Old knowledge and new technologies allow rapid development of model organisms.

Until recently the set of "model" species used commonly for cell biology was limited to a small number of well-understood organisms, and developing a new model was prohibitively expensive or time-consuming. With the current rapid advances in technology, in particular low-cost high-throughput sequencing, it is now possible to develop molecular resources fairly rapidly. Wider sampling of biological diversity can only accelerate progress in addressing cellular mechanisms and shed light on how they are adapted to varied physiological contexts. Here we illustrate how historical knowledge and new technologies can reveal the potential of nonconventional organisms, and we suggest guidelines for selecting new experimental models. We also present examples of nonstandard marine metazoan model species that have made important contributions to our understanding of biological processes.

Effects of low seawater pH on the marine polychaete Platynereis dumerilii.

An important priority for any organism is to maintain internal cellular homeostasis including acid-base balance. Yet, the molecular level impacts of changing environmental conditions, such as low pH, remain uncharacterised. Herein, we isolate partial Na(+)/H(+)exchangers (NHE), carbonic anhydrase (CA), and calmodulin (CaM) genes from a polychaete, Platynereis dumerilii and investigate their relative expression in acidified seawater conditions. mRNA expression of NHE was significantly down-regulated after 1h and up-regulated after 7days under low pH treatment (pH 7.8), indicating changes in acid-base transport. Furthermore, the localisation of NHE expression was also altered. A trend of down regulation in CA after 1h was also observed, suggesting a shift in the CO2 and HCO3(-) balance. No change in CaM expression was detected after 7days exposure to acidified seawater. This study provides insight into the molecular level changes taking place following exposure to acidified seawater in a non-calcifying, ubiquitous, organism.

Interactions of zebrafish peptide YYb with the neuropeptide Y-family receptors Y4, Y7, Y8a, and Y8b.

The neuropeptide Y (NPY) system influences numerous physiological functions including feeding behavior, endocrine regulation, and cardiovascular regulation. In jawed vertebrates it consists of 3-4 peptides and 4-7 receptors. Teleost fishes have unique duplicates of NPY and PYY as well as the Y8 receptor. In the zebrafish, the NPY system consists of the peptides NPYa, PYYa, and PYYb (NPYb appears to have been lost) and at least seven NPY receptors: Y1, Y2, Y2-2, Y4, Y7, Y8a, and Y8b. Previously PYYb binding has been reported for Y2 and Y2-2. To search for peptide-receptor preferences, we have investigated PYYb binding to four of the remaining receptors and compared with NPYa and PYYa. Taken together, the most striking observations are that PYYa displays reduced affinity for Y2 (3 nM) compared to the other peptides and receptors and that all three peptides have higher affinity for Y4 (0.028-0.034 nM) than for the other five receptors. The strongest peptide preference by any receptor selectivity is the one previously reported for PYYb by the Y2 receptor, as compared to NPY and PYYa. These affinity differences may be helpful to elucidate specific details of peptide-receptor interactions. Also, we have investigated the level of mRNA expression in different organs using qPCR. All peptides and receptors have higher expression in heart, kidney, and brain. These quantitative aspects on receptor affinities and mRNA distribution help provide a more complete picture of the NPY system.

Linking micro- and macro-evolution at the cell type level: a view from the lophotrochozoan Platynereis dumerilii.

Ever since the origin of the first metazoans over 600 million years ago, cell type diversification has been driven by micro-evolutionary processes at population level, leading to macro-evolution changes above species level. In this review, we introduce the marine annelid Platynereis dumerilii, a member of the lophotrochozoan clade (a key yet most understudied superphylum of bilaterians), as a suitable model system for the simultaneous study, at cellular resolution, of macro-evolutionary processes across phyla and of micro-evolutionary processes across highly polymorphic populations collected worldwide. Recent advances in molecular and experimental techniques, easy maintenance and breeding, and the fast, synchronous and stereotypical development have facilitated the establishment of Platynereis as one of the leading model species in the eco-evo-devo field. Most importantly, Platynereis allows the combination of expression profiling, morphological and physiological characterization at the single cell level. Here, we discuss recent advances in the collection of -omics data for the lab strain and for natural populations collected world-wide that can be integrated with population-specific cellular analyses to result in a cellular atlas integrating genetic, phenotypic and ecological variation. This makes Platynereis a tractable system to begin understanding the interplay between macro- and micro-evolutionary processes and cell type diversity.

Orthology prediction methods: a quality assessment using curated protein families.

