Evolution of the Neuropeptide Y and Opioid Systems and Their Genomic

Home , Lipotropin, Neuropeptide Y, Opioid receptor, Peptide YY

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Wicked

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Sundström G, Larsson TA, Brenner S, Venkatesh B, Larham- mar D. (2008) Evolution of the neuropeptide Y family: new genes by chromosome duplications in early vertebrates and in teleost fishes. General and Comparative Endocrinology Feb 1;155(3):705-16. II Sundström G, Larsson TA, Xu B, Heldin J, Lundell I, Lar- hammar D. (2010) Interactions of zebrafish peptide YYb with the neuropeptide Y-family receptors Y4, Y7, Y8a and Y8b. Manuscript III Sundström G, Xu B, Larsson TA, Heldin J, Bergqvist CA, Fredriksson R, Conlon JM, Lundell I, Denver RJ, Larhammar D. (2010) Characterization of the neuropeptide Y system's three peptides and six receptors in the frog Silurana tropicalis. Manu- script IV Dreborg S, Sundström G, Larsson TA, Larhammar D. (2008) Evolution of vertebrate opioid receptors. Proc Natl Acad Sci USA Oct 7;105(40):15487-92. V Sundström G, Dreborg S, Larhammar D. (2010) Concomitant duplications of opioid peptide and receptor genes before the origin of jawed vertebrates. PLoS One May 6;5(5):e10512. VI Sundström G, Larsson TA, Larhammar D. (2008) Phylogenet- ic and chromosomal analyses of multiple gene families syntenic with vertebrate Hox clusters. BMC Evolutionary Biology Sep 19;8:254. VII Widmark J, Sundström G, Ocampo Daza D, Larhammar D. (2010) Differential evolution of voltage-gated sodium channels in tetrapods and teleost fishes. Submitted VIII Ocampo Daza D, Sundström G, Bergqvist CA, Duan C, Lar- hammar D. (2010) Evolution of the insulin-like growth factor binding protein (IGFBP) family. Submitted

Additional publications

• Larsson TA, Tay BH, Sundström G, Fredriksson R, Brenner S, Lar- hammar D, Venkatesh B. (2009) Neuropeptide Y-family peptides and receptors in the elephant shark, Callorhinchus milii confirm gene duplications before the gnathostome radiation. Genomics. Mar;93(3):254-60.

• Larsson TA, Olsson F, Sundström G, Lundin LG, Brenner S, Venka- tesh B, Larhammar D. (2008) Early vertebrate chromosome duplications and the evolution of the neuropeptide Y receptor gene regions. BMC Evolutionary Biology Jun 25;8:184.

Contents

Introduction ...... 9 Gene and genome duplication ...... 10 The neuropeptide Y family of receptors ...... 15 The neuropeptide Y family of peptides ...... 19 The opioid receptor family ...... 21 The opioid peptide family ...... 24 Hox-cluster regions ...... 27 Aims ...... 30 Materials and Methods ...... 31 Genomes examined ...... 31 Database searches and sequence identification ...... 34 Sequence alignments and phylogenetic analysis ...... 35 Cloning and Sequencing ...... 35 Receptor binding studies ...... 36 RT-PCR ...... 36 RNA extraction and quantitative real time PCR ...... 36 Results and discussion ...... 38 Paper I ...... 38 Paper II ...... 39 Paper III ...... 39 Paper IV and V ...... 40 Paper VI, VII and VIII ...... 41 Conclusions and perspectives ...... 43 Svensk sammanfattning (Swedish summary) ...... 46 Acknowledgements ...... 49 References ...... 51

Abbreviations

2R Two rounds of genome duplication 3R Third round of genome duplication BLAST Basic Local Alignment Search Tool cAMP Cyclic adenosine monophosphate GPCR G-protein coupled receptor G-protein Guanine nucleotide binding protein HEK Human embryonic kidney cells IGFBP Insulin-like growth factor binding protein NJ Neighbor-joining NPY Neuropeptide Y (Y = tyrosine) PDYN Prodynorphin PENK Proenkephalin PhyML Phylogenetic maximum likelihood PNOC Proorphanin POMC Proopiomelanocortin PP Pancreatic polypeptide PYY, 125I-pPYY Peptide YY (Y = tyrosine), iodinated porcine PYY QP Quartet puzzling maximum likelihood RT-PCR Reverse transcriptase PCR SCN Sodium channel TM Transmembrane Y1, Y2, … Y7, Y8 Neuropeptide Y receptor Yn

Introduction

Cells in our body communicate through numerous systems of receptors and ligands. In some cases, the ligands are exogenous signals such as for the opsins (light receptors), gustatory receptors (salt, sugar etc.) and olfactory receptors (odorants). Ligands used for signalling internally can be small molecules such as acetylcholine and dopamine or peptides that derive from precursor proteins produced from genes. The receptors as well as their ligands often belong to gene families with multiple members, particularly in vertebrates. Several ligands of the same family are frequently able to bind to the same receptor. The repertoire of ligands and receptors often varies between species and sometimes ligand- receptor preferences also differ. Therefore it is important to distinguish between orthologs; genes separated by a speciation event, and paralogs; genes separated by a duplication event, when comparing functions of genes from different organisms. Furthermore, some of the receptor-ligand complexes execute their functions in a wide range of different tissues including the brain as well as peripheral tissues. This distribution may differ between species. Examples of this type of complex systems are the neuropeptide Y system of receptors and peptides with 4 to 7 receptors and 3 to 4 ligands (depending on species) and the opioid system with four receptors and four ligands in mammals. Several receptor and ligand families have expanded in the vertebrate lineage. Gene duplication followed by mutation, in contrast to direct mutation of a gene, allows for change in one gene copy with the other copy still intact and able to perform the original function. The higher complexity of many ligand-receptor systems in vertebrates compared with invertebrates has been attributed to both local gene duplications and whole genome duplications, i.e. tetraploidizations, early in vertebrate evolution.

