR) and V2-type (V2R). On the other hand, only V1aR has so far been identified in teleosts. In the present study, we successfully cloned V2Rs from two ray-finned fish, gray bichir and medaka. Phylogenetic analysis showed that the cloned receptors belong to the V2R group of lobe-finned fish and tetrapods. The amino acid sequences of bichir V2R and medaka V2R were high identity (60-65.5% and 53.2-80.9%, respectively) with other known V2R members, respectively. Reverse transcriptase PCR revealed that ray-finned fish V2R transcripts have been detected in various tissues including brain, gill, heart, liver, kidney and reproductive organs, suggesting that ray-finned fish V2R might mediate multiple functions of VT. In functional analysis, the cells transfected with the cloned receptors responded with the accumulation of intracellular cAMP in a concentration-dependent manner following VT stimulation, but not respond with [Ca(2+)]i. Furthermore, pretreatment with mammalian V2R antagonist (OPC-31260) to the cells transfected with medaka V2R significantly inhibited an increase of the VT-induced intracellular cAMP. These results suggest that ray-finned fish possess a functional V2R linked to adenylate cyclase and the cAMP signaling pathway as well as V2Rs of lobe-finned fish and tetrapods. Thus, the present study suggests that functional V2R evolved prior to the divergence of the ray- and lobe-finned fish lineages.

Summary Polypterid fishes are considered the basal-most group of extant actinopterygians and thus may serve as a direct link to understand the evolution of the first bony fishes. Embryonic and larval specimens, however, are extremely scarce, making it difficult to study their developmental patterns and processes. During the past few years we collected many embryos and larvae of the Senegal bichir Polypterus senegalus and in this paper we describe some novel observations concerning the supposed ecto-endoderm border in the mouth and consecutive initiation of dental development. The mouth of the Senegal bichir does not develop via a classical oropharyngeal membrane but instead, opening the mouth occurs via a separation of the upper and lower jaws that are connected by epithelial bridges. These structures are bilaterally symmetrical and are found invariantly at places of the earliest tooth bud development. It is suggested that the epithelial bridges may represent the ecto-endoderm bordering zone, are both structurally and functionally homologous to the oropharyngeal membrane and consequently it is hypothesized that the epithelial bridges and the developmental factors producing them play a key role in initiation and early distribution of the particular dental domains.

Whole-genome duplication (WGD) is believed to be one of the major evolutionary events that shaped the genome organization of vertebrates. Here, we review recent research on vertebrate genome evolution, specifically on WGD and its consequences for gene and genome evolution in teleost fishes. Recent genome analyses confirmed that all vertebrates experienced two rounds of WGD early in their evolution, and that teleosts experienced a subsequent additional third-round (3R)-WGD. The 3R-WGD was estimated to have occurred 320–400 million years ago in a teleost ancestor, but after its divergence from a common ancestor with living non-teleost actinopterygians (Bichir, Sturgeon, Bowfin, and Gar) based on the analyses of teleost-specific duplicate genes. This 3R-WGD was confirmed by synteny analysis and ancestral karyotype inference using the genome sequences of Tetraodon and medaka. Most of the tetrapods, on the other hand, have not experienced an additional WGD; however, they have experienced repeated chromosomal rearrangements throughout the whole genome. Therefore, different types of chromosomal events have characterized the genomes of teleosts and tetrapods, respectively. The 3R-WGD is useful to investigate the consequences of WGD because it is an evolutionarily recent WGD and thus teleost genomes retain many more WGD-derived duplicates and “traces” of their evolution. In addition, the remarkable morphological, physiological, and ecological diversity of teleosts may facilitate understanding of macrophenotypic evolution on the basis of genetic/genomic information. We highlight the teleosts with 3R-WGD as unique models for future studies on ecology and evolution taking advantage of emerging genomics technologies and systems biology environments.

Vertebrates show diverse sexual characters in sexually attractive and reproductive organs, which are regulated by steroid hormones, particularly androgens. However, the evolutionary history of androgen receptor (AR) gene remains largely unknown on the basis of phylogenic and functional analyses. To elucidate the evolutionary history and functional diversification of AR genes in vertebrates, we cloned the AR cDNAs from a shark, basal ray-finned fishes (Actinopterygii), namely bichir and sturgeon (Acipenseriformes), and teleosts including a basal teleost, arowana (Osteoglossiformes). Molecular phylogenetic analysis revealed that the gene duplication event that gave rise to two different teleost ARs (alpha and beta) likely occurred in the actinopterygian lineage leading to teleosts after the divergence of Acipenseriformes but before the split of Osteoglossiformes, which is compatible with the phylogenetic timing of teleost-specific genome duplication. Searching for AR genes in the medaka genome indicated that the teleost AR gene duplication has been associated with the duplication between chromosomes 10 and 14. Our functional analysis revealed that the shark AR activates the target gene via androgen response element by classical androgens. The teleost ARalpha showed the unique intracellular localization with a significantly higher transactivating capacity than that by teleost ARbeta. These findings indicate that the most ancient type of AR, as activated by the classical androgens as ligands, emerged before the Chondrichthyes-Osteichthyes split, and the AR gene was duplicated during the teleost-specific genome duplication event. We report here for the first time the accurate evolutionary history of AR gene and functional characterization of AR duplicates in teleost lineage.

Amphibian holoblastic cleavage in which all blastomeres contribute to any one of the three primary germ layers has been widely thought to be a developmental pattern in the stem lineage of vertebrates, and meroblastic cleavage to have evolved independently in each vertebrate lineage. In extant primitive vertebrates, agnathan lamprey and basal bony fishes also undergo holoblastic cleavage, and their vegetal blastomeres have been generally thought to contribute to embryonic endoderm. However, the present marker analyses in basal ray-finned fish bichir and agnathan lamprey embryos indicated that their mesoderm and endoderm develop in the equatorial marginal zone, and their vegetal cell mass is extraembryonic nutritive yolk cells, having non-cell autonomous meso-endoderm inducing activity. Eomesodermin (eomes), but not VegT, orthologs are expressed maternally in these animals, suggesting that VegT is a maternal factor for endoderm differentiation only in amphibian. The study raises the viewpoint that the lamprey/bichir type holoblastic development would have been ancestral to extant vertebrates and retained in their stem lineage; amphibian-type holoblastic development would have been acquired secondarily, accompanied by the exploitation of new molecular machinery such as maternal VegT.

