Vertebrate genome evolution and the zebrafish gene map.

In chordate phylogeny, changes in the nervous system, jaws, and appendages transformed meek filter feeders into fearsome predators. Gene duplication is thought to promote such innovation. Vertebrate ancestors probably had single copies of genes now found in multiple copies in vertebrates and gene maps suggest that this occurred by polyploidization. It has been suggested that one genome duplication event occurred before, and one after the divergence of ray-finned and lobe-finned fishes. Holland et al., however, have argued that because various vertebrates have several HOX clusters, two rounds of duplication occurred before the origin of jawed fishes. Such gene-number data, however, do not distinguish between tandem duplications and polyploidization events, nor whether independent duplications occurred in different lineages. To investigate these matters, we mapped 144 zebrafish genes and compared the resulting map with mammalian maps. Comparison revealed large conserved chromosome segments. Because duplicated chromosome segments in zebrafish often correspond with specific chromosome segments in mammals, it is likely that two polyploidization events occurred prior to the divergence of fish and mammal lineages. This zebrafish gene map will facilitate molecular identification of mutated zebrafish genes, which can suggest functions for human genes known only by sequence.

Characterization of the neuropeptide Y system in the frog Silurana tropicalis (Pipidae): three peptides and six receptor subtypes.

Neuropeptide Y and its related peptides PYY and PP (pancreatic polypeptide) are involved in feeding behavior, regulation of the pituitary and the gastrointestinal tract, and numerous other functions. The peptides act on a family of G-protein coupled receptors with 4-7 members in jawed vertebrates. We describe here the NPY system of the Western clawed frog Silurana (Xenopus) tropicalis. Three peptides, NPY, PYY and PP, were identified together with six receptors, namely subtypes Y1, Y2, Y4, Y5, Y7 and Y8. Thus, this frog has all but one of the ancestral seven gnathostome NPY-family receptors, in contrast to mammals which have lost 2-3 of the receptors. Expression levels of mRNA for the peptide and receptor genes were analyzed in a panel of 19 frog tissues using reverse transcriptase quantitative PCR. The peptide mRNAs had broad distribution with highest expression in skin, blood and small intestine. NPY mRNA was present in the three brain regions investigated, but PYY and PP mRNAs were not detectable in any of these. All receptor mRNAs had similar expression profiles with high expression in skin, blood, muscle and heart. Three of the receptors, Y5, Y7 and Y8, could be functionally expressed in HEK-293 cells and characterized with binding studies using the three frog peptides. PYY had the highest affinity for all three receptors (K(i) 0.042-0.34 nM). Also NPY and PP bound to the Y8 receptor with high affinity (0.14 and 0.50 nM). The low affinity of NPY for the Y5 receptor (100-fold lower than PYY) differs from mammals and chicken. This may suggest a less important role of NPY on Y5 in appetite stimulation in the frog compared with amniotes. In conclusion, our characterization of the NPY system in S. tropicalis with its six receptors demonstrates not only greater complexity than in mammals but also some interesting differences in ligand-receptor preferences.

Birth and death of neuropeptide Y receptor genes in relation to the teleost fish tetraploidization.

Extensive evidence exists for a genome duplication in the fish lineage leading to the species-rich clade of the teleosts, comprising > 99% 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.

Uneven evolutionary rates of bradykinin B1 and B2 receptors in vertebrate lineages.

Bradykinin acts through two receptor subtypes in mammals and generates a variety of responses including pain, inflammation and hypotension. The evolutionary history of the bradykinin system has been unclear due to shortage of information outside mammals. We describe here two receptor subtypes and the bradykinin precursor in three species of bony fish (the zebrafish Danio rerio, the Japanese pufferfish Takifugu rubripes, and the green spotted pufferfish Tetraodon nigroviridis) and chicken and analyze the relationships to mammals by a combination of phylogeny, conserved synteny and exon-intron organization. All of these species have two receptor genes located close to each other in a tandem formation, with the B2 gene 5' to the B1 gene, in chromosomal regions displaying conserved synteny between the species (albeit conservation of synteny in zebrafish is still unclear due to poor genome assembly). The evolutionary rate differs between the two genes as well as between lineages leading to differing pharmacological properties for both B1 and B2 across vertebrate classes. Also the bradykinin precursor gene was identified in all of these species in a chromosome region with conserved synteny. The tissue distribution of mRNA in T. rubripes is similar for B1 and B2, suggesting more similar regulation for the two genes than in mammals. In conclusion, the receptor tandem duplication predates the divergence of ray-finned fish and tetrapods and no additional duplicates of the receptors or bradykinin seem to have survived the ray-finned fish tetraploidization.