9 Gene and genome duplication Gene and genome duplications have been promoted as the main mechanisms that can lead to the emergence of new genes and gene functions. In the 1970s Susumu Ohno argued that big leaps in evolution required new (duplicate) sets of genes. However, even before him there had been suggestions that it was not enough to modify and mutate already existing genes (Ohno, 1970). There are several well known molecular mechanisms by which new genes can be formed, including classical gene duplication and retroposition (Long et al., 2003). Data from several eukaryotic species have shown that gene duplications occur frequently and that the probability of duplication of a gene is at least 1% per million years (Lynch and Conery, 2003). Estimations of the number of gene duplications in different lineages have also been conducted. The duplication rates range from 0.27 and 0.8 per million years in chicken and frog up to 7.27 and 9.05 per million years in mouse and zebrafish. These estimates are however based on predicted genes in the genome databases and the extreme values might reflect the quality of genome assembly and not the true duplication rate. A low value might also be an indication of a high rate of gene loss and not only a low duplication rate (Blomme et al., 2006). Other studies have shown that humans and chimpanzees have a three-fold higher rate of gene gain and loss than other mammals (Hahn et al., 2007), and both segmental duplication and copy number variation seem to have played important roles in the evolution of the primate genomes (Bailey and Eichler, 2006; Demuth and Hahn, 2009). When a gene has been duplicated, one of the gene copies is sufficient to maintain the function, while the other copy may evolve new functions (see below). Alternatively, the duplicate could easily become a pseudogene through accumulation of deleterious mutations due to reduced selective pressure (Kellogg, 2003; Semon and Wolfe, 2007a). It has been calculated that a duplicated gene has an average half-life of approximately 4 million years before it will be silenced (Lynch and Conery, 2003). However, there are examples of genes that have become pseudogenes recently even if the duplication took place a long time ago. One example is the HoxB7a gene in the teleost fugu (Takifugu rubripes) (Amores et al., 2004). Also many of the olfactory genes in hominids have been inactivated long after they originated (Zhang, 2003).

Tetraploidization and polyploidy Polyploidy is a well known and well-studied phenomenon in plants and several ancient whole genome duplications seem to have occurred during the evolution of angiosperms (Adams and Wendel, 2005; Van de Peer et al., 2009a). The outcome of whole-genome duplication in yeast has also been thoroughly investigated (Byrne and Wolfe, 2005; Scannell et al., 2007). Po-

10 lyploidy has been viewed as uncommon in animals but it is known for some amphibian, cyprinid and salmonid species (David et al., 2003; Evans et al., 2004; Hordvik et al., 1997). Genome duplications can occur in different ways; autopolyploidy involves genes from the same species and is caused by failure during mitosis. In the cases of allopolyploidy two different species merge their genomes (Chen and Ni, 2006; Comai, 2005; Panopoulou and Poustka, 2005; Wolfe, 2001). One outcome of whole-genome duplications that has been discussed is its importance in the radiation of species. After duplication differential losses of genes can occur in different populations. These losses might lead to repro- ductive isolation and generation of new species (Hoegg et al., 2004; Jaillon et al., 2009; Meyer and Van de Peer, 2005; Scannell et al., 2006; Semon and Wolfe, 2007c; Taylor et al., 2001; Van de Peer et al., 2009b). The euteleost lineage is species rich and displays a morphological diversity that might be related to the event of genome duplication in ancient teleosts, even though the time span between the duplication event and the actual radiation seems to be long (Crow and Wagner, 2006; Donoghue and Purnell, 2005; Hurley et al., 2007; Meyer and Schartl, 1999). Some fishes, such as Salmonidae and Cyprinidae, are tetraploid due to later tetraploidization events and often used as examples of genome duplication that lead to species radiation (David et al., 2003; Hordvik et al., 1997). Both the existence of tetraploids and evidence of independent genome duplications indicate that the fish genomes are in some sense more plastic than genomes in other vertebrates (Venkatesh, 2003). The rearrangement rate after whole genome duplications in teleosts is also higher compared to other vertebrates investigated (Ravi and Venkatesh, 2008; Semon and Wolfe, 2007b). Some studies have also argued that a period of significant rearrangements have preceded whole genome duplications in vertebrate evolution and that it was possible for the duplications to occur because the genome was unstable, it was not the genome duplications that caused the insta- bility (Hufton et al., 2008). Most species return to a diploid state after the polyploidization event. The mechanisms responsible for the process of diploidization are complex and involve several different components. Studies in plants have shown that loss and retention of different genes seems to be important, as well as different methylation patterns that might change the regulation of genes. Also large chromosomal rearrangements and transposable elements contribute to in- creasing the divergence of homologous chromosomes and facilitate a cytological diploidization (Ma and Gustafson, 2005). Computer simulations have also pinpointed genetic drift as an important factor (Le Comber et al., 2010).

11 Non-, sub- and neofunctionalization One advantage of whole genome duplications compared to local gene duplications is that the biochemical balance is preserved, the proportions of proteins that interact with each other are the same as before the duplication event (Otto, 2007; Papp et al., 2003). Different functional categories of genes seem also to be retained after whole genome duplications versus local gene duplications (Maere et al., 2005; Paterson et al., 2006). After duplication there are three main fates for the duplicated genes. The most common one is non-functionalization i.e. the accumulation of mutations leading to pseudogenization and loss of the gene. The second most common consequence is subfunctionalization, often described in a duplication-degeneration-complementation model (Force et al., 1999): many proteins have multiple functions, i.e. are involved in more than one process in the organism. After duplication, different mutations can occur in the regula- tory regions of the genes, leading to expression in different tissues or at different time points (Force et al., 1999; Hahn, 2009). Subfunctionalization has also been seen as a step on the path towards neofunctionalization (Conant and Wolfe, 2008; He and Zhang, 2005; Rastogi and Liberles, 2005). Neo- functionalization is the least common fate and implies that one of the copies acquires a completely new function (Conant and Wolfe, 2008; Lynch and Conery, 2000). A fourth outcome is that both duplicates might preserve the original function of the gene, this is called conservation or originalization. Gene dosage effects have been proposed as the main evolutionary advantage of two genes with identical functions (Hahn, 2009; Xue and Fu, 2009). Given that non-functionalization is the most common fate for gene duplicates, one aspect that has been studied is the retention rate after whole genome duplications. Losses of genes are frequent and the degrees of retention differs between species, 15-24% after the teleost-specific duplication, see below (Brunet et al., 2006; Jaillon et al., 2004; Woods et al., 2005).

Two rounds of genome duplication, 2R Ohno suggested that the evolution of vertebrates included either whole genome duplication (tetraploidization) or at least large chromosome block duplications (Ohno, 1970). The duplications were thought to have occurred in two rounds, one on each side of the branch leading to the agnathans (see Fig. 1) commonly referred to as the 2R-hypothesis (Hughes, 1999; Kasahara, 2007; Meyer and Schartl, 1999; Panopoulou and Poustka, 2005). The precise timing of the duplications is still elusive and it is not clear if the agnathan lineage experienced one or two rounds of duplication (Escriva et al., 2002; Kuraku et al., 2008).