Diplogyrodactylus martini n. g. et sp., a viviparous monogenean, is described from the gills of gray bichir Polypterus senegalus Cuvier, 1829, in Senegal. This new genus can be readily distinguished from the other described viviparous genera based on the morphology of the attachment apparatus. The haptor is equipped with a pair of centrally positioned hamuli, a simple ventral bar without membrane and 8 pairs of marginal hooks of 2 types, 5 pairs of hooks with well-defined large falculate sickles, and 3 pairs of smaller hooks with well-articulated sickles. The new genus is unique in having a teardrop-shaped muscular tubular male copulatory organ which lacks spines. A partial sequence of the internal transcribed spacer (ITS rDNA) was obtained from 4 specimens of D. martini. The relationship among D. martini and other genera of the Gyrodactylidae is discussed based on the similarities of the attachment apparatus.

The natriuretic peptide (NP) family consists of multiple subtypes in teleosts, including atrial, B-type, ventricular, and C-type NPs (ANP, BNP, VNP, CNP-1–4, respectively), but only ANP, BNP, CNP-3, and CNP-4 have been identified in tetrapods. As part of understanding the molecular evolution of NPs in the tetrapod lineage, we identified NP genes in the chicken genome. Previously, only BNP and CNP-3 have been identified in birds, but we characterized two new chicken NP genes by cDNA cloning, synteny and phylogenetic analyses. One gene is an orthologue of CNP-1, which has only ever been reported in teleostei and bichir. The second gene could not be assigned to a particular NP subtype because of high sequence divergence and was named renal NP (RNP) due to its predominant expression in the kidney. CNP-1 mRNA was only detected in brain, while CNP-3 mRNA was expressed in kidney, heart, and brain. In the developing embryo, BNP and RNP transcripts were most abundant 24 h post-fertilization, while CNP mRNA increased in a stage-dependant manner. Synthetic chicken RNP stimulated an increase in cGMP production above basal level in chicken kidney membrane preparations and caused a potent dose-dependant vasodilation of pre-constricted dorsal aortic rings. From conserved chromosomal synteny, we propose that the CNP-4 and ANP genes have been lost in chicken, and that RNP may have evolved from a VNP-like gene. Furthermore, we have demonstrated for the first time that CNP-1 is retained in the tetrapod lineage.

Anatomical and functional studies of the autonomic innervation and the putative oxygen receptors-the neuroepithelial (NEC)-like cells of the bichirs are lacking. The present paper describes the distribution of both NEC-like cells and the polymorphous granular cells (PGCs) that populate the mucociliated epithelium of the lung in the air breathing fish Polypterus senegalus. By using confocal immunohistochemistry we determined the coexpression of specific neurochemical markers. Colocalization studies showed that 5HT is coexpressed with calbindin and nNOS in the NEC-like cells and PGCs, and choline acetyltransferase (ChAT) is coexpressed with nNOS in both the two types of cells. Distribution of neurotransmitters (5HT, NO) and neurochemical marker ChAT is also investigated in the lung muscle. The role of these transmitters may be the autonomic control of circulation and respiration. However, the importance of these signals for the respiratory responses in the species studied is still not known. The present 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 defense, more research is useful to understand a possible interaction of PGCs with immune response in the bichir lung.

Natriuretic peptides (NPs) have diversified from a single NP in cyclostomes and elasmobranchs to multiple NPs in ray-finned fishes where ANP, BNP, VNP, and/or up to four CNPs (CNP-1, 2, 3, and/or 4) have been identified. To trace the evolutionary diversification of NPs in fishes, we analyzed the bichir (Polypterus endlicheri), believed to be the most primitive extant ray-finned fish, for the presence of any NPs by a PCR-based method using primers that amplify all NP cDNAs identified to date. We have cloned cDNAs encoding ANP, BNP, VNP from the heart and three CNPs (CNP-1, 3, and 4) from the brain. An extensive search for CNP-2 from the brain was not successful. The C-terminus of bichir ANP presented an amidation signal as in ray-finned fish ANP. The bichir BNP mRNA had AUUUA repeats in the 3'-untranslated region as observed in all BNP cDNAs of vertebrates. The bichir VNP had a long C-terminal 'tail' sequence extending from the intramolecular ring as does teleost VNP. The three bichir CNPs are structurally similar to each teleost counterpart and are grouped after molecular phylogenetic analyses. ANP was most abundantly expressed in the atrium, BNP in the ventricle, and VNP was expressed in both atrium and ventricle. The three CNPs are most abundantly expressed in the brain, and CNP-4 transcripts were found in small amounts in the ventricle and kidney. Taken together, it is clear that all major NPs exist prior to the whole genome duplication that occurred in the teleost lineage. Furthermore, this is the first observation that CNP-3, ANP, BNP, and VNP, whose genes are colocalized in the same chromosome, coexist in a single fish species including teleosts, thereby confirming that CNP-3 is not an ortholog of VNP, and that ANP, BNP, and VNP genes were generated by tandem duplication from the CNP-3 gene.

The study of Hox clusters and genes provides insights into the evolution of genomic regulation of development. Derived ray-finned fishes (Actinopterygii, Teleostei) such as zebrafish and pufferfish possess duplicated Hox clusters that have undergone considerable sequence evolution. Whether these changes are associated with the duplication(s) that produced extra Hox clusters is unresolved because comparison with basal lineages is unavailable. We sequenced and analyzed the HoxA cluster of the bichir (Polypterus senegalus), a phylogenetically basal actinopterygian. Independent lines of evidence indicate that bichir has one HoxA cluster that is mosaic in its patterns of noncoding sequence conservation and gene retention relative to the HoxA clusters of human and shark, and the HoxAα and HoxAβ clusters of zebrafish, pufferfish, and striped bass. HoxA cluster noncoding sequences conserved between bichir and euteleosts indicate that novel cis-sequences were acquired in the stem actinopterygians and maintained after cluster duplication. Hence, in the earliest actinopterygians, evolution of the single HoxA cluster was already more dynamic than in human and shark. This tendency peaked among teleosts after HoxA cluster duplication.