The increasing number of sequenced genomes has prompted the development of several automated orthology prediction methods. Tests to evaluate the accuracy of predictions and to explore biases caused by biological and technical factors are therefore required. We used 70 manually curated families to analyze the performance of five public methods in Metazoa. We analyzed the strengths and weaknesses of the methods and quantified the impact of biological and technical challenges. From the latter part of the analysis, genome annotation emerged as the largest single influencer, affecting up to 30% of the performance. Generally, most methods did well in assigning orthologous group but they failed to assign the exact number of genes for half of the groups. The publicly available benchmark set (http://eggnog.embl.de/orthobench/) should facilitate the improvement of current orthology assignment protocols, which is of utmost importance for many fields of biology and should be tackled by a broad scientific community.

Evolution of vertebrate rod and cone phototransduction genes.

Vertebrate cones and rods in several cases use separate but related components for their signal transduction (opsins, G-proteins, ion channels, etc.). Some of these proteins are also used differentially in other cell types in the retina. Because cones, rods and other retinal cell types originated in early vertebrate evolution, it is of interest to see if their specific genes arose in the extensive gene duplications that took place in the ancestor of the jawed vertebrates (gnathostomes) by two tetraploidizations (genome doublings). The ancestor of teleost fishes subsequently underwent a third tetraploidization. Our previously reported analyses showed that several gene families in the vertebrate visual phototransduction cascade received new members in the basal tetraploidizations. We here expand these data with studies of additional gene families and vertebrate species. We conclude that no less than 10 of the 13 studied phototransduction gene families received additional members in the two basal vertebrate tetraploidizations. Also the remaining three families seem to have undergone duplications during the same time period but it is unclear if this happened as a result of the tetraploidizations. The implications of the many early vertebrate gene duplications for functional specialization of specific retinal cell types, particularly cones and rods, are discussed.

Major genomic events and their consequences for vertebrate evolution and endocrinology.

Comparative studies of proteins often face the problem of distinguishing a true orthologue (species homologue) from a paralogue (a gene duplicate). This identification task is particularly challenging for endocrine peptides and neuropeptides because they are short and usually have several invariant positions. For some peptide families, this has led to a terminology with peptide names relating to the first species where a specific peptide sequence was determined, such as chicken or salmon gonadotropin-releasing hormone, or names that highlight amino acid differences, e.g., Lys-vasopressin. With accumulating information from multiple species, such a terminology becomes almost impenetrable for nonexperts and difficult even for aficionados. The sequenced genomes offer a new way to distinguish orthologues and paralogues, namely by location of the genes relative to neighboring genes on the chromosomes. In addition, the genome databases can ideally provide a complete listing of the family members in each species. Many vertebrate gene families have expanded in the two basal tetraploidizations (2R) and the teleost fish third tetraploidization (3R), after which some vertebrate lineages have lost some of the duplicates. We review here some peptide families (neuropeptide Y, oxytocin-vasopressin, and somatostatin) where genomic information helps simplify nomenclature. This approach is useful also for other gene families, such as peptide receptors.

Early duplications of opioid receptor and Peptide genes in vertebrate evolution.

The opioid receptor family in mammals has four members called delta, kappa, mu, and NOP (the nociceptin/orphanin receptor). We show here that they arose from a common ancestral gene through quadruplication of a large chromosomal region, presumably in the two basal vertebrate tetraploidizations. The four opioid peptide precursor genes have a more complicated evolutionary history involving chromosomal rearrangements but nevertheless seem to have arisen in the same time period as the receptors. Thus the system of opioid peptides and receptors was already established approximately 450 Ma at the dawn of gnathostome evolution.

Evolution of the growth hormone-prolactin-somatolactin system in relation to vertebrate tetraploidizations.

Gene sequences from several species representing major vertebrate groups were used to create phylogenetic trees for the growth hormone family of peptide hormones as well as the growth hormone receptor family. These analyses show that both the peptide and receptor families were formed through local duplications in early vertebrate evolution and chromosome duplications.

Neuropeptide Y-family peptides and receptors in the elephant shark, Callorhinchus milii confirm gene duplications before the gnathostome radiation.