Figure 1. Schematic overview of chordate evolution. Time scale indicates million years before present. Arrows indicate proposed time points for the two tetraploidizations, 2R, according to (Panopoulou and Poustka, 2005) and the fish specific tetraploidization, 3R, (Christoffels et al., 2004; Jaillon et al., 2004; Panopoulou and Poustka, 2005). The dashed line for cyclostomes (lampreys and hagfishes) indicates lack of fossil representation of hagfishes in their early evolution. Proposed divergence times are based on paleontological data and fossil minimum times (Benton and Donoghue, 2007; Blair and Hedges, 2005; Sansom et al., 1996).

Before the era of the genome sequencing projects the tetraploidization theory was based on differences in genome size (DNA content in the cell nucleus), chromosome number and isozyme activity. However, these findings are not sufficient to support tetraploidizations because genes are also generated by local duplications (Furlong and Holland, 2002). Therefore both the phylogenies and the chromosomal positions of genes are important in the study of genome duplication. In the 1990s more genes and whole gene families were mapped and cloned and the connection between the early tetraploidizations and the groups of paralogous chromosomes visible in the genomes of both human and mouse was made (Lundin, 1993). By molecular cloning it was revealed that genes with one copy in the cephalochordate amphioxus (Bran- chiostoma floridae) often have several copies in vertebrate species. One well studied gene family where this has been shown is the Hox family (Furlong and Holland, 2002; Garcia-Fernandez and Holland, 1994; Hoegg and Meyer, 2005)

13 Paralogous genes are genes that have arisen from a duplication of an ancestral gene. Large regions of paralogous genes for multiple gene families, clustered together in a similar way on different chromosomes, have been interpreted as an indication of chromosome duplications in the past. Degene- ration (non-functionalization) of duplicates is common and therefore it is in most cases not possible to see all four paralogs that would be expected after two tetraploidizations. Numerous evolutionary mechanisms have also affected the regions of paralogous genes, for instance rearrangements by inversions, fissions and translocations of chromosome blocks (Lundin et al., 2003). Despite these complications it is possible to identify paralogous regions particulary by considering multiple species, since differential gains and losses may have occurred in different lineage. A set (usually a quartet) of paralogous chromosome regions is called a paralagon (Coulier et al., 2000) and several of these have been found in the human genome through in depth studies of specific gene families and/or regions (Abi-Rached et al., 2002; Kasahara et al., 1996; Katsanis et al., 1996; Larhammar et al., 2002; Olinski et al., 2006; Pebusque et al., 1998; Popovici et al., 2001a; Popovici et al., 2001b; Vienne et al., 2003). And subsequently in large scale analyses using whole genomes (Dehal and Boore, 2005; Nakatani et al., 2007; Putnam et al., 2008). It has been argued that a phylogenetic tree of a gene family that has undergone duplications through two tetraploidizations should display a symme- trical topology as the one shown in figure 2A (Hughes and Friedman, 2003). However, trees with such an appearance are rarely found in the analysis of vertebrate gene families, instead the result is often trees with asymmetrical topology, as exemplified in figure 2B and 2C. The asymmetrical topologies are to be expected since different evolutionary rates of genes, crossing-over, gene conversion often affect the evolution of gene families. Additionally tree-construction artifacts like long branch attraction may disrupt a symme- trical topology (Taylor et al., 2003; Taylor et al., 2001).

Figure 2. Panel A shows the (A,B)(C,D) topology while B and C displays asymmetrical topologies.

14 There has been some controversy over the validity of the 2R hypothesis. The history of the debate and references can be found in (Panopoulou and Poustka, 2005; Van de Peer et al., 2010; Vandepoele et al., 2004). Large scale studies seem to have settled the issue (Dehal and Boore, 2005; Nakatani et al., 2007) and with the release of the genome sequence of the cephalochordate Branchiostoma floridae it was possible to clearly visualize the one to four relationship between genes in the cephalochordate and vertebrates (Putnam et al., 2008).

A third round of genome duplication, 3R The presence of a third whole genome duplication in teleost fishes was first suggested with the Hox clusters as an example. Zebrafish (Danio rerio) was found to have seven Hox clusters, and the same was true for several other species such as medaka (Oryzias latipes) and fugu (Takifugu rubripes) (Amores et al., 1998; Hoegg and Meyer, 2005). Although these teleosts have seven Hox clusters, the presence of different clusters in different species indicates lineage-specific losses and that the ancestral euteleost probably had eight clusters right after the tetraploidization, just as the 3R theory predicts. Also other gene families, besides Hox, have been investigated in order to study the 3R event (Meyer and Schartl, 1999; Postlethwait et al., 2000; Taylor et al., 2003; Taylor et al., 2001; Van de Peer et al., 2003). Using whole genome data available from tetraodon (Tetraodon nigroviridis) and fugu, Jallion et al. showed that many gene families display double conserved synteny (one gene from any mammal is often represented by two teleost orthologs) and other large scale analysis has confirmed the 3R hypothesis (Christoffels et al., 2004; Jaillon et al., 2004; Van de Peer, 2004). The acknowledgement of a tetraploidization event in the teleost fish lineage generated greater acceptance of the 2R hypothesis. Using a whole genome assembly from medaka it was possible to confirm the pattern of double conserved synteny seen in the other species and also to delineate chromosomal rearrangement in the teleost lineage (Kasahara et al., 2007).