There is general agreement that the polypteriform fishes, like Polypterus senegalus, constitute a unique lineage in the evolution of the vertebrates. However, the precise position of these fishes had been a point of controversy since the time of Darwin and Huxley. There is now consensus that the polypteriform fishes are members of superorder Actinopterygii. However, within the Actinopterygii, it is still debatable as to whether the polypteriform fishes are an early offshoot of the Actinopterygii or a more recent sister group to the sturgeon and other extant chondrostean fishes. In this study the sequence of proopiomelanocortin (POMC), the common precursor for the melanocortins and beta-endorphin, was used to evaluate the phylogenetic position of the polypteriform fishes relative to other bony fishes. 3(')RACE and 5(')RACE protocols were used to amplify overlapping regions of a POMC cDNA from the brain of P. senegalus. The full-length POMC cDNA had an open reading frame that encoded 259 amino acids. As seen in most gnathostomes, P. senegalus POMC has three melanocortin sequences (ACTH/alpha-MSH, gamma-MSH, and beta-MSH), and a beta-endorphin region. For phylogenetic analysis, the following POMC sequences were aligned at the amino acid level and analyzed using a maximum parsimony algorithm: P. senegalus, dogfish, sturgeon A, paddlefish A, sockeye salmon A, tilapia, and gar. The dogfish POMC sequence was used as the out-group. In this analysis the P. senegalus POMC sequence formed a clade with the chondrostean POMC sequences (sturgeon A and paddlefish A), and not with the neopterygian sequences (sockeye salmon A, tilapia, and gar). P. senegalus POMC is remarkably similar to sturgeon POMC A. In particular, in both precursors there is evidence for degeneration at the proteolytic cleavage site that precedes the gamma-MSH sequence. Based on the analysis of this nuclear gene it would appear that P. senegalus belongs to a branch of the chrondrostean lineage rather than representing a lineage of ray-finned fish that is ancestral to the chondrostean and neoptyergian ray-finned fishes. Alternatively, if the polypteriform fishes are in fact an early offshoot of the Actinopterygii (the traditional view), then the observations made for P. senegalus POMC relative to the chondrostean POMC sequences is the result of convergence.

The connections of the dorsal telencephalon (pallium) of the Senegal bichir (Polypterus senegalus) were investigated in order to test the hypothesis that the simple everted pallium of polypterid fishes is directly comparable to the evaginated pallium of most vertebrates. Neuroanatomical tracers (DiI or biotinylinated dextran amines) were injected into the three traditionally recognized divisions of the bichir pallium to determine their afferent and efferent connections. The connections were corroborated with injections into regions identified as projecting to, or receiving input from, one or more pallial divisions. The results suggest that the bichir pallium consists not of three, but only two primary divisions: a dorsomedial one and a dorsolateral one. This reinterpretation refutes the hypothesis of a direct one-to-one comparison with the tripartite pallium of most vertebrates. Homologues of the bichir dorsomedial and dorsolateral pallium are recognized in the lateral and medial pallium of anuran amphibians, respectively. The pallium of bichirs is compared to that of derived ray-finned fishes as a link between derived ray-finned fishes and other vertebrates. The available information on the connections of the pallium of teleosts suggests that only the olfactory recipient (pars posterior) of the pallium can be directly compared to bichirs and amphibians and that the remaining divisions of the pallium in teleosts are uniquely derived features of the teleost telencephalon.

The phylogenetic position of Polypteriforme (bichirs) remains elusive, despite extensive research both on morphological and molecular datasets. Unfortunately morphological cladistic analyses and molecular phylogenetic analyses had reached conflicting conclusions, as Polypteriformes were either grouped with lobe-finned fishes (Sarcopterygii) or ray-finned fishes (Actinopterygii), or even classified as their own group, the Brachiopterygii. In this study, we applied a third independent source of datasets, the presence versus absence of microRNAs, to re-investigate the phylogenetic relationship of bichirs. Through deep sequencing of small RNA library, we showed that bichirs should be grouped into ray-finned fishes rather than lobe-finned fishes. Phylogenetic analysis confirmed that bichirs were placed as the most basal member of the ray-finned fishes. Bichirs shared five unique microRNA families with teleostei, which were not found in any other species investigated to date. Bichirs have also retained three of five microRNAs that were previously deemed to be lost exclusively in teleostei. Furthermore, we report more than one hundred novel microRNAs that are unique to bichir. The identification of microRNAs in bichir provides overwhelming evidence for their affinity of ray-finned fishes. The sarcopterygian-like characteristics such as lobed fins, paired ventral lungs, and external gills in juveniles should not be considered as derived traits shared with those of sarcopterygians.

Bichirs, Polypteriformes, are a basal group of vertebrates with many unique characters as ganoid scales, fleshy pectoral fin, spiraculum or paired lung originated from ventral outpocketing from floor of pharynx. Among the most notable is also the dentition, which contatin teeth among whole area of oropharyngeal cavity. This arrangement is not easily call oral and pharyngeal, what is recently used e.g. for medaka, because it is only one dention with teeth on majority of oral as well as pharyngeal elements. This diploma thesis utilizes expression patterns of Dlx genes to visualize morphogenesis of dentition and pharyngeal arches in the Senegal Bichir (Polypterus senegalus). The very first developing teeth of oral dentition, as well as its further patterning were detected and accurately located using gene expression analyses. It was also discovered an interesting histological context connected with unique mouth opening mechanism during early development of teeth in oral cavity. Another discussion point is establishment of dorsoventral polarization in pharyngeal arches development of bichir based on results from Dlx gene expression analysis. Powered by TCPDF (www.tcpdf.org)

Cartilaginous fishes (chondrichthyans) have traditionally been taken as an early offshoot among jawed vertebrates. To examine some crucial chondrichthyan relationships, we have sequenced the mitochondrial genomes of the holocephalan Chimaera monstrosa (ratfish) and the basal galeomorph species Heterodontus francisci (horn shark) and analysed them together with the corresponding data set of several other chondrichthyans, teleosts, the coelacanth, the African lungfish and the bichir. The rooting point of the tree was established using unequivocal outgroups, the sea lamprey , the sea lancelet or echinoderms. The phylogenetic analyses identified monophyletic Chondrichthyes in a terminal position in the piscine tree, lending no support to the traditionally accepted basal position of cartilaginous fishes among extant gnathostomes. The findings suggest that the cartilage characterizing extant chondrichthyans is a retention of an embryonic condition, thus representing a derived rather than a primitive phylogenetic and developmental stage. Similarly, the analyses suggest that the open gill slits of neoselachians (sharks and rays) constitute a derived state compared to the operculum (gill cover) characterizing bony fishes and holocephalans. The analyses did not support the so-called Squalea/Galea hypothesis which posits that batomorphs (sharks, rays) have arisen from recent selachians (sharks). Inconsistent with the common understanding of piscine and gnathostome evolution, the two taxa having lungs, the African lungfish and the bichir, had a basal position in the piscine tree. The findings put into question the phylogenetic validity of the taxonomic nomenclature attributed to various vertebrate, notably piscine, clades.

The distribution of serotonin-immunoreactive (5HT-IR) neurons was studied in the brain of a primitive bony fish, Polypterus senegalus. The diencephalon contained a prominent 5HT-IR cell group (G2) consisting mainly of liquor-contacting neurons in the hypothalamic periventricular wall including the paraventricular organ. Their ependymofugal processes arborized profusely and gave rise to dense networks of varicose fibers in the hypothalamus. Other groups of 5HT-IR cells were situated in the preoptic area including the periventricular preoptic nucleus (G1) and in the dorsal thalamus (G3). In the brain stem, 5HT-IR cells were found in the medial part, mainly in the superior raphe nucleus (G4). No 5HT-IR cells were demonstrated in other regions of the brain. The cell groups referred to as G1 and G4 in the present study seem to be new components comprising the serotonergic systems in the Polypterus brain.