We describe here the repertoire of neuropeptide Y (NPY) peptides and receptors in the elephant shark Callorhinchus milii, belonging to the chondrichthyans that diverged from the rest of the gnathostome (jawed vertebrate) lineage about 450 million years ago and the first chondrichthyan with a genome project. We have identified two peptide genes that are orthologous to NPY and PYY (peptide YY) in other vertebrates, and seven receptor genes orthologous to the Y1, Y2, Y4, Y5, Y6, Y7 and Y8 subtypes found in tetrapods and teleost fishes. The repertoire of peptides and receptors seems to reflect the ancestral configuration in the predecessor of all gnathostomes, whereas other lineages such as mammals and teleosts have lost one or more receptor genes or have acquired 1-2 additional peptide genes. Both the peptides and receptors showed broad and overlapping mRNA expression which may explain why some receptor gene losses could take place in some lineages, but leaves open the question why all the known ancestral receptors have been retained in the elephant shark.

Evolution of vertebrate opioid receptors.

The opioid peptides and receptors have prominent roles in pain transmission and reward mechanisms in mammals. The evolution of the opioid receptors has so far been little studied, with only a few reports on species other than tetrapods. We have investigated species representing a broader range of vertebrates and found that the four opioid receptor types (delta, kappa, mu, and NOP) are present in most of the species. The gene relationships were deduced by using both phylogenetic analyses and chromosomal location relative to 20 neighboring gene families in databases of assembled genomes. The combined results show that the vertebrate opioid receptor gene family arose by quadruplication of a large chromosomal block containing at least 14 other gene families. The quadruplication seems to coincide with, and, therefore, probably resulted from, the two proposed genome duplications in early vertebrate evolution. We conclude that the quartet of opioid receptors was already present at the origin of jawed vertebrates approximately 450 million years ago. A few additional opioid receptor gene duplications have occurred in bony fishes. Interestingly, the ancestral receptor gene duplications coincide with the origin of the four opioid peptide precursor genes. Thus, the complete vertebrate opioid system was already established in the first jawed vertebrates.

Phylogenetic and chromosomal analyses of multiple gene families syntenic with vertebrate Hox clusters.

BACKGROUND: Ever since the theory about two rounds of genome duplication (2R) in the vertebrate lineage was proposed, the Hox gene clusters have served as the prime example of quadruplicate paralogy in mammalian genomes. In teleost fishes, the observation of additional Hox clusters absent in other vertebrate lineages suggested a third tetraploidization (3R). Because the Hox clusters occupy a quite limited part of each chromosome, and are special in having position-dependent regulation within the multi-gene cluster, studies of syntenic gene families are needed to determine the extent of the duplicated chromosome segments. We have analyzed in detail 14 gene families that are syntenic with the Hox clusters to see if their phylogenies are compatible with the Hox duplications and the 2R/3R scenario. Our starting point was the gene family for the NPY family of peptides located near the Hox clusters in the pufferfish Takifugu rubripes, the zebrafish Danio rerio, and human. RESULTS: Seven of the gene families have members on at least three of the human Hox chromosomes and two families are present on all four. Using both neighbor-joining and quartet-puzzling maximum likelihood methods we found that 13 families have a phylogeny that supports duplications coinciding with the Hox cluster duplications. One additional family also has a topology consistent with 2R but due to lack of urochordate or cephalochordate sequences the time window when these duplications could have occurred is wider. All but two gene families also show teleost-specific duplicates. CONCLUSION: Based on this analysis we conclude that the Hox cluster duplications involved a large number of adjacent gene families, supporting expansion of these families in the 2R, as well as in the teleost 3R tetraploidization. The gene duplicates presumably provided raw material in early vertebrate evolution for neofunctionalization and subfunctionalization.

Early vertebrate chromosome duplications and the evolution of the neuropeptide Y receptor gene regions.

BACKGROUND: One of the many gene families that expanded in early vertebrate evolution is the neuropeptide (NPY) receptor family of G-protein coupled receptors. Earlier work by our lab suggested that several of the NPY receptor genes found in extant vertebrates resulted from two genome duplications before the origin of jawed vertebrates (gnathostomes) and one additional genome duplication in the actinopterygian lineage, based on their location on chromosomes sharing several gene families. In this study we have investigated, in five vertebrate genomes, 45 gene families with members close to the NPY receptor genes in the compact genomes of the teleost fishes Tetraodon nigroviridis and Takifugu rubripes. These correspond to Homo sapiens chromosomes 4, 5, 8 and 10. RESULTS: Chromosome regions with conserved synteny were identified and confirmed by phylogenetic analyses in H. sapiens, M. musculus, D. rerio, T. rubripes and T. nigroviridis. 26 gene families, including the NPY receptor genes, (plus 3 described recently by other labs) showed a tree topology consistent with duplications in early vertebrate evolution and in the actinopterygian lineage, thereby supporting expansion through block duplications. Eight gene families had complications that precluded analysis (such as short sequence length or variable number of repeated domains) and another eight families did not support block duplications (because the paralogs in these families seem to have originated in another time window than the proposed genome duplication events). RT-PCR carried out with several tissues in T. rubripes revealed that all five NPY receptors were expressed in the brain and subtypes Y2, Y4 and Y8 were also expressed in peripheral organs. CONCLUSION: We conclude that the phylogenetic analyses and chromosomal locations of these gene families support duplications of large blocks of genes or even entire chromosomes. Thus, these results are consistent with two early vertebrate tetraploidizations forming a paralogon comprising human chromosomes 4, 5, 8 and 10 and one teleost tetraploidization. The combination of positional and phylogenetic data further strengthens the identification of orthologs and paralogs in the NPY receptor family.