The neuropeptide Y family of receptors G-protein coupled receptors (GPCRs) constitute the largest gene family in many genomes. They can be classified into five superfamilies or clans where the neuropeptide Y receptors belong to the rhodopsin clan (Fredriksson et al., 2003). The GPCRs span the cell membrane seven times and consist of three extra- and intracellular loops respectively ending with a carboxyterminal intracellular tail. Ligand binding to NPY receptors results in a con- formational change of the receptor that triggers intracellular signals through

15 the G-proteins Gi and Go which inhibit adenylyl cyclase, resulting in a reduc- tion of cAMP (Holliday et al., 2004). The NPY receptors are expressed in both brain and periphery. In mammals they are involved in the regulation of appetite and have also been shown to have functions in the regulation of blood pressure, anxiety, pituitary release of hormones, bone formation and pain, just to name a few of the many observed effects (Lee and Herzog, 2009; Morales-Medina et al., 2010; Pedrazzini et al., 2003). Besides the 5 to 8 receptors found in vertebrates, NPY-like receptors have been discovered in some invertebrates, such as pond snail (Lymnea stagnalis), fruitfly (Drosophila melanogaster) and malaria mosquito (Anopheles gambiae ) (Garczynski et al., 2002; Hill et al., 2002; Tensen et al., 1998). In vertebrates, the receptor family has evolved through both local gene duplications and large scale genome duplications. An ancestral triplet is thought to have been formed by local duplications; the three receptor genes, Y1, Y2 and Y5 are still located close to each other on the same chromosome (chromosome 4 in the human genome) (Larhammar et al., 2004). These three genes have thereafter been duplicated in both 2R and 3R with differential gene losses in various lineages resulting in a setup of seven genes in the ancestral gnathostome (Fig. 3) (Larhammar and Salaneck, 2004; Larsson et al., 2008; Salaneck et al., 2008; Wraith et al., 2000). The receptors are divided into three clades or subfamilies based on phylogenetic analysis. The Y2 subfamily consist of the Y2 and Y7 receptors, the Y1 subfamily is the most numerous with five members: Y1, Y4, Y6 and Y8, of which the Y8 receptor gene has been duplicated in the teleost fish ancestor generating Y8a and Y8b. The Y5 receptor is the only member in the third subfamily. The amino acid identities between the three subfamilies are low, only around 30%, but all receptors in mammals bind to all peptides of the neuropeptide Y family (Larhammar et al., 2001), although with different affinities. One receptor subtype, Y3, has been proposed to exist based on pharmacological data but has not been found in any genome and probably does not exist as a separate gene (Herzog et al., 1993; Jazin et al., 1993; Larhammar et al., 2001).

Figure 3. Schematic drawing of the NPY receptor repertoire found in different vertebrate lineages and the inferred ancestral gnathostome. Multiple duplication events as well as lineage-specific losses have led to the different subtypes seen in extant species. Proposed divergence times are based on paleontological data (Benton and Donoghue, 2007; Sansom et al., 1996).

NPY-receptors in mammals In mammals, the receptor family consists of five members, Y1, Y2, Y4, Y5 and Y6. The Y6 receptor has a complex evolutionary history within the mammalian lineage. It is a pseudogene in humans and other primates, as well as in pig (Wraith et al., 2000) and guinea pig (Starback et al., 2000). It is absent in rats but seems to be functional in for instance mouse and rabbit (Larhammar et al., 2004). The binding profile and physiological functions of the Y6 receptor are not well studied. In humans, the Y1 and Y2 receptors have opposing functions in many cases, one example is that Y1 increases food intake after stimulation by NPY, while Y2 functions as a presynaptic autoreceptor (Pedrazzini et al., 2003). The Y5 receptor also is appetite stimulating just as the Y1 receptor but studies in rodents indicate that Y1 is more involved in the food-seeking phase, while Y5 seems to be responsible for the consummatory phase (Day et al., 2005; Lecklin et al., 2002; Lecklin et al., 2003). It has been suggested that Y4 (the fastest evolving NPY receptor in mammals) has some appetite inhibiting functions (Huda et al., 2006). The

17 Y4 receptor seems to have co-evolved together with the tetrapod-specific peptide PP and shows a high binding preference for that peptide, in contrast to the Y1, Y2 and Y5 receptors that in humans all prefer NPY and PYY over PP (Lundell et al., 1995).

NPY-receptors in birds Six members in the family, Y1, Y2, Y4, Y5, Y6 and Y7 have been cloned and pharmacologically characterized in chicken (Gallus gallus) (Bromee et al., 2006; Holmberg et al., 2002; Lundell et al., 2002; Salaneck et al., 2000) and no other receptors have been identified in the chicken genome database. The same set up of receptors can also be identified in the genomes of zebra finch (Taeniopygia guttata), duck (Anas platyrhynchos) and turkey (Melea- gris gallopavo). Some of the chicken receptors display functional differences compared to their mammalian orthologs, such as a reduced affinity for truncated peptides for the Y2 receptor and a more widespread tissue distribution and less selective pharmacological binding profile for the Y4 receptor (Lundell et al., 2002; Salaneck et al., 2000). Notably, the Y6 receptor appears to be functional in chicken (Bromee et al., 2006) and the other three birds with sequenced genomes. Some studies of the importance of the NPY system in feeding in broilers have been conducted (Boswell et al., 1999; Tachibana et al., 2006; Zhou et al., 2005), but the roles of the different receptor subtypes are not known.

NPY-receptors in amphibians A couple of NPY receptors have been cloned from different frog species by our laboratory, such as Y1 in the African clawed frog Xenopus laevis, Y7 in the marsh frog, Pelophylax ridibundus (previously called Rana ridibunda) and the western clawed frog Silurana (Xenopus) tropicalis (Blomqvist et al., 1995; Fredriksson et al., 2004). The genome of the diploid Silurana tropicalis has been sequenced and we have identified six receptor genes in that genome (Paper III). The seventh receptor (Y6) has been cloned in Pelophy- lax ridibundus. This implies that the amphibian lineage has the same repertoire of receptors as the common ancestor of tetrapods and teleosts (Fig. 3).

NPY-receptors in teleost fishes Members of the NPY receptor family have been cloned and pharmacologically characterized in zebrafish (Berglund et al., 2000; Fredriksson et al., 2004; Fredriksson et al., 2006; Lundell et al., 1997; Ringvall et al., 1997; Starback et al., 1999). Parts of several receptors have also been cloned in basal teleosts in order to resolve the early evolution of the family and the

18 importance of 3R in forming the repertoire seen in teleosts (Salaneck et al., 2008). The whole genomes of five teleosts (zebrafish, tetraodon, fugu, medaka and stickleback (Gasterosteus aculeatus)) are available and have helped identify the receptors in these species (Larsson et al., 2008). Teleosts display a slightly different repertoire of receptors than tetrapods, including Y2, Y4 (formerly called Ya), Y7, Y8a (Yc) and Y8b (Yb). Some basal teleosts also have one or more of the subtypes Y1, Y5 and Y6 (Salaneck et al., 2008). Using a phylogenetic approach and analyzing the genes around the NPY receptors in tetraodon we came to the conclusion that Y8a and Y8b (formerly called Yc and Yb) arose through the 3R (Larsson et al., 2008). Interestingly, they seem to be the only surviving copies in the NPY receptor family from the teleost tetraploidization. Most of the teleosts seem to have lost the appetite stimulating receptors Y1 and Y5. That has been puzzling because feeding studies in goldfish (Carassius auratus) have shown that administration of NPY enhances feeding (Narnaware & Peter, 2001; Narnaware et al., 2000). The effects seen in these studies have been attributed to some other receptor subtype. However, recently the Y1 receptor was identified in the zebrafish genome and this new information may explain the results seen in the feeding experiments (Larsson et al., 2008; Salaneck et al., 2008).