Peptide tyrosine–tyrosine (PYY) has been isolated from the intestines of two species of reptile, the desert tortoise Gopherus agassizii (Testudines) and the Burmese python Python molurus (Squamata), from the primitive Actinopterygian fish, the bichir Polypterus senegalis (Polypteriformes) and from two agnathans, the Southern-hemisphere lamprey Geotria australis (Geotriidae) and the holarctic lamprey Lampetra fluviatilis (Petromyzontidae). The primary structure of bichir PYY is identical to the proposed ancestral sequence of gnathostome PYY (YPPKPENPGE10 | DAPPEELAKY20 | YSALR HYINL30 | ITRQRY). Tortoise and python PYY differ by six and seven residues, respectively, from the ancestral sequence consistent with the traditional view that the Testudines represent an earlier divergence from the primitive reptilian stock than the Squamates. The current views of agnathan phylogeny favor the hypothesis that the Southern-hemisphere lampreys and the holarctic lampreys arose from a common ancestral stock but their divergence is of a relatively ancient (pre-Tertiary) origin. The Geotria PYY-related peptide shows only two amino acid substitutions (Pro10→Gln and Leu22→Ser) compared with PYY from the holarctic lamprey Petromyzon marinus. This result was unexpected as Petromyzon PYY differs from Lampetra PYY deduced from the nucleotide sequence of a cDNA (Soderberg et al. J. Neurosci. Res. 1994;37:633–640) by 10 residues. However, a re-examination of an extract of Lampetra intestine revealed the presence of a PYY that differed in primary structure from Petromyzon PYY by only one amino acid residue (Pro10→Ser). This result suggests that the structure of PYY has been strongly conserved during the evolution of Agnatha and that at least two genes encoding PYY-related peptides are expressed in Lampetra tissues.

The distribution of neuropeptide Y (NPY)-like immunoreactivity in the brain of the bichir, Polypterus senegalus, was examined immunohistochemically. NPY-like immunoreactivity was distributed widely in the brain, with the highest density in the diencephalon. NPY-positive perikarya were found in various areas, including the terminal nerve, the pallial zones of the telencephalon, the periventricular preoptic nucleus, the thalamic nucleus, the ventral hypothalamus of the diencephalon, the tegmentum of the mesencephalon, and the area intermedioventralis of the rhombencephalon. In the hypothalamus, NPY-positive liquor-contacting neurons were frequently observed. Immunoreactive neuron-like cells also appeared in the distal lobe of the hypophysis. NPY fibers were densely distributed in the ventral telencephalon, the hypothalamus, and the ventrolateral area of the rhombencephalon. They were also demonstrated in the terminal nerve. In the hypophysis, NPY fibers were dense in the median eminence, but sparse in the neural lobe. Electron-microscopic double immunostaining of the terminal nerve revealed the coexistence of NPY-like antigen with gonadotropin-releasing hormone-like and molluscan cardioexcitatory tetrapeptide-like antigens in the same cytoplasmic granules. These results suggest that NPY or a related substance is involved in neuroregulation of various areas of the bichir brain, by mainly acting as a neurotransmitter or neuromodulator.

Extensively migrating population of neural crest cells, which contributes to many tissues and builds up most of craniofacial vertebrate structures, has a crucial role in embryonic development of vertebrate body. The migratory pathways of neural crest cells are thought to be very conserved throughout the vertebrates and cranial neural crest migration is defined by progression of three migratory streams: trigeminal, hyoid and a common branchial stream. In this diploma thesis, migration of cranial neural crest was analysed using embryos of the Senegal bichir (Polypterus senegalus) and of sterlet (Acipenser ruthenus), which represent two basal-most lineages of extant ray-finned fishes. A combination of several techniques was used in both species in order to study cranial neural crest cells from their sites of origin to post- migratory stages and the pattern of migration was compared and discussed in revealed embryonic context. In the Senegal bichir the hyoid neural crest stream was shown to migrate first and it is also the most abundant; this heterochrony shift is apparently related to formation of external gills, which in bichir are situated on the hyoid arch only. In sterlet, neural crest cells migrate in a classic pattern of three progressive streams but their dynamics and patterning is influenced by...

The organization of the roots, ganglia and peripheral distribution of the rami of the cranial nerves of larval and juvenile Senegal bichirs was examined with a wide range of techniques, including gross dissection and histological preparations. The profundal nerve of bichirs is completely separate from the trigeminal nerve and innervates the skin overlying the orbit and snout, and there is no evidence that a superficial ophthalmic ramus of the trigeminal nerve exists. Bichirs appear to retain all six pairs of lateral line nerves that characterized the earliest gnathostomes, but the sensory ganglia of the anterodorsal and otic lateral line nerves have fused, and the sensory ganglia of the anterodorsal and anteroventral lateral line nerves are partially fused, which appears to be a shared derived character of ray-finned fishes. The anteroventral lateral line nerve of bichirs retains mandibular and opercular rami, a primitive feature shared with other bony fishes, and the fibers of the opercular ramus innervate all postotic ampullary organs. Surprizingly, bichirs, unlike most nonteleost bony fishes, have lost the spiracular organ. The lateral ramus of the posterior lateral line nerve of bichirs is divided into a pars dorsalis and a pars ventralis, which appears to be a shared derived character of bony fishes. The glossopharyngeal and vagal nerves of bichirs exhibit lateral and medial sensory ganglia, and this appears to be a shared derived character of bony fishes. The posttrematic rami of the glossopharyngeal nerve and both the pre- and posttrematic rami of the vagal nerve of bichirs are divided, which appears to be a shared derived feature of bony fishes. The hypobranchial nerve of bichirs is unusual in that it is formed by only two spinal nerves, a pattern also seen in gars, but not other bony fishes, and apparently a uniquely derived character.

