Identification of three somatostatin genes in lampreys.

Somatostatins (SSs) are a structurally diverse family of neuropeptides that play important roles in the regulation of growth, development and metabolism in vertebrates. It has been recently proposed that the common ancestor of gnathostomes possessed three SS genes, namely SS1, SS2 and SS5. SS1 and SS2 are still present in most extant gnathostome species investigated so far while SS5 primarily occurs in chondrichthyes, actinopterygians and actinistia but not in tetrapods. Very little is known about the repertoire of SSs in cyclostomes, which are extant jawless vertebrates. In the present study, we report the cloning of the cDNAs encoding three distinct lamprey SS variants that we call SSa, SSb and SSc. SSa and SSb correspond to the two SS variants previously characterized in lamprey, while SSc appears to be a totally novel one. SSa exhibits the same sequence as gnathostome SS1. SSb differs from SSa by only one substitution (Thr12→Ser). SSc exhibits a totally unique structure (ANCRMFYWKTMAAC) that shares only 50% identity with SSa and SSb. SSa, SSb and SSc precursors do not exhibit any appreciable sequence similarity outside the C-terminal region containing the SS sequence. Phylogenetic analyses failed to clearly assign orthology relationships between lamprey and gnathostome SS genes. Synteny analysis suggests that the SSc gene arose before the split of the three gnathostome genes SS1, SS2 and SS5.

Molecular cloning of the cDNAs encoding three somatostatin variants in the dogfish (Scylorhinus canicula).

It has been recently shown that the somatostatin gene family was likely composed of at least three paralogous genes in the common ancestor of all extant jawed vertebrates. These three genes, namely SS1, SS2 and SS5, are thought to have been generated through the two rounds of whole-genome duplications (2R) that took place early during the vertebrate evolution. In the present study, we report the cloning of three distinct somatostatin cDNAs from the dogfish Scylorhinus canicula, a member of the group of cartilaginous fish. We decided to call these cDNAs, at least provisionally, SSa, SSb and SSc, respectively. Two of them, SSa and SSb, encode proteins that both contain the same tetradecapeptide sequence at their C-terminal extremity (AGCKNFFWKTFTSC). This putative peptide is identical to that generated by the SS1 gene in other vertebrate species. The last cDNA, SSc, encodes a protein that contains at its C-terminal extremity the same peptide sequence as that generated by the SS2 gene in teleosts (APCKNFFWKTFTSC). Phylogenetic analysis showed that the SSa and SSc genes likely correspond to the dogfish counterparts of the SS1 and SS2 genes, respectively. In contrast, the phylogenetic status of the SSb gene is less clear. Several lines of evidence suggest that it could correspond to the SS5 gene, but this view will need to be confirmed, for example by synteny analysis. Finally, RT-PCR analysis revealed that SSa, SSb and SSc genes are differentially expressed in dogfish tissues, suggesting that the corresponding peptides may exert distinct functions.

Impact of gene/genome duplications on the evolution of the urotensin II and somatostatin families.

The present review describes the molecular evolution of two phylogenetically related families of neuropeptides, the urotensin II (UII) and somatatostatin (SS) families. The UII family consists of four paralogous genes called UII, URP, URP1 and URP2 and the SS family is composed of six paralogous genes named SS1, SS2, SS3, SS4, SS5 and SS6. All these paralogs are present in teleosts, while only four of them, UII, URP, SS1 and SS2 are detected in tetrapods. Comparative genomics showed that most of these genes, namely UII, URP, URP1 and URP2 on the one hand and SS1, SS2 and SS5 on the other hand arose through the 2R. In contrast, the teleost-specific 3R had a much more moderate impact since it only concerned the UII and SS1 genes, which once duplicated, generated a second UII copy and SS4, respectively. The two remaining genes, SS3 and SS6, arose through tandem duplications of the SS1 and SS2 genes respectively, probably in the stem lineage of actinopterygians, before the emergence of teleosts. The history of the UII and SS families has also been marked by massive gene lost, both in tetrapods and in teleosts, but only after the 3R in this latter lineage. Finally, ancestral UII and SS genes are thought to have arisen through tandem duplication of a single ancestral gene, largely before the 1R. An important challenge for the future will be to understand the physiological significance of the molecular diversity of these two families.

Characterization of the true ortholog of the urotensin II-related peptide (URP) gene in teleosts.