NPY-receptors in cartilaginous fishes Several NPY receptors have been cloned in cartilaginous fishes such as the spiny dogfish (Squalus acanthias) (Salaneck et al., 2003) but the complete repertoire has been troublesome to obtain using degenerate primers. The genome of a cartilaginous fish, the chimera Callorhinchus milii (elephant shark) is partly sequenced and we have identified the neuropeptide Y system in this species. The elephant shark has all members of the proposed gnathostome ancestral repertoire with seven receptors as well as the two peptides NPY and PYY (Larsson et al., 2009).

The neuropeptide Y family of peptides Neuropeptide Y (NPY) and peptide YY (PYY) are two structurally related peptides that have been found in all investigated vertebrate species. Two separate genes code for the peptides, these genes arose from a common ancestral peptide gene in one of the tetraploidizations in the vertebrate 2R event (Larhammar, 1996; Soderberg et al., 2000 and Paper I). They were later duplicated in the teleost-specific whole genome duplication, generating NPYa, NPYb, PYYa and PYYb (Paper I). PYY was duplicated locally in tetrapods and gave rise to pancreatic polypeptide (PP) (Cerda-Reverter and Larhammar, 2000). Some other lineage-specific duplications also seem to

19 have occured in lamprey (Petromyzon marinus) (Montpetit et al., 2005), African clawed frog (Xenopus laevis) (Griffin et al., 1994; van Riel et al., 1993) and sturgeon (Scaphirhynchus albus) (Kim et al., 2000). There have also been reports of extra copies of the genes for PYY and PP in primates and ruminants (Couzens et al., 2000; Herzog et al., 1995; Lewis et al., 1985). Some NPY-like peptides have been identified in invertebrates such as sea slug (Aplysia californica) (Rajpara et al., 1992) and pond snail (Lymnaea stagnalis) (De Jong-Brink et al., 1999) and there have been several candidates for NPY in fruitfly (Drosophila melanogaster) of which one, called NPF, seems to be the ortholog (Brown et al., 1999). We were able to identify a putative NPY ortholog in the genome of amphioxus (Branchiostoma floridae) and have synthesized several variants of the peptide in order to identify its receptor among a handful of candidates. The NPY gene in human translates into a 97 amino acid long protein precursor, encompassing a signal peptide, the 36 amino acid long mature peptide, and a carboxyterminal extension. This structure is similar for the other peptides in the family. However, chicken has a mature PYY peptide with 37 amino acids (Cerda-Reverter and Larhammar, 2000). All peptides are ami- dated at the carboxy terminus and have the ability to form the same three- dimensional structure, the PP-fold (Darbon et al., 1992; Keire et al., 2000; Li et al., 1992). The mature peptides bind to the members in the NPY receptor family. In mammals, endogenous truncated peptides for NPY and PYY have been identified and one of them, PYY 3-36, has been found to have an in- hibitory effect on appetite (Batterham et al., 2003). NPY is expressed in both the brain and peripheral nervous system while PYY and PP are mainly expressed in endocrine cells in the gastro-intestinal tract in mammals (Larhammar et al., 2004). In non-tetrapod vertebrates, PYY is expressed in the nervous system (Cerdá-Reverter et al., 2000; Larsson et al., 2009; Soderberg et al., 1994). The sequences of NPY and PYY are well conserved in many species, but PP shows a higher evolutionary rate and is less conserved (Cerda-Reverter and Larhammar, 2000; Larhammar, 1996). The duplicates of NPY and PYY in teleosts display amino acid differences that render them difficult to assign as either NPY or PYY from sequence comparison alone. Another complica- tion in this identification process is the short length of the peptides; it is not possible to make reliable phylogenetic trees based on such short sequences, due to repeated mutations (saturation) at informative sites. For compilations of NPY-family sequences, see the review by Cerda-Reverter and Larham- mar, 2000 and Paper I.

NPYa and NPYb NPY is, as mentioned above, found in all vertebrate species studied and is the most conserved peptide in the family, with 20 invariant amino acid posi-

20 tions. The NPY gene is located close to the HoxA cluster on chromosome 7 in the human genome, which has helped elucidate the evolutionary history of the peptide (Larhammar et al., 2004; Soderberg et al., 2000) and Paper I. Database searches in the fugu genome revealed four peptides belonging to this family. By studying only sequence similarity it was not possible to con- fidently designate all of them as either NPY or PYY. However, by using the combination of chromosomal location and phylogeny of neighbouring genes as described in Paper I we were able to identify one of them as NPYb. We also concluded that NPYa and NPYb were formed during the teleost specific whole genome duplication. The orthologs are also found in the genomes of tetraodon, stickleback and medaka but NPYb is missing in the zebrafish genome database (Paper I). The functions and receptor preference of NPYb are not known for any species where it has been found, but RT-PCR in fugu shows a tissue expression similar to that of NPYa (Paper I).

PYYa, PYYb and PP PYY and PP are located close to the HoxB cluster on chromosome 17 in the human genome and these peptides were the first ones to be isolated in the family. PP is only present in tetrapods and seems to be a duplicate of PYY located in tandem with PYY in all species where the chromosomal location is known. It is also the peptide with the highest evolutionary rate (Larhammar, 1996). In 1985 a new peptide, PY, was isolated from the anglerfish (Lophius americanus) (Andrews et al., 1985). Its relationship to the three mammalian peptides was unclear, hence it was named PY for peptide tyrosine. Later this peptide has been found in several fish species and it was given different names by different investigators. Using the chromosomal location provided by the whole genome of several species, it was possible for us to identify this peptide as a 3R copy of PYYa, hence it was renamed PYYb (Paper I). Just like in the case for NPYb, the function of PYYb is not known, the expression pattern of PYYb in fasted and fed yellowtail (Seriola quinqueradiata) have however been investigated (Murashita et al., 2006). Zebrafish PYYb has been used as a ligand in competition assays for the zebrafish Y receptors. It binds with high affinity to zebrafish Y2 but has similar affinity as NPY and PYYa for the other receptors studied (Fredriksson et al., 2006 and Paper II).