Polypterid bony fishes are believed to be basal to other living ray-finned fishes, and their brain organization is therefore critical in providing information as to primitive neural characters that existed in the earliest ray-finned fishes. The cholinergic system has been characterized in more advanced ray-finned fishes, but not in polypterids. In order to establish which cholinergic neural centers characterized the earliest ray-finned fishes, the distribution of choline acetyltransferase (ChAT) is described in Polypterus and compared with the distribution of this molecule in other ray-finned fishes. Cell groups immunoreactive for ChAT were observed in the hypothalamus, the habenula, the optic tectum, the isthmus, the cranial motor nuclei, and the spinal motor column. Cholinergic fibers were observed in both the telencephalic pallium and the subpallium, in the thalamus and pretectum, in the optic tectum and torus semicircularis, in the mesencephalic tegmentum, in the cerebellar crest, in the solitary nucleus, and in the dorsal column nuclei. Comparison of the data within a segmental neuromeric context indicates that the cholinergic system in polypterid fishes is generally similar to that in other ray-finned fishes, but cholinergic-positive neurons in the pallium and subpallium, and in the thalamus and cerebellum, of teleosts appear to have evolved following the separation of polypterids and other ray-finned fishes.

The traditionally accepted relationships among basal jawed vertebrates have been challenged by some molecular phylogenetic analyses based on mitochondrial sequences. Those studies split extant gnathostomes into two monophyletic groups: tetrapods and piscine branch, including Chondrichthyes, Actinopterygii and sarcopterygian fishes. Lungfish and bichir are found in a basal position on the piscine branch. Based on transcriptomes of an armored bichir (Polypterus delhezi) and an African lungfish (Protopterus sp.) we generated, expressed sequences and whole genome sequences available from public databases, we obtained 111 genes to reconstruct the phylogenetic tree of basal jawed vertebrates and estimated their times of divergence. Our phylogenomic study supports the traditional relationship. We found that gnathostomes are divided into Chondrichthyes and the Osteichthyes, both with 100% support values (posterior probabilities and bootstrap values). Chimaeras were found to have a basal position among cartilaginous fishes with a 100% support value. Osteichthyes were divided into Actinopterygii and Sarcopterygii with 100% support value. Lungfish and tetrapods form a monophyletic group with 100% posterior probability. Bichir and two teleost species form a monophyletic group with 100% support value. The previous tree, based on mitochondrial data, was significantly rejected by an approximately unbiased test (AU test, p = 0). The time of divergence between lungfish and tetrapods was estimated to be 391.8 Ma and the divergence of bichir from pufferfish and medaka was estimated to be 330.6 Ma. These estimates closely match the fossil record. In conclusion, our phylogenomic study successfully resolved the relationship of basal jawed vertebrates based on transtriptomes, EST and whole genome sequences.

The chemical components of nerve membranes of primitive bony fishes (coelacanth (Latimeria chalumnae), lungfish (Lepidosiren paradoxa and Protopterus aetiopicus), bichir (Polypterus senegalus), and sturgeon (Acipenser ruthenus)) were analyzed to investigate their phylogenetic relations. Representative myelin components such as cerebroside and myelin proteins and enzyme exhibited characteristic profiles in the different species. Coelacanth was characterized by an abundance of hydroxy components of cerebroside fatty acids and by the occurrence of a 28 000 molecular weight band (possibly P0-like protein) and proteolipid protein (PLP). Lungfish possessed glycosylated PLP, but lacked PLP; bichir and sturgeon showed the same protein compositions, PLP and P0-like protein being major components. The activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP), a myelin marker enzyme, was detected in coelacanth myelin, but was almost negligible in the others. The ganglioside compositions were simple in coelacanth and lungfish compared with those in bichir and sturgeon, which were abundant in polysialo components. These findings suggest that coelacanth nerve membranes are the most developed in these species; thus, coelacanth may be more advanced phylogenetically than the others. Bichir and sturgeon were suggested to occupy unique phylogenetic positions, showing similar chemical compositions of nerve membranes which were distinct from those of coelacanth and lungfish.

The proteasome subunit beta type-8 (PSMB8) gene encodes a catalytic subunit of the immunoproteasome, which is involved in the generation of peptides presented by MHC class I molecules. To date, highly diverged dichotomous alleles of PSMB8 have been reported in Oryzias species (actinopterygian teleosts) and Xenopus species (sarcopterygian amphibians). These dimorphic alleles share a similar substitution (A/V(31)F/Y) at the 31st position of the mature protein, which is most probably involved in formation of the S1 pocket. This substitution likely confers different cleavage specificities on the dimorphic PSMB8s. In addition, two paralogous PSMB8 genes possessing the A and F residues at the 31st position have been reported in sharks. Phylogenetic analysis indicated that the two types of PSMB8 of Oryzias, Xenopus, and sharks arose by independent evolutionary events. Here, we identified another pair of dimorphic alleles of PSMB8, which have the A and F residues at the 31st position of the mature protein, from bichir, Polypterus senegalus, a basal actinopterygian. The sequences of the mature proteins-encoding region of the dimorphic alleles of bichir PSMB8, the A and F types, showed only 72.7% and 77.5% identities at the nucleotide and the deduced amino acid levels, respectively. Their intronic sequences show almost no similarity, indicating that the dimorphic alleles of bichir PSMB8 have a very ancient origin. However, phylogenetic analysis showed that the dimorphisms of PSMB8 of bichir, Xenopus, and Oryzias arose by independent evolutionary events, suggesting the presence of a strong selective pressure for possessing the dimorphism.

The interferon regulatory factor (IRF) with its 10 members is a very important gene family related to innate immunity. Currently, most fish IRFs reported are from bony fish (teleosts). Cloning and sequencing of IRFs from chondrosteans, the so-called "ancient fish" including sturgeon, paddlefish, bichir and gar, are absent from the literature. In this study, three IRF genes PsIRF-1, PsIRF-2 and PsIRF-5, were cloned and characterized from the paddlefish (Polyodon spathula). PsIRF-1 includes an open reading frame (ORF) of 972 bp that encodes a putative protein of 324 amino acids; PsIRF-2 includes an ORF of 1023 bp encoding 341 amino acids and P5IRF-5 includes an ORF of 1491 bp that encodes 497 amino acids. The P5IRF-5 gene structure is similar to those in mammals but differs from those in teleosts in the first and second exons. Phylogenetic studies of the putative amino acid sequences of PsIRF-1, PsIRF-2 and PsIRF-5 based on the neighbor-joining and Bayesian inference method for Osteichthyes found widely accepted inter-relationships among actinopterygians and tetrapods. Reverse Transcription Polymerase Chain Reaction (RT-PCR) analysis of PsIRF-1, PsIRF-2 and P5IRF-5 in different paddlefish tissues shows higher levels of expression in gill, spleen and head kidney. Poly (I: C) (polyinosinic-polycytidylic acid) stimulation in vivo up-regulated PsIRF-1 and PsIRF-2 expression, while P5IRF-5 gene expression did not respond to the challenge of Poly (I: C). (C) 2011 Elsevier Ltd. All rights reserved.