Cloning and characterization of a zebrafish Y2 receptor.

The NPY receptors belong to the superfamily of G-protein coupled receptors and in mammals this family has five members, named Y1, Y2, Y4, Y5, and Y6. In bony fish, four receptors have been identified, named Ya, Yb, Yc and Y7. Yb and Y7 arose prior to the split between ray-fined fishes and tetrapods and have been lost in mammals. Yc appeared as a copy of Yb in teleost fishes. Ya may be an ortholog of Y4, but surprisingly no unambiguous receptor ortholog to any of the mammalian subtypes has yet been identified in bony fishes. Here we present the cloning and pharmacological characterization of a Y2 receptor in zebrafish, Danio rerio. To date, this is the first Y2 receptor outside mammals and birds that has been characterized pharmacologically. Phylogenetic analysis and synteny confirmed that this receptor is orthologous to mammalian Y2. We show that the receptor is pharmacologically most similar to chicken Y2 which leads to the conclusion that Y2 has acquired several novel characteristics in mammals. Y2 from zebrafish binds very poorly to the Y2-specific antagonist BIIE0246. Our pharmacological characterization supports our previous conclusions regarding the binding pocket of BIIE0246 in the human Y2 receptor.

Cloning and sequence analysis of a neuropeptide Y/peptide YY receptor Y1 cDNA from Xenopus laevis.

Neuropeptide Y (NPY) and peptide YY (PYY) are structurally related peptides that share at least two distinct receptors denoted Y1 and Y2. The Y1 receptor has previously been cloned in man, rat and mouse. We describe here the cloning and sequence of a Xenopus laevis Y1 receptor that shares 81% amino acid sequence identity with the human receptor in the region spanning transmembrane (TM) regions I to VII. The extracellular amino-terminal part, TM IV and the second extracellular loop contain several replacements suggesting that these portions have no or limited direct interactions with the peptide ligands. The intracellular regions including the carboxy-terminal tail are nearly identical between Xenopus and mammals, suggesting strong structural constraints on the portions that may interact with G proteins.

Variations in base pair composition and associated long-range correlations in DNA sequences--computer simulation results.

Recently, the possible occurrence of long-range correlations between nucleotides in DNA sequences of living organisms has excited considerable interest. Of particular importance is the claim that only intron-containing sequences exhibit these correlations. Different investigations, however, have disproved the claimed difference between intron-containing and intron-less DNA sequences. Moreover, very recent investigations pointed out that the long-range correlations appear only if relatively large variations in nucleotide composition along the DNA sequence are present. Furthermore, some examples demonstrated that these variations may have clear biological reasons. In this paper we investigate in detail, with the aid of computer simulations, the connection between compositional heterogeneity of a DNA sequence and the appearance of long-range correlations. As an explicit example, the DNA sequence of the lambda-phage is compared with different artificial sequences of similar compositional heterogeneity. The results demonstrate that the variations of the nucleotide composition along the sequence can fully account for all properties of the claimed long-range correlations. New results of extensive computer simulations are presented which clearly demonstrate how the apparent 'fractal' (or 'long-range-correlated') character of a sequence gradually evaporates, as the frequency of the compositional variations of a simulated sequence continuously increases.

Inhibition of neurotransmitter release in the lamprey reticulospinal synapse by antibody-mediated disruption of SNAP-25 function.

Exocytosis - syntaxin - synaptobrevin - SNARE synaptic vesicle The lamprey giant reticulospinal synapse can be used to manipulate the molecular machinery of synaptic vesicle exocytosis by presynaptic microinjection. Here we test the effect of disrupting the function of the SNARE protein SNAP-25. Polyclonal SNAP-25 antibodies were shown in an in vitro assay to inhibit the binding between syntaxin and SNAP-25. When microinjected presynaptically, these antibodies produced a potent inhibition of the synaptic response. Ba2+ spikes recorded in the presynaptic axon were not altered, indicating that the effect was not due to a reduced presynaptic Ca2+ entry. Electron microscopic analysis showed that synaptic vesicle clusters had a similar organization in synapses of antibody-injected axons as in control axons, and the number of synaptic vesicles in apparent contact with the presynaptic plasma membrane was also similar. Clathrin-coated pits, which normally occur at the plasma membrane around stimulated synapses, were not detected after injection of SNAP-25 antibodies, consistent with a blockade of vesicle cycling. Thus, SNAP-25 antibodies, which disrupt the interaction with syntaxin, inhibit neurotransmitter release without affecting the number of synaptic vesicles at the plasma membrane. These results provide further support to the view that the formation of SNARE complexes is critical for membrane fusion, but not for the targeting of synaptic vesicles to the presynaptic membrane.