The opioid receptor family The opioid receptors (mu, kappa, delta and orphanin) are GPCRs that just like the NPY receptors belong to the rhodopsin clan. They are involved in

21 various functions in the body including nociception, memory, food intake and reward mechanisms (Bodnar, 2009; Leknes and Tracey, 2008; Terenius and Johansson, 2010). The classical opioid receptor family has three members, mu, delta and kappa. They were cloned in the early nineties (Chen et al., 1993; Evans et al., 1992; Kieffer et al., 1992; Yasuda et al., 1993) but their existence had been suggested before since specific binding sites in the brain for opioid compounds had been identified twenty years earlier (Martin et al., 1976; Pert and Snyder, 1973; Simon et al., 1973; Terenius, 1973). After the cloning of the classical receptors, two research groups independently identified a fourth receptor with sequence similarity to the known receptors and this was called LC132 or ORL1 (Bunzow et al., 1994; Mollereau et al., 1994). This fourth receptor has then been found to display a different binding pattern compared to known opioid receptor agonists and antagonists in the species investigated. There have also been contradictions regarding the physiological effect after administration of orphanin receptor agonists (Mogil and Pasternak, 2001). The receptor does, however, display sequence similarity to the classical receptors and inclusion of this receptor in the opioid receptor family has been suggested since it was cloned. The receptor repertoire in other species than mammals has not been investigated to a great extent. Partial sequences of some receptors have been ob- tained by cloning in several vertebrate species including a cartilaginous fish, Alopias vulpinus and one agnathan, Eptatretus stoutii (Li et al., 1996a; Li et al., 1996b). A thorough investigation of mu receptor sequences available in the whole genome sequences of several vertebrates has also been conducted (Herrero-Turrion and Rodriguez, 2008). The evolutionary history of the opioid receptors has not previously been investigated in detail even if expansion of the family through genome duplications has been suggested (Stevens, 2005; Stevens, 2009). One opioid receptor channel has been proposed to exist in a mollusc (Mytilus edulis), but due to remarkably high identity with the human receptor its presence is questionable (Cadet and Stefano, 1999; Cadet et al., 2002). The endogenous ligands for the receptors are peptides generated from four different precursors, proenkephalin, prodynorphin, proorphanin and proopiomelanocortin. The binding profiles have been investigated in mammals and the mu and delta receptors both have high affinity for enkephalin peptides and β-endorphin, while the kappa receptor prefers dynorphins and the orphanin receptor binds to the orphanin peptide (Dores et al., 2002; Terenius and Johansson, 2010). Sequence comparisons of the known components of the opioid system in lampreys and sturgeon indicate that the ex- clusive binding of the orphanin peptide to the orphanin receptor is a late evolutionary event and that a more mixed binding profile might be ancestral (McClendon et al., 2010).

22 It has been suggested that the non-mammalian receptors are less type- selective than the mammalian ones based on both sequence similarity and pharmacological characterization (Stevens, 2009; Stevens et al., 2007). Most binding studies in non-mammalian tetrapods have however been conducted using brain homogenate (Stevens, 2009) and pharmacological profiles for the different receptors are therefore unknown. Studies with cloned receptors have been performed (Alvarez et al., 2006; Bradford et al., 2005; Bradford et al., 2006; Brasel et al., 2008; Pinal-Seoane et al., 2006; Rodriguez et al., 2000; Walthers et al., 2005) however, the endogenous ligands have only been used in a few cases (de Velasco et al., 2009; Gonzalez-Nunez et al., 2007a; Gonzalez-Nunez et al., 2007b). It is therefore hard to draw any clear conclusions regarding any differences in binding preferences.

Opioid receptors in tetrapods All four receptors are present in mammals, they are located on chromosome 1 (delta receptor), 6 (mu receptor), 8 (kappa receptor) and 20 (orphanin receptor) in the human genome. Several splice variants have been identified for the different receptors (for both human and mouse), with the highest number seen for the mu receptor type. It is however not clear if this difference reflects the true situation or is just because the mu receptor has been studied more extensively (Stevens, 2009). Knockout mice for the three classical receptors have been made in order to determine the distinct roles for the different receptors in physiology and behavior. The mu receptor seems to be the main receptor in the opioid system regarding promotion and initiation of reward while the kappa receptor seems to have the opposite function. The delta receptor is less involved in this behavior as opposed to pain where all three play important roles (Kieffer and Evans, 2009). The orphanin receptor has been found to be able to gener- ate both analgesic and hyperalgesic effects and the role of this receptor in reward mechanisms and drug dependence have also shown converse effects (Civelli, 2008). Several behavioral tests have been conducted in various amphibian, reptile and bird species showing antinociceptive effects as the result of administration of morphine (Stevens, 2004; Stevens, 2009). The presence of three opioid receptors in the songbird Junco hyemalis was suggested after binding studies using brain homogenates (Deviche et al., 1993). This study was conducted before the cloning of the orphanin receptor in mammals. All four receptors are present in the genomes of chicken, zebra finch, duck and turkey. A complete repertoire of four receptors has been cloned in the Northern grass frog Rana pipiens and the binding profile for the mu receptor has been elucidated, however no endogenous ligands where investigated (Brasel et al., 2008; Stevens et al., 2007). The same is true for the four receptors in the rough-skinned newt, Taricha granulosa (Bradford et al., 2005; Bradford et al., 2006; Walthers et al., 2005).