INTRODUCTIONAxis and germ-layer formations are central issues in vertebrate embryology. An intriguing question is how the mechanisms that existed in an ancestral vertebrate have been modified during vertebrate evolution. A major stream of vertebrates (Osteichthyes) evolved in two monophyletic lineages: the Sarcopterygii and Actinopterygii. There are many differences in the molecular and cellular mechanisms of embryogenesis between teleosts (actinopterygians) and amphibians (sarcopterygians), including the differences in bauplan (body plan). Polypterus diverged from all other actinopterygians ~400 million years ago (Mya) during the Devonian period, soon after the divarication of an ancestral bony fish into Actinopterygii and Sarcopterygii. Polypterus is thus uniquely well suited for studies assessing the ancestral state or bauplan of Osteichthyes and Actinopterygii, as well as the divergence of embryogenetic processes in teleosts and amphibians. This protocol describes the collection of Polypterus embryos and the method for microinjection.

INTRODUCTIONAxis and germ-layer formations are central issues in vertebrate embryology. An intriguing question is how the mechanisms that existed in an ancestral vertebrate have been modified during vertebrate evolution. A major stream of vertebrates (Osteichthyes) evolved in two monophyletic lineages: the Sarcopterygii and Actinopterygii. There are many differences in the molecular and cellular mechanisms of embryogenesis between teleosts (actinopterygians) and amphibians (sarcopterygians), including the differences in bauplan (body plan). Polypterus diverged from all other actinopterygians ~400 million years ago (Mya) during the Devonian period, soon after the divarication of an ancestral bony fish into Actinopterygii and Sarcopterygii. Polypterus is thus uniquely well suited for studies assessing the ancestral state or bauplan of Osteichthyes and Actinopterygii, as well as the divergence of embryogenetic processes in teleosts and amphibians. This protocol describes a method for in situ hybridization in Polypterus embryos.

Ryanodine receptors (RyRs) are large homotetrameric protein complexes that mediate the release of intracellular stores of calcium. Mammals possess three gene copies, RyR1, RyR2, and RyR3 that are expressed in a variety of tissue types. Teleost fish express RyR1a and RyR1b genes that are expressed in slow twitch skeletal muscle and fast twitch skeletal muscles respectively. Here we report the results of a survey of the genome of bichir (Polypterus ornatipinnis), considered the most basal ray-finned fish, for its RyR genes. The bichir genome encodes four RyR genes, RyR1a, RyR1b, RyR2, and RyR3 that phylogenetically cluster with their vertebrate orthologs. Quantitative real time PCR shows fibre type-specific expression of the RyR1a and RyR1b genes. The RyR3 gene, however, is down regulated in bichir in contrast to derived teleosts including zebrafish in which the RyR1 and RyR3 genes are co-expressed at equivalent levels.

Anatomical and functional studies of the autonomic innervation in the lung and the heart of the bichirs are lacking. The present review paper describes the presence of nerve fibers located in the muscle layers of the lung and its submucosa, the collection of unipolar neurons found in the submucosal and muscle layers of the glottis in a bichir species (Polypterus bichir bichir). Putative oxygen chemoreceptive, neuroepithelial cells (NECs) in the lung mucosa are also included. The latter share many immunohistochemical characteristics similar to those observed in the carotid body and neuroepithelial bodies of mammals. A packed collection of paraganglion cells is located within the trunk of the pulmonary vagus nerves. The paper also examines the occurrence of intracardiac neurons and nerve fibers in the heart of the above species. These studies show that various neurotransmitters may indicate different patterns of innervation in the lung and the heart of the bichirs. However, there is still much to be discovered about the lung and cardiovascular nervous control of these primitive fishes.

Anatomical and physiological studies of cardiovascular control are lacking in the ray-finned fish, the bichirs. The present immunohistochemical studies on the bichir (Polypterus bichir bichir) demonstrated the occurrence of intracardiac neurons and nerve fibers in the heart. Immunoreactivity to tyrosine hydroxylase (TH) and acetylcholinesterase (AchE) and various neuropeptides (substance P, galanin, vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP)), including neuronal nitric oxide synthase (nNOS), was found in the nerve cell bodies lying close to the Sinus venosus and the sino-atrial region. The main intracardiac localization of the nervous tissue is a network of nerve fibers, presumably corresponding to the postganglionic outflow giving rise to nerve terminals and the nerve cell bodies. In addition, the heart is innervated by extrinsic monoamine-containing nerve fibers supplying the Conus arteriosus and Sinus venosus, and substance P and galanin immunopositive fibers probably originating from cranial and spinal ganglia. The adrenergic innervation of the heart of the bichir is similar to that of the teleosts, but further studies are required on nervous control of the heart.

To assess evolutional changes in the expression pattern of Otx paralogues, expression analyses were undertaken in fugu, bichir, skate and lamprey. Together with those in model vertebrates, the comparison suggested that a gnathostome ancestor would have utilized all of Otx1, Otx2 and Otx5 paralogues in organizer and anterior mesendoderm for head development. In this animal, Otx1 and Otx2 would have also functioned in specification of the anterior neuroectoderm at presomite stage and subsequent development of forebrain/midbrain at somite stage, while Otx5 expression would have already been specialized in epiphysis and eyes. Otx1 and Otx2 functions in anterior neuroectoderm and brain of the gnathostome ancestor would have been differentially maintained by Otx1 in a basal actinopterygian and by Otx2 in a basal sarcopterygian. Otx5 expression in head organizer and anterior mesendoderm seems to have been lost in the teleost lineage after divergence of bichir, and also from the amniotes after divergence of amphibians as independent events. Otx1 expression was lost from the organizer in the tetrapod lineage. In contrast, in a teleost ancestor prior to whole genome duplication, Otx1 and Otx2 would have both been expressed in the dorsal margin of blastoderm, embryonic shield, anterior mesendoderm, anterior neuroectoderm and forebrain/midbrain, at respective stages of head development. Subsequent whole genome duplication and the following genome changes would have caused different Otx paralogue usages in each teleost lineage. Lampreys also have three Otx paralogues; their sequences are highly diverged from gnathostome cognates, but their expression pattern is well related to those of skate Otx cognates.