It has been recently established that the urotensin II (UII) family consists of four distinct paralogs in bony vertebrates, namely UII, and the three UII-related peptides (URPs) called URP, URP1 and URP2. These four peptides are encoded by genes which arose from the two rounds of tetraploidization (2R) which took place early during vertebrate evolution. Up to now, three of them, UII, URP1 and URP2, have been identified in teleosts, while only two, UII and URP, have been reported in tetrapods. The fact that fish URP has not been found in previous studies led to the suggestion that the corresponding gene had been lost in the teleost lineage. In the present study, we show that this view is not correct. A search of the most recent release of the Ensembl genome database led us to identify a novel UII/URP-like gene in teleosts. Using synteny analysis, we demonstrate that this gene corresponds to the true ortholog of the tetrapod URP gene. Molecular cloning of the corresponding cDNA in medaka revealed that URP gene encodes a putative peptide, with the primary structure GEPCFWKYCV. In stickleback, tilapia and takifugu, URP exhibited the same sequence while, in tetraodon, it differed by only one amino acid substitution Gly ↔ Ser. In zebrafish, URP appeared totally divergent at its N-terminus with the structure DDTCFWKYCV. In conclusion, the occurrence of a true URP in teleosts shows that the quartet of UII-related genes which arose from 2R has been integrally preserved in this lineage.

Tectorotundal connections in turtles: an electron microscopic tracing and GABA-immunocytochemical study.

The nucleus rotundus of the turtles Emys orbicularis and Testudo horsfieldi was analysed by axonal tracing methods and post-embedding GABA immunocytochemistry. After injections of horseradish peroxidase or biotinylated dextran amine into the optic tectum, electron microscopic observations showed that the vast majority of ipsilateral tectorotundal axon terminals were small in size, had smooth contours and contained small, round, densely packed synaptic vesicles. These terminals were GABA-immunonegative, often gathered in clusters, and established asymmetrical synaptic contacts with either small- or medium-sized GABA-negative dendritic profiles and with GABA-immunoreactive (GABA-ir) dendrites, which did not contain synaptic vesicles. Occasional GABA-ir-labelled axon terminals were observed; these may arise from the rare GABAergic neurons in the central tectal layer, or from neurons in the ventral pretectal nucleus, which projects both to the optic tectum and nucleus rotundus. In addition to tracer-labelled axon terminals, we observed both GABA-negative and GABA-ir cell bodies and dendrites also labelled by the tracer. No GABA-ir presynaptic dendritic profiles containing synaptic vesicles were observed. The existence in reptiles of reciprocal connections between the nucleus rotundus and the optic tectum as a phylogenetically ancient feedback system is discussed.

Distribution of calcium-binding proteins in the pigeon visual thalamic centers and related pretectal and mesencephalic nuclei. Phylogenetic and functional determinants.

Multichannel processing of environmental information constitutes a fundamental basis of functioning of sensory systems in the vertebrate brain. Two distinct parallel visual systems - the tectofugal and thalamofugal exist in all amniotes. The vertebrate central nervous system contains high concentrations of intracellular calcium-binding proteins (CaBPrs) and each of them has a restricted expression pattern in different brain regions and specific neuronal subpopulations. This study aimed at describing the patterns of distribution of parvalbumin (PV) and calbindin (CB) in the visual thalamic and mesencephalic centers of the pigeon (Columba livia). We used a combination of immunohistochemistry and double labeling immunofluorescent technique. Structures studied included the thalamic relay centers involved in the tectofugal (nucleus rotundus, Rot) and thalamofugal (nucleus geniculatus lateralis, pars dorsalis, GLd) visual pathways as well as pretectal, mesencephalic, isthmic and thalamic structures inducing the driver and/or modulatory action to the visual processing. We showed that neither of these proteins was unique to the Rot or GLd. The Rot contained i) numerous PV-immunoreactive (ir) neurons and a dense neuropil, and ii) a few CB-ir neurons mostly located in the anterior dorsal part and associated with a light neuropil. These latter neurons partially overlapped with the former and some of them colocalized both proteins. The distinct subnuclei of the GLd were also characterized by different patterns of distribution of CaBPrs. Some (nucleus dorsolateralis anterior, pars magnocellularis, DLAmc; pars lateralis, DLL; pars rostrolateralis, DLAlr; nucleus lateralis anterior thalami, LA) contained both CB- and PV-ir neurons in different proportions with a predominance of the former in the DLAmc and DLL. The nucleus lateralis dorsalis of nuclei optici principalis thalami only contained PV-ir neurons and a neuropil similar to the interstitial pretectal/thalamic nuclei of the tectothalamic tract, nucleus pretectalis and thalamic reticular nucleus. The overlapping distribution of PV and CB immunoreactivity was typical for the pretectal nucleus lentiformis mesencephali and the nucleus ectomamillaris as well as for the visual isthmic nuclei. The findings are discussed in the light of the contributive role of the phylogenetic and functional factors determining the circuits׳ specificity of the different CaBPr types.