Evolution of the neuropeptide Y family: new genes by chromosome duplications in early vertebrates and in teleost fishes.

Despite sequence information from many vertebrates the evolution of the neuropeptide Y (NPY) family of peptides has been difficult to resolve, particularly among ray-finned fishes. We have used chromosomal location and sequence analyses to identify orthologs and gene duplicates in teleost fish genomes. Our analyses support origin of NPY and peptide YY (PYY) from a common ancestor in early vertebrate evolution through a chromosome duplication. We report here that the teleost tetraploidization generated duplicates of both NPY and PYY and that all four genes are still present in the two sequenced pufferfish genomes Tetraodon nigroviridis and Takifugu rubripes as well as three-spined stickleback, Gasterosteus aculeatus. The zebrafish Danio rerio NPYb gene has probably been lost whereas medaka, Oryzias latipes seems to lack PYYb. Some of the previously published PYY sequences were misidentified and actually constitute NPYb. Our analyses confirm that the peptide previously named PY in some fish species is a duplicate of the PYY gene and hence should be called PYYb. The NPYa and NPYb genes in Takifugu rubripes are predominantly expressed in brain, as detected by RT-PCR, whereas PYYa and PYYb are expressed in several organs including brain, intestine and gonads. Thus, also the resemblance in expression pattern supports the fish gene duplication scenario. Our study shows that when sequence comparisons give ambiguous results, chromosomal location can serve as a useful criterion to identify orthologs. This strategy may help to resolve relationships in several families of short peptides.

Cloning and sequence analysis of the neuropeptide Y receptors Y5 and Y6 in the coelacanth Latimeria chalumnae.

Two coelacanth species, Latimeria chalumnae and Latimeria menadoensis, the recently discovered second species, have a key evolutionary position at the divergence of bony fishes and tetrapods. Together with lungfishes, they are the only living species separating the species-rich tetrapods from the other major group of vertebrates, the ray-finned fishes. The coelacanth is therefore of great importance for comparisons of gene families that differ between these two groups, such as the neuropeptide Y (NPY) receptor family. In this work we have sequenced the full-length genes for two NPY receptors in Latimeria chalumnae. Phylogenetic analysis indicated that the two sequences are orthologs of the mammalian Y5 and Y6 receptors. The Y5 gene has been implicated in appetite stimulation in mammals but is absent from teleost fishes. The presence of the Y5 receptor in Latimeria together with phylogenetic analysis shows that Y5 existed before the separation of bony fishes and tetrapods. The Latimeria receptor has about 62% identity to tetrapod Y5 sequences and contains the extended third intracellular loop with several highly conserved motifs that may be involved in signal transduction. The Latimeria Y6 receptor has about 60% identity to tetrapod Y6 sequences. The functional role of Y6 is unclear as the gene is seemingly functional in some mammals but is non-functional in others. The Y6 receptor is also missing in teleost fishes. Our results confirm an early vertebrate origin for all NPY receptor subtypes presently found in mammals followed by differential gene loss in the different classes of vertebrates.

Neuropeptide Y-family receptors Y6 and Y7 in chicken. Cloning, pharmacological characterization, tissue distribution and conserved synteny with human chromosome region.