Leu- and Met-enkephalin were the first endogenous opioid peptides identified in different mammalian species including the human. Comparative biochemical and bioinformatic evidence indicates that enkephalins are not limited to mammals. Various prodynorphin (PDYN) sequences in lower vertebrates revealed the presence of other enkephalin fingerprints in these precursor polypeptides. Among the novel enkephalins Ile-enkephalin (Tyr-Gly-Gly-Phe-Ile) was primarily observed in the African clawed frog (Xenopus laevis) PDYNs, while the structure of Phe-enkephalin (Tyr-Gly-Gly-Phe-Phe) was predicted by analyzing brain cDNA sequences encoding a PDYN of the African lungfish (Protopterus annectens). Ile-enkephalin can also be found in the PDYNs of four other fish species including the eel, bichir, zebrafish and tilapia, but no further occurrence for the Phe-enkephalin motif is available as yet. Based on sequencing data, the biological relevance of Phe- and Ile-enkephalin is suggested, because both of them can arise by regular posttranslational enzymatic processing of the respective neuropeptide precursors. In various receptor binding assays performed on rat brain membrane preparations both of the new peptides turned out to be moderate affinity opioids with a weak preference for the delta-opioid receptor (DOP) sites. Phe-enkephalin of the lungfish displayed rather unexpectedly low affinities toward the mu-opioid receptor (MOP) and DOP, while exhibiting moderate affinity toward the kappa-opioid receptor (KOP). In receptor-mediated G-protein activation assays measured by the stimulation of [(35)S]GTPgammaS binding, Met-enkephalin produced the highest stimulation followed by Leu-enkephalin, Ile-enkephalin and Phe-enkephalin, whereas the least efficacious among these endogenous peptides was still more effective than the prototype opiate agonist morphine in these functional tests.

All extant vertebrates have a blood-brain barrier (BBB), a specialized layer of cells that controls molecular traffic between blood and brain, and contributes to the regulation (homeostasis) of the brain microenvironment. Such homeostasis is critical for the stable function of synapses and neural networks. The barrier is formed by vascular endothelial cells in most groups, but by perivascular glial cells (astrocytes) in elasmobranch fish (sharks, skates, and rays). It has been unclear which is the ancestral form, but this information is important, as it could offer insights into the roles of the endothelium and perivascular glia in the modern mammalian BBB. We have used electron microscopic techniques to examine three further ancient fish groups, with intravascular horseradish peroxidase as permeability tracer. We find that in bichir and lungfish the barrier is formed by brain endothelial cells, while in sturgeon it is formed by a complex perivascular glial sheath, but with no detectable tight junctions. From their BBB pattern, and position on the vertebrate family tree, we conclude that the ancestral vertebrate had a glial BBB. This means that an endothelial barrier would have arisen independently several times during evolution, and implies that an endothelial barrier gave strong selective advantage. The selective advantage may derive partly from greater separation of function between endothelium and astrocytic glia. There are important implications for the development, physiology, and pathology of the mammalian BBB, and for the roles of endothelium and glia in CNS barrier layers.

Polypterid fishes are considered the basalmost group of extant actinopterygians and may be a direct link to understanding the systematics and evolution of the first bony fishes. Several investigations have been conducted on one member genus, Polypterus; however, since the first specimens of its sister taxon Erpetoichthys calabaricus were described, remarkably little work has been done on the species. We review terminology critical to understanding cranial morphology in polypterids and present a new description of the skull of E. calabaricus as observed through classical methods of skeletal preparation, X-radiographic microfocus computed tomography, and 3D-digital reconstruction. Differences among E. calabaricus and at least three species of Polypterus (P. bichir, P. senegalus, and P. endlicheri), besides the gross variation in size, include an overall elongation of the skull roof observable in most elements of E. calabaricus with a shortening of most associated processes. In addition, several elements present in species of Polypterus are absent in E. calabaricus. As a result, Polypterus should not be used as a proxy for the family Polypteridae to the exclusion of E. calabaricus in phylogenetic studies, which examine early actinopterygians. Each should be treated separately, to resolve inter- and intrarelationships of Polypteridae.

Anatomical and functional studies of the autonomic innervation in the lung of dipnoan fishes and the bichirs are lacking. The present immunohistochemical studies demonstrated the presence of nerve fibers in the muscle layers of the lung of the bichir, Polypterus bichir bichir, and identified the immunoreactive elements of this innervation. Tyrosine hydroxylase, acetylcholinesterase, and peptide immunoreactivity was detected in the intramural nerve fibers. Extensive innervation was present in the submucosa where adenylatecyclase/activating polypeptide 38, substance P, P2X2, and 5-hydroxytryptamine (5-HT)–immunoreactive nerve fibers mainly supplied blood vessels. A collection of monopolar neurons located in the submucosal and the muscular layers of the glottis expressed a variety of various transmitters. These neurons may be homologous to ganglion cells in the branchial and pharyngeal rami of the vagus in fishes. Nerves containing 5-HT and P2X2 receptor immunoreactivity projected to the lung epithelium. Associated with neuroepithelial cells in mucociliated epithelium, were neuronal nitric oxide synthase–immunopositive axons. The physiological function of this innervation is not known. The present study shows that the pattern of autonomic innervation of the bichir lung may by similar in its elements to that in tetrapods. Anat Rec, 290:1166–1177, 2007. © 2007 Wiley-Liss, Inc.

Extensive evidence exists for a genome duplication in the fish lineage leading to the species-rich clade of the teleosts, comprising99% of the known actinopterygian (ray-finned) fish species. Our previous studies of the neuropeptide Y receptor (NPYR) gene family suggested an ancestral gnathostome repertoire of 7 genes in 3 subfamilies. However, studies in the zebrafish have earlier identified only 5 NPYR genes, despite the expected increase in gene number due to the teleost tetraploidization. Notably, receptors Y(1), Y(5) and Y(6) were missing in the zebrafish genome database and only Y(8) had been duplicated. We report here an investigation of the evolutionary history of the Y(1) subfamily (Y(1), Y(4), Y(6) and Y(8)) and the Y(5) receptor. Seven basal actinopterygian species and a shark were investigated and a total of 22 gene fragments were cloned and analyzed. Our results show that subtypes Y(1), Y(5) and Y(6) still exist in species representing basal actinopterygian lineages (bichir, sturgeon, gar and bowfin) as well as in some basal teleost lineages. Surprisingly we identified a zebrafish Y(1) receptor, the first Y(1) receptor found in euteleosts. Thus, these findings confirm the ancestral gnathostome repertoire of 7 NPYR genes and show that many of these receptors are present in basal actinopterygians as well as some basal teleosts. NPYR losses seem to have occurred relatively recently in euteleosts because Y(1), Y(5) and Y(6) are absent in the genome databases of two pufferfishes as well as medaka and stickleback and Y(5) and Y(6) are absent in the zebrafish database. A duplicate of Y(8) seems to be the only remaining receptor gene resulting from the teleost tetraploidization. The unexpected absence of the two appetite-stimulating receptors Y(1) and Y(5) in some euteleosts, along with our discovery of duplicates of the peptide ligands NPY and PYY, has implications for the role of the NPY system in euteleost feeding behavior.