Comparative distribution and in vitro activities of the urotensin II-related peptides URP1 and URP2 in zebrafish: evidence for their colocalization in spinal cerebrospinal fluid-contacting neurons.

Urotensin II (UII) is an evolutionarily conserved neuropeptide initially isolated from teleost fish on the basis of its smooth muscle-contracting activity. Subsequent studies have demonstrated the occurrence of several UII-related peptides (URPs), such that the UII family is now known to include four paralogue genes called UII, URP, URP1 and URP2. These genes probably arose through the two rounds of whole genome duplication that occurred during early vertebrate evolution. URP has been identified both in tetrapods and teleosts. In contrast, URP1 and URP2 have only been observed in ray-finned and cartilaginous fishes, suggesting that both genes were lost in the tetrapod lineage. In the present study, the distribution of urp1 mRNA compared to urp2 mRNA is reported in the central nervous system of zebrafish. In the spinal cord, urp1 and urp2 mRNAs were mainly colocalized in the same cells. These cells were also shown to be GABAergic and express the gene encoding the polycystic kidney disease 2-like 1 (pkd2l1) channel, indicating that they likely correspond to cerebrospinal fluid-contacting neurons. In the hindbrain, urp1-expressing cells were found in the intermediate reticular formation and the glossopharyngeal-vagal motor nerve nuclei. We also showed that synthetic URP1 and URP2 were able to induce intracellular calcium mobilization in human UII receptor (hUT)-transfected CHO cells with similar potencies (pEC50=7.99 and 7.52, respectively) albeit at slightly lower potencies than human UII and mammalian URP (pEC50=9.44 and 8.61, respectively). The functional redundancy of URP1 and URP2 as well as the colocalization of their mRNAs in the spinal cord suggest the robustness of this peptidic system and its physiological importance in zebrafish.

Differences in parvalbumin and calbindin chemospecificity in the centers of the turtle ascending auditory pathway revealed by double immunofluorescence labeling.

Using double immunofluorescence labeling, quantitative ratio between parvalbumin- and calbindin-containing neurons, neurons that co-localize both peptides, as well as the intensity of their immunoreactivities were studied in the brainstem, midbrain and forebrain auditory centers of two chelonian species, Testudo horsfieldi and Emys orbicularis. In the spiral ganglion and first-order cochlear nuclei, highly immunoreactive parvalbumin-containing neurons predominated, and almost all neurons in these nuclei also exhibited weak immunoreactivity to calbindin. The number of strongly calbindin-immunoreactive (-ir) cells increased in the second-order brainstem auditory centers (the laminar cochlear nucleus, superior olivary complex, lateral lemniscal nucleus), and co-localization with parvalbumin in some of them was observed. In the midbrain, a complementary distribution of parvalbumin and calbindin immunoreactivity was found: the central (core) region of the torus semicircularis showed strong parvalbumin immunoreactivity, while the laminar (belt) nucleus was strongly calbindin-ir. In the thalamic nucleus reuniens, almost complete topographic overlapping of the parvalbumin-ir and calbindin-ir neurons was shown in its dorsomedial region (core), with the intensity of immunoreactivity to calbindin being much higher than that to parvalbumin. The predominance of calbindin immunoreactivity in neurons of the dorsomedial region of the nucleus reuniens is correlated with the existence of the dense calbindin-ir terminal field in its projection area in the telencephalon. We conclude that the turtle auditory pathway is chemically heterogeneous with respect to calcium-binding proteins, the predominance of parvalbumin in the brainstem and midbrain centers giving way to that of calbindin in the forebrain centers; the portion of neurons co-localizing both peptides nonlinearly decreases from lower to higher order centers.

Core-and-belt organisation of the mesencephalic and forebrain auditory centres in turtles: expression of calcium-binding proteins and metabolic activity.