23 Opioid receptors in teleost fishes Six fragments from different opioid receptors have been cloned from the teleost fish Catostomus commersoni, suckerfish. It was possible to retrieve a full-length sequence from one of them, an ortholog to the mu receptor. This receptor was also pharmacologically characterized using classical opioid receptor ligands and shown to have a binding profile similar to that of mammalian mu receptors (Darlison et al., 1997). It should be noted that this fish species belongs to a lineage, Catostomidae, that has probably undergone a fourth and independent tetraploidization (Uyeno and Smith, 1972). Five full-length receptors from zebrafish have been cloned and pharmacologically characterized using both endogenous opioid peptides and synthetic ligands (Alvarez et al., 2006; Barrallo et al., 2000; Barrallo et al., 1998; Pinal-Seoane et al., 2006; Rodriguez et al., 2000). The delta receptor is duplicated and the topology of the tree and chromosomal location of the genes indicate that they are the results of the teleost-specific whole genome duplication (Pinal-Seoane et al., 2006 and Paper IV). The involvement of the opioid system in nociception in teleost fishes has been studied using several different fish species and behavioral tests (Sneddon, 2004).

The opioid peptide family The endogenous opioid peptides, met- and leu-enkephalins, α-neoendorphin, dynorphin A, dynorphin B, β-endorphin, orphanin/nociceptin and nociceptin-like are cleaved from four different precursors coded for by four distinct genes. Three of the genes, proenkephalin, prodynorphin and proopiomelanocortin, were cloned in mammals (Kakidani et al., 1982; Nakanishi et al., 1979; Noda et al., 1982) and subsequently the similarities in gene structure lead to suggestions that gene duplications mechanisms had played a part in the evolution of this family (Herbert et al., 1983). The orphanin peptide was identified from porcine brain in screening for the endogenous ligand of the orphanin receptor (Reinscheid et al., 1995). The gene was cloned and had a similar structure as the other precursor genes, with a grouping of the mature peptides toward the C-terminus and conserved cysteines in the N-terminus (Nothacker et al., 1996). All propeptides have in common that they include one or more enkephalin sequences, YGGFM/L. The degree of preservation of this motif differs between species and peptides, and this is also true for the number of motifs present in each precursor (Fig. 4). The enkephalin propeptide includes seven cleavable peptides that contain the motif while the proopiomelanocortin precursor has only one motif. The presence of the enkephalin motif in all four of the propeptides lead to the hypothesis that the propeptide gene family

24 has expanded by duplications (Dores et al., 2002). The mature peptides, which in most cases are longer than the enkephalin core motif, are flanked by single or dibasic amino acid residues to enable cleavage from the propeptide. The genes for all four opioid peptide precursors have been cloned in several species in attempts to elucidate their evolutionary history. They are found in all vertebrate lineages while the physiological functions mainly have been investigated in mammals (Dores et al., 2002). Some invertebrate peptides have been reported, although none has been cloned which leaves the question of the presence of opioid peptides outside vertebrates unre- solved (Ewadinger et al., 1996; Luschen et al., 1991; Stefano et al., 1993).

Figure 4. Schematic picture of the repertoire of opioid precursors in vertebrates. Black bars indicates enkephalin motifs, a star beneath the bar symbolize degenerated enkephalin motif. Note that the PDYN gene is missing in chicken and the structure of the precursor in turkey is shown in the figure. Proposed divergence times are based on paleontological data (Benton and Donoghue, 2007).

Proenkephalin Seven peptides can be cleaved from the enkephalin propeptide. In mammals five pentapeptides, one heptapeptide and one octapeptide are cleaved (Dores et al., 2002). Zebrafish have a duplicate of this gene and one of the two duplicates has only four putative pentapeptides while the fifth is degenerated (Gonzalez Nunez et al., 2003). The pentapetides can be either met-

25 enkephalin (YGGFM) or leu-enkephalin (YGGFL); there is no difference in binding affinity for met- and leu-enkephalin in mammals (Jordan et al., 2000) and shifts between these forms seems to have occurred several time during evolution (Dores et al., 2002).

Prodynorphin Three peptides are cleaved from the dynorhin precursor in mammals, α- neoendorphin, dynorphin A and dynorphin B (Khalap et al., 2005). Addi- tional enkephalin motifs are present in teleost fishes, lungfish and frog (Alrubaian et al., 2006; Danielson et al., 2002; Dores et al., 2004; Khalap et al., 2005; Pattee et al., 2003) but the knowledge of processed peptides in non-mammalian tetrapods is limited. The dynorphin propeptide in frogs was first designated the name Xen-dorphin and suggested to be a new member in the peptide family, but sequence alignment showed that it is the dynorphin precursor gene (Khalap et al., 2005; Pattee et al., 2003).

Proorphanin One peptide is cleaved from the proorhanin precursor in mammals, a 17 amino acid long peptide with a degenerated enkephalin motif in the begin- ning, FGGFT. The corresponding motif is YGGFI in a majority of the ray- finned fishes studied and these species also have a second cleavable peptide within the precursor (Danielson et al., 2001; Gonzalez-Nunez et al., 2003). We were able to identify this second peptide in the frog genome, as described in Paper V. Duplicates of the proorphanin gene have been retained after 3R.

Proopiomelanocortin One opioid peptide, β-endorphin, is cleaved from the proopioimelanocortin precursor. Depending on the species, 2-4 melanocortin peptides are also cleaved from this precursor. The β-endorphin shows sequence similarity throughout the gnathostome lineage (Dores and Lecaude, 2005). Two proopiomelanocortin genes, POC and POM, have been identified in several lamprey species. Both genes contains a β-endorphin peptide but the number of melaoncortin sequences differs between them (Takahashi and Kawauchi, 2006; Takahashi et al., 2006). Zebrafish and other teleost fishes have duplicates of the POMC gene which were generated in 3R (de Souza et al., 2005 and paper V) and duplicates of the POMC gene have also been found in other species that have undergone independent tetraploidizations (Deen et al., 1992; Takahashi et al., 2009).