1. Background Vertebrate epithelial cells typically express a specific set of keratins. In teleosts, keratins are also present in a variety of mesenchymal cells, which usually express vimentin. Significantly, our previous studies revealed that virtually all known teleost keratins evolved independently from those present in terrestrial vertebrates. To further elucidate the evolutionary scenario that led to the large variety of keratins and their complex expression patterns in present day teleosts, we have investigated their presence in bichir, sturgeon and gar. 2. Results We have discovered a novel group of type I keratins with members in all three of these ancient ray-finned fish, but apparently no counterparts are present in any other vertebrate class so far investigated, including the modern teleost fish. From sturgeon and gar we sequenced one and from bichir two members of this novel keratin group. By complementary keratin blot-binding assays and peptide mass fingerprinting using MALDI-TOF mass spectrometry, in sturgeon we were able to assign the sequence to a prominent protein spot, present exclusively in a two-dimensionally separated cytoskeletal preparation of skin, thus identifying it as an epidermally expressed type I keratin. In contrast to the other keratins we have so far sequenced from bichir, sturgeon and gar, these new sequences occupy a rather basal position within the phylogenetic tree of type I keratins, in a close vicinity to the keratins we previously cloned from river lamprey. 3. Conclusion Thus, this new K14 group seem to belong to a very ancient keratin branch, whose functional role has still to be further elucidated. Furthermore, the exclusive presence of this keratin group in bichir, sturgeon and gar points to the close phylogenetic relationship of these ray- finned fish, an issue still under debate among taxonomists.

Immunohistochemical localisation of neurotransmitters was used to determine the distribution of unipolar neurons and neuroepithelial cells (NECs) in the respiratory tract of the bichir, Polypterus bichir bichir. NECs were commonly encountered in the mucociliated epithelium of the lung. Unipolar neurons were located in the submucosal and muscle layers of the glottis. The results suggest the presence of tyrosine hydroxylase (TH) and nNOS immunoreactivities in NECs. In addition, ACh-E/nNOS and TH/nNOS nerve fibers were also found associated with these cells. Unipolar neuronal cells showed a chemical code including the presence of 5-HT, ACh-E, peptides and P2×2 receptors. The present findings indicate that nitric oxide (NO) is a primitive transmitter of neuroepithelial oxygen-sensitive chemoreceptor cells together with acetylcholine. The coexistence of ACh-E with other substances in the unipolar neurons, but not with NO, may be a property of vagal postganglionic neurons since the emergence of the cranial autonomic pathways in the earliest vertebrates. It would be interesting to know about the provenance of the nerves in contact with NECs, which appear to have a complex innervation pattern.

The Sarcopterygii are considered to be the living species most closely related to the ancestors of the tetrapods: they include the extinct rhipidistians, the coelacanths and the dipnoans. Furthermore, many Authors debate whether the Polypteriformes should be assigned a very peculiar place in the phylogeny of the bony fishes. To investigate the group of Polypteriformes and the Dipnoans and to provide new support for the classic morphological and molecular data and previous karyological evidence, we examined the DNA sequences of the mitochondrial genes 16S, 12S and cyt-b in two polypterids (Polypterus palmas and Erpetoichthys calabaricus) and two lungfish (Protopterus annectens and P. aethiopicus). In all the trees, Polypteriformes and Dipnoi are grouped together, while coelacanths remain as a sister group of these two. This molecular evidence supports the earliest hypotheses in which Polypteriformes were grouped in the same subclass together with the coelacanths and Dipnoi. © 2004 Taylor & Francis Group, LLC.

It has been suggested that the increase in the number of Hox genes may have been one of the key events in vertebrate evolution. Invertebrates have one Hox cluster, while mammals have four. Interestingly, the number of Hox gene clusters is greater in the teleost fishes, zebrafish and medaka, than in mouse and human. The greater number of Hox clusters in the teleosts suggests that Hox gene duplication events have occurred during the radiation of ray-finned fishes. The question is when the Hox gene duplication event(s) that lead to seven Hox clusters in the teleosts actually occurred. We have addressed this question by studying the Hox genes in the bichir, Polypterus palmas. A preliminary PCR-estimation of the number of Hox genes suggests that Polypterus has five different Hox9 cognate group genes, which may be an indication of more than four Hox clusters in the bichir.

SUMMARYThe fast-start escape response is the primary reflexive escape mechanism in a wide phylogenetic range of fishes. To add detail to previously reported novel muscle activity patterns during the escape response of the bichir, Polypterus, we analyzed escape kinematics and muscle activity patterns in Polypterus senegalus using high-speed video and electromyography (EMG). Five fish were filmed at 250 Hz while synchronously recording white muscle activity at five sites on both sides of the body simultaneously (10 sites in total). Body wave speed and center of mass velocity, acceleration and curvature were calculated from digitized outlines. Six EMG variables per channel were also measured to characterize the motor pattern. P. senegalus shows a wide range of activity patterns, from very strong responses, in which the head often touched the tail, to very weak responses. This variation in strength is significantly correlated with the stimulus and is mechanically driven by changes in stage 1 muscle activity duration. Besides these changes in duration, the stage 1 muscle activity is unusual because it has strong bilateral activity, although the observed contralateral activity is significantly weaker and shorter in duration than ipsilateral activity. Bilateral activity may stiffen the body, but it does so by a constant amount over the variation we observed; therefore, P. senegalus does not modulate fast-start wave speed by changing body stiffness. Escape responses almost always have stage 2 contralateral muscle activity, often only in the anterior third of the body. The magnitude of the stage 2 activity is the primary predictor of final escape velocity.

The evolutionary relationships of gnathostomes (jawed vertebrates), which comprise chondrichthyans (cartilaginous fishes), lobe-finned fishes (coelacanths and lungfishes), tetrapods, and actinopterygians (ray-finned fishes), have been debated for almost a century. Phylogenetic analyses based on fossils, morphology, and molecular sequences have generated different models of relationships that remain unresolved. We identified 13 derived shared molecular markers (synapomorphies) that define clades in the vertebrate lineage and used them to resolve the phylogenetic relationships of extant jawed vertebrates. Our markers include the presence or absence of insertions and deletions in coding sequences, nuclear introns, and alternatively spliced transcripts. The synapomorphies identified by us are congruent with each other and give rise to a single phylogenetic tree. This tree confirms that chondrichthyans are basal to all living gnathostomes, that lungfishes (Dipnoi) are the closest living relatives of tetrapods, and that bichirs (Cladistia) are the living members of the most ancient family of ray-finned fishes. Our study also provides molecular evidence to support the monophyly of living tetrapods and teleosts.