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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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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 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.

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.

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.

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.

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 Polypteriformes (bichirs and reedfish) are a family of ray-finned fishes of ancient lineage. Insulin has been isolated from an extract of the pancreas and upper gastrointestinal tract of the bichir Polypterus senegalis and its primary structure established as A-chain: Gly-Ile-Val-Glu-Gln-Cys-Cys-Asp-Thr-Pro10-Cys-Ser- Leu-Tyr-Asp-Leu-Glu-Asn-Tyr-Cys20-Asn: B-chain: Ala-Ala-Asn-Arg-His-Leu-Cys-Gly-Ser-His10-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly20-Asn-Arg-Gly-Phe- Phe-Tyr-Ile-Pro-Ser-Lys30-Met. Despite the fact that Polypterus insulin contains several unusual structural features that are not found in insulins from other jawed fish (Asp at A-8, Thr at A-9, Arg at B-4, Asn at B-21, Ile at B-27, Met at B-31), all the residues in human insulin that are involved in receptor binding, dimerization, and hexamerization have been conserved. A comparison of the structures of insulins from a range of species indicates that Polypterus insulin most closely resembles paddlefish insulin II (seven amino acid substitutions). In contrast, Polypterus glucagon (His-Ser- Gln-Gly-Thr-Phe-Thr-Asn-Asp-Tyr10-Thr-Lys-Tyr- Gln-Asp-Ser-Arg-Arg-Ala-Gln20-Asp-Phe-Val-Gln- Trp-Leu-Met-Ser-Asn) most closely resembles the glucagons from the gar Lepisosteus spatula and the bowfin Amia calva (four amino acid substitutions). The data are consistent with the conclusion based on comparison of morphological characteristics that the Polypterids are the most basal living group of the Actinopterygians with evolutionary connections to both the Acipenserids and the Neopterygians.

The telencephalon in ray-finned fish (actinopterygians) is everted, in contrast to the evaginated telencephalic hemispheres in all other vertebrates. In the more derived ray-finned fish, the teleosts, proliferation of neurons and their migration from the ependymal zone of the pallium renders comparisons between telencephalic cell groups of the teleosts and members of other vertebrate groups extremely difficult. The telencephalon of Polypterus (a primitive living ray-finned fish), although everted, is cytoarchitecturally much simpler than that of teleosts. We have thus applied immunohistochemical techniques to the study of the telencephalon of Polypterus to help clarify the evolution of the telencephalon in teleosts and facilitate comparisons between the telencephalon in ray-finned fish and other vertebrates. Antisera against the following neuroactive substances were used: 1) serotonin (5HT), 2) tyrosine hydroxylase (TH), 3) substance P (SP), 4) leucine-enkephalin (ENK), 5) neuropeptide Y (NPY), and 6) the neurotensin-related hexapeptide LANT6. Several features of the labeling patterns obtained suggested that the dorsal and ventral subdivisions of the area ventralis are homologous as a field to the basal ganglia and septum plus other basal telencephalic regions of land vertebrates, sharks and lungfish: 1) an abundance of SP +, NPY +, and ENK+ fibers; 2) an abundance of TH+ fibers, possibly of posterior tubercle/tegmental origin; 3) the presence of an SP + fiber bundle that appeared to descend from basal telencephalic levels and terminate in the posterior tubercle/tegmentum, which contain TH + (possibly dopaminergic) neurons; and 4) an abundance of 5HT+ fibers, presumably of posterior tubercle/tegmental origin. It was not possible, however, to recognize distinct pallidal and striatal subdivisions within the area ventralis of Polypterus. The olfactory pallium (Pl) was generally poor in most of the substances examined, except for the presence of LANT6+ fibers. The P3 pallid field was conspicuously rich in SP+ and ENK+ fibers throughout its extent, and the caudal and lateral parts of the P2 field were rich in SP+ fibers and ENK+ fibers. Since this is characteristic of the medial pallial and/or dorsomedial pallial walls of the telencephalon in lungfish, sharks, frogs, and reptiles, the P3 field and caudolateral part of the P2 field may be homologous to these portions of the telencephalon in other vertebrates. More rostromedial parts of P2 may correspond to those parts of the pallium in land vertebrates that are in receipt of specific sensory input from the thalamus, since low neuropeptide levels are characteristic of these regions. These results indicate that the dorsal part of the ventral area of the telencephalon in ray-finned fish may be homologous to the basal ganglia in other vertebrates. These results also suggest that the pallial portions of the telencephalon in Polypterus may include a region comparable to part of the isocortex or its homologues in amniotes. o 1992 Wiley-Liss, Inc.