The peptides of the neuropeptide Y (NPY) family exert their functions, including regulation of appetite and circadian rhythm, by binding to G-protein coupled receptors. Mammals have five subtypes, named Y1, Y2, Y4, Y5 and Y6, and recently Y7 has been discovered in fish and amphibians. In chicken we have previously characterized the first four subtypes and here we describe Y6 and Y7. The genes for Y6 and Y7 are located 1 megabase apart on chromosome 13, which displays conserved synteny with human chromosome 5 that harbours the Y6 gene. The porcine PYY radioligand bound the chicken Y6 receptor with a K(d) of 0.80 +/- 0.36 nm. No functional coupling was demonstrated. The Y6 mRNA is expressed in hypothalamus, gastrointestinal tract and adipose tissue. Porcine PYY bound chicken Y7 with a K(d) of 0.14 +/- 0.01 nm (mean +/- SEM), whereas chicken PYY surprisingly had a much lower affinity, with a Ki of 41 nm, perhaps as a result of its additional amino acid at the N terminus. Truncated peptide fragments had greatly reduced affinity for Y7, in agreement with its closest relative, Y2, in chicken and fish, but in contrast to Y2 in mammals. This suggests that in mammals Y2 has only recently acquired the ability to bind truncated PYY. Chicken Y7 has a much more restricted tissue distribution than other subtypes and was only detected in adrenal gland. Y7 seems to have been lost in mammals. The physiological roles of Y6 and Y7 remain to be identified, but our phylogenetic and chromosomal analyses support the ancient origin of these Y receptor genes by chromosome duplications in an early (pregnathostome) vertebrate ancestor.

Characterization of NPY receptor subtypes Y2 and Y7 in rainbow trout Oncorhynchus mykiss.

We report the cloning and pharmacological characterization of two neuropeptide Y (NPY) receptor subtypes, Y2 and Y7, in rainbow trout (Oncorhynchus mykiss). These subtypes are approximately 50% identical to each other and belong to the Y2 subfamily of NPY receptors. The binding properties of the receptors were investigated after expression in human HEK-293 EBNA cells. Both receptors bound the three zebrafish peptides NPY, PYYa, and PYYb, as well as porcine NPY and PYY, with affinities in the nanomolar range that are similar to mammalian Y2. The affinity of the truncated porcine NPY fragments, NPY 13-36 and NPY 18-36 was markedly lower compared to mammalian and chicken Y2. This suggests that mammalian and chicken Y2 are unique among NPY receptors in their ability to bind truncated peptide fragments. The antagonist BIIE0246, developed for mammalian Y2, did not bind either of the two rainbow trout receptors. Our results support the proposed expansion of this gene family by duplications before the gnathostome radiation. They also reveal appreciable differences in the repertoire and characteristics of NPY receptors between fish and tetrapods stressing the importance of lineage-specific gene loss as well as sequence divergence after duplication.

Pufferfish and zebrafish have five distinct NPY receptor subtypes, but have lost appetite receptors Y1 and Y5.

The two neuropeptide Y (NPY) receptors Y1 and Y5 stimulate feeding in mammals, but are missing in the euteleosts, zebrafish and pufferfish (Takifugu rubripes). Both species have five other subtypes called Y2, Y7, Ya, Yb, and Yc. RT-PCR studies in pufferfish show that all five are expressed in the brain and may mediate NPY effects on feeding. Y2, Ya, and Yb are also broadly expressed in peripheral organs. These results reveal interesting differences in the NPY system of teleosts and mammals that may have arisen in the genetic turmoil involving the basal ray-fin fish tetraploidization.

Effects of a teleost tetraploidization on neuropeptide Y receptor gene repertoire in ray-finned fishes.

The ancestral vertebrate repertoire for neuropeptide Y receptor genes of the Y1 subfamily probably included four subtypes: Y1, Y4, Y6, and Y8. There was probably a single gene in the Y5 category. Both Y1 and Y5 stimulate food intake in mammals. As the genome seems to have duplicated during the evolution of ray-finned fishes, we have investigated the gene repertoire in species that diverged prior to the appearance of teleosts, as well as a basal teleost and a shark. Our results show that the genes Y1, Y5, and Y6, which are missing in many teleosts, are present in basal actinopterygians. These dramatic alterations of the teleost receptor repertoire may be related to the tetraploidization in a teleost ancestor.

Ray-fin fish tetraploidization gave rise to pufferfish duplicates of NPY and PYY, but zebrafish NPY duplicate was lost.

We have used sequence information and gene location to identify NPY family genes in the pufferfish, Takifugu rubripes (fugu), and zebrafish. Fugu has two copies of NPY, presumably resulting from the ray-fin fish tetraploidization. Zebrafish has probably lost one of the copies. Both species have two copies of PYY, the second of which was previously named PY. The two fugu NPY genes are predominantly expressed in brain. The two PYY genes are expressed in a broad range of tissues including brain and gonads. Thus, the NPY system appears to be more complex in teleosts than in tetrapods.