The distribution of immunoreactivity to the calcium-binding proteins parvalbumin, calbindin and calretinin and of cytochrome oxidase activity was studied in the mesencephalic (torus semicircularis), thalamic (nucleus reuniens) and telencephalic (ventromedial part of the anterior dorsal ventricular ridge) auditory centres of two chelonian species Emys orbicularis and Testudo horsfieldi. In the torus semicircularis, the central nucleus (core) showed intense parvalbumin immunoreactivity and high cytochrome oxidase activity, whereas the laminar nucleus (belt) showed low cytochrome oxidase activity and dense calbindin/calretinin immunoreactivity. Within the central nucleus, the central and peripheral areas could be distinguished by a higher density of parvalbumin immunoreactivity and cytochrome oxidase activity in the core than in the peripheral area. In the nucleus reuniens, the dorsal and ventromedial (core) regions showed high cytochrome oxidase activity and immunoreactivity to all three calcium-binding proteins, while its ventrolateral part (belt) was weakly immunoreactive and showed lower cytochrome oxidase activity. In the telencephalic auditory centre, on the other hand, no particular region differed in either immunoreactivity or cytochrome oxidase activity. Our findings provide additional arguments in favour of the hypothesis of a core-and-belt organisation of the auditory sensory centres in non-mammalian amniotes though this organisation is less evident in higher order centres. The data are discussed in terms of the evolution of the auditory system in amniotes.

Synaptic circuitry in the retinorecipient layers of the optic tectum of the lamprey (Lampetra fluviatilis). A combined hodological, GABA and glutamate immunocytochemical study.

The ultrastructure of the retinorecipient layers of the lamprey optic tectum was analysed using tract tracing techniques combined with GABA and glutamate immunocytochemistry. Two types of neurons were identified; a population of large GABA-immunonegative cells, and a population of smaller, highly GABA-immunoreactive interneurons, some of whose dendrites contain synaptic vesicles (DCSV). Five types of axon terminals were identified and divided into two major categories. The first of these are GABA-immunonegative, highly glutamate-immunoreactive, contain round synaptic vesicles, make asymmetrical synaptic contacts, and can in turn be divided into AT1 and AT2 terminals. The AT1 terminals are those of the retinotectal projection. The origin of the nonretinal AT2 terminals could not be determined. AT1 and AT2 terminals establish synaptic contacts with DCSV, with dendrites of the retinopetal neurons (DRN), and with conventional dendritic (D) profiles. The terminals of the second category are GABA-immunoreactive and can similarly be divided into AT3 and AT4 terminals. The AT3 terminals contain pleiomorphic synaptic vesicles and make symmetrical synaptic contacts for the most part with glutamate-immunoreactive D profiles. The AT4 terminals contain rounded synaptic vesicles and make asymmetrical synaptic contacts with DRN, with DCSV, and with D profiles. A fifth, rarely observed category of terminals (AT5) contain both clear synaptic vesicles and a large number of dense-core vesicles. Synaptic triads involving AT1, AT2 or AT4 terminals are rare. Our findings are compared to these of previous studies of the fine structure and immunochemical properties of the retinorecipient layers of the optic tectum or superior colliculus of Gnathostomes.

Fine structure of the visual dorsolateral anterior thalamic nucleus of the pigeon (Columba livia): a hodological and GABA-immunocytochemical study.

The ultrastructure of the lateroventral subcomponent of the visual dorsolateral anterior thalamic nucleus of the pigeon (DLLv) was analyzed using hodological techniques and GABA-immunocytochemistry. Two types of GABA-immunonegative hyperpalliopetal neurons and a single type of strongly GABA-immunoreactive (-ir) interneuron were identified, the latter displaying long dendrites with some containing synaptic vesicles (DCSV). Ten types of axon terminal were identified and divided into two categories. The first, GABA-immunonegative and making asymmetrical synaptic contact, contain round (RT1, RT2, RT3) or pleiomorphic synaptic and many dense-core vesicles (DCT). RT1 terminals are retinothalamic and RT2 terminals hyperpalliothalamic; both mainly contact dendrites of projection neurons (72% and 78% respectively), less frequently dendrites of interneurons and sometimes DCSV; RT1 terminals are rarely involved in synaptic triads. The second category are consistently GABA-immunopositive. Four types (PT1-4), distinguished by their pleiomorphic synaptic vesicles, make symmetrical synaptic contact essentially with dendrites of projection neurons, more rarely on dendrites of interneurons (PT2). PT1 terminals are very probably those of interneurons, whereas the rare PT4 terminals are of retinal origin. A fifth type (RgT) contains round synaptic vesicles and makes asymmetrical synaptic contact with dendrites of projection neurons and interneurons. PT2 and RgT terminals occasionally contact DCSV of interneurons, which are sometimes involved in synaptic triads. Two final subcategories (DCgT1-2) contain many dense-core vesicles. Our findings are compared with those of previous studies concerning the fine structure and neurochemical properties of the GLd of reptiles and mammals, with special reference to the origin of the extraretinal and extracortical projections to this structure.