The presence and function of tachykinins were studied in the intestine of hagfish (Myxine glutinosa), lamprey (Lampetra fluviatilis), starry ray (Raja radiata), lungfish (Lepidosiren paradoxa), bichir (Polypterus senegalensis), and rainbow trout (Oncorhynchus mykiss), which represent different systematic groups of fish. Immunohistochemistry revealed cells containing substance P (SP)-like material in the intestine of lamprey and lungfish, and in the stomach of the ray. The intestinal motility was studied using isolated muscle strip preparations. SP had no effect on hagfish or lamprey intestine. In the other four species SP produced intestinal contractions. In ray, bichir, and lungfish the tachykinins may be released from endocrine cells and act, at least in the bichir and lungfish, directly onto the smooth muscle cells. In the rainbow trout intestine, where SP-like material may be released from both nerve fibres and endocrine cells, it is indicated that the contractile effect is in part direct upon the smooth muscle and in part via stimulation of cholinergic and serotonergic neurons.

The ampullary organs of the bichir were examined by light and electron microscopy. Unlike most other ampullary organs, they are exclusively found in the epidermis and are never sunk into the subepidermal connective tissue. The sensory epithelium consists of sensory cells and supporting cells surrounded by mantle cells. The luminal surface of the sensory cell is provided with a cilium surrounded by several microvilli. In the apical cytoplasm are found numerous mitochondria and microtubules. In the basal part of the cell synaptic sheets or synaptic bodies opposite to afferent nerve endings are frequent.

The anatomy and ultrastructure of the sacculus, lagena, and utriculus of the ear of Polypterus bichir and Scaphirhynchus platorynchus were studied using the scanning electron microscope. The otolithic organs each contain a single dense calcareous otolith in close contact with a sensory epithelium (macula). The maculae have sensory hair cells typical of those found in other vertebrates, surrounded by microvilli-covered supporting cells. The hair cells on each macula are divided into several groups, with all of the cells in each group morphologically polarized in the same direction. The cells of the utricular macula in both species are divided into opposing groups in a pattern similar to that found in other vertebrates. The saccular and lagenar maculae are located in a single large chamber in both species. In Scaphirhychus the two maculae are on the same plane, while in Polypterus they are at right angles to one another. The hair cells on the saccular maculae of both species are divided into two oppositely oriented groups. In Scaphirhynchus the cells on the posterior half of the macula are oriented dorsally on the dorsal half of the macula and ventrally on the ventral half. The anterior region of the macula is rotated and the cells of the dorsal and ventral groups are shifted so that they are oriented on the animal's horizon plane. A similar pattern is found in Polypterus, except that this macula is shaped like a "J" with the vertical portion of the J having horizontal cells and the bottom portion vertical cells. The lagenar maculae in both species have dorsally oriented cells on the anterior side of the macula and ventrally oriented cells on the posterior half of the macula. While these data are not sufficient for clarifying the taxonomic relationship between the two species studied, it is clear that the ears in these species have a number of significant differences from the teleost ear that could have functional and/or taxonomic significance.

Myelin, defined as an arrangement of spirally fused unit membranes, is an acquisition of vertebrates and first appeared during evolution in Gnathostomata. In all species studied PNS and CNS myelins contain the myelin-associated glycoprotein (MAG) and the myelin basic protein (MBP). Throughout phylogeny PNS myelin is characterized by the major P(0) glycoprotein which is called IP in fishes. The PNS myelin proteins did not evolve further except for the addition of P(2) protein from reptiles onward. In Elasmobranchii and Chondrostei, PNS and CNS myelin proteins are similar. CNS myelin of actinopterygian fishes possesses a 36,000 Da protein (36K) in addition to P(0)-like IP glycoproteins. In tetrapod CNS myelin, P(0) is replaced by the proteolipid protein (PLP) and the Wolfgram protein (WP). Of particular interest in a transitional phylogenetic sense are the lungfish Protopterus, carrying glycosylated PLP (g-PLP) but no P(0), 36K or WP, and the bichir Polypterus, showing simultaneous presence of P(0), 36K and PLP. These results indicate that myelin proteins could be valuable molecular markers in establishing vertebrate phylogenetic relationships and in reconstructing the fish-tetrapod transition.

SUMMARY Active neurohypophysial principles were separated from pituitary glands from a primitive actinopterygian, Polypterus senegalis, and the African lungfish, Protopterus aethiopicus. Both species appeared to possess arginine vasotocin (8-arginine oxytocin) and a more nearly neutral principle resembling oxytocin. In the case of Polypterus the neutral principle was pharmacologically indistinguishable from isotocin (4-serine, 8-isoleucine oxytocin). When subjected to partition chromatography on a Sephadex column the neutral Polypterus principle appeared homogeneous and emerged at an elution volume identical with that of isotocin. The neutral principle from Protopterus neurohypophysial extracts resembled mesotocin (8-isoleucine oxytocin) pharmacologically. On partition chromatography it appeared to be a single peptide with mobility comparable to that of mesotocin. No evidence was found for the presence of oxytocin despite earlier reports that this principle occurs in Protopterus and in the South American lungfish, Lepidosiren. From these studies it appears that Polypterus has neurohypophysial principles (arginine vasotocin and isotocin) identical with those found among higher actinopterygians, the Holostei and Teleostei. On the other hand, Protopterus principles resemble those found among Amphibia (arginine vasotocin, mesotocin, and possibly oxytocin).