Neuropeptide-Y gene expression in the goldfish brain: distribution and regulation by ovarian steroids.

Neuropeptide-Y (NPY) has been recently cloned from a goldfish cDNA library. Using a probe derived from this cDNA clone, the distribution and regulation of NPY mRNA in the goldfish brain were examined in the present study by in situ hybridization, Northern blot analysis, and ribonuclease protection assay. Frozen tissue sections from the goldfish brain and pituitary were hybridized with a digoxigenin-labeled RNA probe. Hybridization signal was detected mainly in fore-brain regions, particularly in the nucleus entopeduncularis of the ventral telencephalon, the preoptic area (POA), the olfactory bulbs, and various thalamic regions. In the midbrain, NPY mRNA was found in the optic-tectum and locus coeruleus. Northern blot hybridization of total RNA extracted from different brain areas with a 32P-labeled RNA probe detected a single mRNA species and confirmed that most NPY mRNA was present in telencephalon/POA and optic-tectum/thalamus. Our previous physiological studies showed that the ovarian steroids testosterone (T) and estradiol (E2) have potentiating effects on the actions of NPY on gonadotropin and GH release. Therefore, we also tested the possibility that T and E2 may modulate NPY gene expression. Fish were implanted ip with pellets containing T, E2, or no steroid (control) for 5 days, RNA was extracted, and NPY mRNA levels were estimated using a ribonuclease protection assay. Pretreatment with T or E2 induced a 2- to 3-fold increase in NPY mRNA levels in the telencephalon/POA, but not in the optic-tectum/thalamus. In situ hybridization using brains taken from T-implanted fish demonstrated that the site of steroid action is the POA. This report represents the first study on NPY gene expression in a nonmammalian species and demonstrates that 1) NPY mRNA is present in the neuroendocrine regulatory centers; and 2) ovarian steroids stimulate NPY gene expression in the POA.

Spontaneous insertions into cosmid vector: a warning.

During the course of characterization of a human genomic library we found that some of the selected pNNL cosmid clones carried only very short inserts. In addition, several clones that did contain full-length inserts were found to be unstable and generated repeated deletions of various portions of their inserts. Moreover, all the clones examined displayed rearrangements in the vector portions. The rearranged clones that were characterized by restriction mapping were found to have deletions starting between ClaI and HindIII in the region of the cos segment of the pNNL vector used in the construction of the library. We determined the nucleotide sequence of the 1300-bp EcoRI-PvuII segment located in the cos region, and by the computer search we found that it contains Escherichia coli insertion elements IS1.

Complex gene organization of synaptic protein SNAP-25 in Drosophila melanogaster.

The evolutionarily conserved protein SNAP-25 (synaptosome-associated protein 25 kDa (kilodaltons)) is a component of the protein complex involved in the docking and/or fusion of synaptic vesicles in nerve terminals. We report here that the SNAP-25 gene (Snap) in the fruit fly Drosophila melanogaster has a complex organization with eight exons spanning more than 120 kb (kilobases). The exon boundaries coincide with those of the chicken SNAP-25 gene (Bark, 1993). Only a single exon 5 has been found in Drosophila, whereas human, rat, chicken, zebrafish and goldfish have two alternatively spliced versions of this exon. In situ hybridization and immunocytochemistry to whole mount embryos show that SNAP-25 mRNA and protein are detected in stage 14 and later developmental stages, and are mainly localized to the ventral nerve cord. Thus, Snap has an evolutionarily conserved and complex gene organization, and its onset of expression in Drosophila melanogaster correlates with a time in neuronal development when synapses begin to be formed and when other synapse-specific genes are switched on.

Identification of a conserved protein motif in a group of growth factor receptors.

Residues 370-383 (helix C) of the human nerve growth factor receptor (NGF-R) are highly similar to the sequence of the 14 residue wasp toxin, mastoparan. Both regions are predicted to form amphiphilic alpha-helices, as is the amino-terminal region of the third intracytoplasmic loop (i3) of the beta 2-adrenergic receptor (beta 2AR). As both mastoparan and the beta 2AR i3 interact with G-proteins, it is suggested that helix C of the NGF-R may facilitate interactions with a cytoplasmic protein. A similar structural motif was identified in the cytoplasmic domains of a number of other growth factor receptors, suggesting an important role for this motif in signal transduction mechanisms.

Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y2(1).

Here we report the molecular cloning of the chicken (Gallus gallus) neuropeptide Y (NPY) receptor Y2, the first non-mammalian Y2 receptor. It displays 75-80% identity to mammalian Y2 and has a surprisingly divergent cytoplasmic tail. Expression of the receptor protein in a cell line showed that the receptor did not bind the mammalian Y2 selective antagonist BIIE0246. Furthermore, porcine [Leu(31), Pro(34)]NPY, which binds poorly to mammalian Y2, exhibited an unexpectedly high affinity for chicken Y2. In situ hybridisation revealed expression in the hippocampus. Thus, the chicken Y2 receptor exhibits substantial differences with regard to sequence and pharmacological profile in comparison to mammalian Y2 receptors, while the expression pattern in the central nervous system resembles that observed in mammals.

Preprocholecystokinin mRNA-expressing neurons in the rat parabrachial nucleus: subnuclear localization, efferent projection, and expression of nociceptive-related intracellular signaling substances.

The pontine parabrachial nucleus (PB) is a major target for ascending fibers from nociresponsive dorsal horn neurons. Several different neuropeptides have been identified in the PB. By using double-labeling methods that combine in situ hybridization histochemistry with retrograde tract tracing and immunohistochemistry, we have examined the subnuclear localization of preprocholecystokinin mRNA (ppCCK)-containing neurons, investigated their efferent projection, and analyzed their expression of intracellular signaling substances that may be of importance for nociceptive processing. The results show that neurons containing ppCCK are preferentially localized to the superior lateral subnucleus (PBsl), whereas other subnuclei, such as the dorsal lateral, external lateral, central lateral, and ventral lateral subnuclei, and the Kölliker-Fuse nucleus, contain only moderate to small numbers of such neurons. Injections of the retrograde tracer cholera toxin subunit b into the ventromedial hypothalamus demonstrated that ppCCK-containing neurons in PBsl were projection neurons. Following nociceptive stimulation, the ppCCK-containing neurons expressed FOS protein as well as phosphorylated cyclic AMP-responsive element-binding protein (CREB). In addition, Ca2+/calmodulin-dependent kinase II (CaMKII) was heavily and rather selectively expressed in PBsl and was co-localized to ppCCK-containing neurons. These observations show that nociceptive stimuli activate a cholecystokinin pathway from the parabrachial nucleus to the ventromedial hypothalamus that may be important for homeostatic responses to tissue damage, and point to a putative intracellular route for Ca2+-mediated FOS transcription via CaMKII and CREB for the regulation of ppCCK transcription.

Neuropeptide expression in rat paraventricular hypothalamic neurons that project to the spinal cord.

The paraventricular hypothalamic nucleus (PVH) exerts many of its regulatory functions through projections to spinal cord neurons that control autonomic and sensory functions. By using in situ hybridization histochemistry in combination with retrograde tract tracing, we analyzed the peptide expression among neurons in the rat PVH that send axons to the spinal cord. Projection neurons were labeled by immunohistochemical detection of retrogradely transported cholera toxin subunit B, and radiolabeled long riboprobes were used to identify neurons containing dynorphin, enkephalin, or oxytocin mRNA. Of the spinally projecting neurons in the PVH, approximately 40% expressed dynorphin mRNA, 40% expressed oxytocin mRNA, and 20% expressed enkephalin mRNA. Taken together with our previous findings on the distribution of vasopressin-expressing neurons in the PVH (Hallbeck and Blomqvist [1999] J. Comp. Neurol. 411:201-211), the results demonstrated that the different PVH subdivisions display distinct peptide expression patterns among the spinal cord-projecting neurons. Thus, the lateral parvocellular subdivision contained large numbers of spinal cord-projecting neurons that express any of the four investigated peptides, whereas the ventral part of the medial parvocellular subdivision displayed a strong preponderance for dynorphin- and vasopressin-expressing cells. The dorsal parvocellular subdivision almost exclusively contained dynorphin- and oxytocin-expressing spinal cord-projecting neurons. This parcellation of the peptide-expressing neurons suggested a functional diversity among the spinal cord-projecting subdivisions of the PVH that provide an anatomic basis for its various and distinct influences on autonomic and sensory processing at the spinal level.

Chicken neuropeptide Y-family receptor Y4: a receptor with equal affinity for pancreatic polypeptide, neuropeptide Y and peptide YY.

Within the neuropeptide Y (NPY) family of peptides, pancreatic polypeptide is the most divergent across species. It differs in 20 of 36 positions between human and chicken. In mammals, it binds primarily to the Y4 receptor, to which NPY and peptide YY (PYY) bind with lower affinities. Because of these large sequence differences in pancreatic polypeptide, we decided to characterise the chicken Y4 receptor. We report here that Y4 displays the least sequence conservation among the Y-family receptors, with only 57-60% overall amino acid identity between chicken and mammals, compared with 64-83% for the Y1, Y2 and Y5 receptors. After expression of the chicken Y4 receptor in COS-7 cells, (125)I-labelled porcine (p) PYY bound with a K(d) of 20 pM. In competition with (125)I-pPYY, chicken pancreatic polypeptide bound with high affinity at 140 pM. Interestingly, chicken PYY bound with even greater affinity at 68 pM. The affinity of NPY, 160 pM, was similar to that of pancreatic polypeptide. Chicken Y4 is less sensitive than is mammalian Y4 to truncation of the amino terminus of the NPY molecule. RT-PCR revealed expression in several peripheral organs, including adipose tissue and oviduct. In brain, Y4 mRNA was detected in the brainstem, cerebellum and hippocampus. In situ hybridisation to brain sections showed expression in the dorsal motor nucleus of the vagus in the brainstem. Thus the chicken Y4 receptor is less selective and anatomically more widespread than that in mammals, probably reflecting the original properties of the Y4 receptor.

Embryonic expression of the mRNA for the rat homologue of the fusin/CXCR-4 HIV-1 co-receptor.

We have previously cloned a human receptor recently shown to be a cofactor for entry of T-tropic HIV-1 strains into CD4+ cells, now named fusin. Stromal derived factor-1 (SDF-1) is an endogenous ligand for fusin, also called CXCR-4. Here we show the distribution of fusin/CXCR-4 mRNA during ontogeny in the rat. The onset of mRNA expression is around embryonic day 9 and the mRNA expression is high in the thymus as well as proliferative areas of the brain during development. Our results suggest: (1) that fusin/CXCR-4 might have a dual role in both brain development and the immune system; (2) that SDF-1 has a role in brain development or that additional physiological ligands exist for this receptor; (3) co-expression of CD4 and fusin/CXCR-4 may make fetuses susceptible to HIV infection during development.

Differential distribution of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs in the entopeduncular nucleus of the rat.

The entopeduncular nucleus is one of the major output nuclei of the basal ganglia, with topographically organized projections to both motor and limbic structures. Neurons of the entopeduncular nucleus use GABA as the principal transmitter, and glutamic acid decarboxylase (the GABA synthetic enzyme) is widely distributed throughout the region. Previous studies have shown that glutamate decarboxylase exists in two forms (glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67), and that the messenger RNAs for these different enzymes are widely distributed in rat brain. The purpose of the present experiment was to describe the distribution of glutamic acid decarboxylase-65 and glutamic decarboxylase-67 messenger RNAs throughout the entopeduncular nucleus using recently developed oligodeoxynucleotide probes and in situ hybridization histochemical methods. In agreement with previous studies, northern analysis of rat brain poly(A)+ messenger RNA preparations showed that the glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 probes used in the present study hybridized to messenger RNAs of approximately 5.7 and 3.7 kb, respectively. Film autoradiographic analysis revealed large region-dependent, isoform-specific differences in the levels of expression of the two messenger RNAs, with glutamic acid decarboxylase-65 messenger RNA predominating in rostral and medial regions of the entopeduncular nucleus and glutamic acid decarboxylase-67 messenger RNA most abundant in the caudal region. Cellular analysis showed that these region-dependent differences in labelling were due to differences in the relative amounts of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs expressed per cell rather than the number of cells expressing each form of glutamic acid decarboxylase messenger RNA. The differences in the distribution of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs are closely related to the organization of limbic and motor circuits of the entopeduncular nucleus, suggesting that GABAergic transmission through the limbic pathway is regulated predominantly by glutamic acid decarboxylase-65, whereas glutamic acid decarboxylase-67 is of principal importance in the motor pathway. These data provide additional evidence that the neurons of the limbic and motor subregions of the entopeduncular nucleus are neurochemically distinct.