26 Hox-cluster regions The Hox cluster of homebox-containing transcription factors is important for the segmental patterning in development of the animal body in both deute- rostomes and protostomes. The genes are collinear and located on the chromosomes in the same order as they are expressed in the body (Kmita and Duboule, 2003; Mallo et al., 2010). In addition to the importance of these for developmental and gene regulation, the gene clusters have also been used as a model for evolutionary events (Furlong and Holland, 2002; Hoegg and Meyer, 2005; Holland and GarciaFernandez, 1996). Protostome model species such as the fruitfly Drosophila melanogaster have a single cluster, which has been split in fruitfly but not in other insects such as the red flour beetle (Tribolium castaneum) (Lemons and McGinnis, 2006) while humans and other investigated tetrapods have four clusters. The cephalochordate amphioxus also has only one Hox cluster and this has been considered strong evidence to support the two whole genome doublings early in vertebrate evolution (2R) (Amemiya et al., 2008). The urochordate Ciona intestinalis (and other tunicates) have only one set-up of the Hox- cluster genes and these genes are not located in a single cluster but have been split into two half-clusters (Ikuta and Saiga, 2005). The agnathan lineage, with hagfishes and lampreys, has three to four Hox-clusters each. These clusters seem to be the result of different duplication events when compared to other vertebrates (Fried et al., 2003; Stadler et al., 2004). All teleost fishes with sequenced genomes have seven clusters, however different clusters are missing in different species indicating an ancestral teleost set-up of eight clusters. This is the expected outcome if the expansion is the result of 3R. Cloning of the Hox-clusters from several teleost fishes also confirm this picture (Crow et al., 2006; Guo et al., 2010; Hoegg et al., 2007). Members of all eight clusters have been identified in the japanese eel (Anguilla japonica) and goldeye (Hiodon alosoides), two representative of basal teleost fishes (Chambers et al., 2009; Guo et al., 2010). In teleost fishes with known late tetraploidization events, such as the salmonids, up to 118 Hox genes distributed in 13-14 clusters have been identified (Moghadam et al., 2005; Mungpakdee et al., 2008). The number of genes in each cluster varies to some degree depending of species and cluster. Before the sequencing of the amphioxus genome 14 Hox genes were identified in this species and this was thought to be the repertoire of the ancestral chordate (Amemiya et al., 2008; Minguillon et al., 2005). A fifteenth gene was identified in the genome sequence and confirmed by PCR (Holland et al., 2008). In human the HoxA clusters is comprised of 11 genes, B of 10, C of 9 and D of 9. Other lineages have had different (and fewer) losses, the elephant shark has 11 genes in their HoxA cluster, 11 in B, 11 in C and 12 in D (Ravi et al., 2009).

27 The quadrupled Hox-cluster in mammals as compared to the single one in amphioxus has been used as one argument in support of genome duplication events in the vertebrate lineage. Some attempts have also been made to use this region (the Hox clusters themselves and neighboring genes on the same chromosomes) as examples to prove that no genome doublings have occurred in the vertebrate lineage (Abbasi and Grzeschik, 2007; Hughes et al., 2001). The gene families and interpretation of topologies of the trees used to support this hypothesis has however not survived detailed scrutiny (Larhammar et al., 2002; Sundstrom et al., 2008; Van de Peer et al., 2010). The NPY peptide family is one of many gene families that is located in the same chromosomal regions as the Hox cluster and might therefore been involved in the same chromosomal events as the Hox cluster, as described above. The Hox clusters are located on chromosome 2, 7, 12 and 17 in the human genome, and this group of chromosomes is called the Hox paralogon. In the early nineties parts of a fifth chromosome was included in the paralogon, chromosome 3 (Lundin, 1993; Lundin and Larhammar, 1998). Studies of several gene families showed that a number of genes seemed to have been translocated from one of the chromosomes in the original quartet, most probably from chromosome 7, to chromosome 3 (Plummer and Meisler, 1999). This translocation seems to be a mammalian event because the chromosomal regions in chicken do display fourfold paralogy (Paper VII).

SCN One gene family subject to this translocation from chromosome 7 to chromosome 3 in humans is the sodium channel alpha subunit gene family. Each sodium channel (SCN) consists of an alpha subunit of approximately 2,000 amino acids and a smaller beta subunit that modifies the properties of the alpha subunit (Ruan et al., 2009). The SCN alpha subunits, also called Nav1, consist of four domains connected by intracellular loops. Each domain has six transmembrane regions. Many phylogenetic analyses have been conducted for this gene family using parts of the nucleotide or protein sequences (Goldin, 2002; Goldin et al., 2000; Lopreato et al., 2001; Novak et al., 2006; Piontkivska and Hughes, 2003; Plummer and Meisler, 1999; Yu and Catterall, 2003; Yu and Catterall, 2004) however these produced quite varying topologies. Humans and other eutherian mammals have ten genes coding for the sodium channel alpha subunits, each gene consists of up to 26 exons and two unusual AT-AC introns can be identified in the genes (Kohrman et al., 1996; Wu and Krainer, 1999), see also Paper VII. Five of the genes are located in a cluster on one chromosome (chromosome 2 in the human genome), three of them are together on another chromosome (chromosome 3 in human) and the remaining two on two different chromosomes (chromosome 12 and 17)

28 (Plummer and Meisler, 1999). Teleost fishes have eight genes, none of which is located next to another SCN alpha gene on the chromosome (Novak et al., 2006). Experimentally confirmed sodium channels have also been identified in a few insect species (Dong, 2007). Several studies have investigated the evolutionary relationships among vertebrate SCN alpha genes. The syntenic relationship with the Hox-clusters was observed early on (Lundin, 1993; Lundin and Larhammar, 1998). The chromosomal locations of the genes suggest that the genome duplications in 2R together with local duplications in the tetrapod lineage formed this family (Plummer and Meisler, 1999). The family has also expanded in 3R as indi- cated by the chromosomal locations of the eight SCN alpha genes in zebrafish (Novak et al., 2006).

IGFBP Members of the insulin-like growth factor binding protein (IGFBP) family function as carrier proteins for the insulin-like growth factors, IGF-1 and IGF-2, and are able to regulate the functions of these IGF:s (Duan and Xu, 2005). Humans have a repertoire of six IGFBPs, all between 240 and 328 amino acid residues and with the same main structure with an IGF-binding site in the N-terminal and a thyroglobulin type-1 domain in the C-terminal. The middle part of the protein is variable. The location of the IGFBP genes on the same chromosomes as the Hox- clusters was noted early on, and since then the IGFBP genes have been used (together with other gene families) to support that the Hox-cluster bearing chromosomes arose through block duplications (Larhammar et al., 2002; Lundin, 1993 and Paper VI) but also the opposite; that they contradict such a scenario (Abbasi and Grzeschik, 2007; Hughes et al., 2001). Several alternative duplication scenarios for IGFBP genes themselves have also been pub- lished (Rodgers et al., 2008). A pair of IGFBP genes seems to have been present before the genome duplications. This pair was duplicated and two genes were lost, resulting in the set-up of six genes in eutherian mammals, the repertoire is however slightly different in opossum and birds (Paper VIII). Several IGFBP genes have been cloned in teleost fishes and duplicates of the genes have also been identified (Chen et al., 2009; Dai et al., 2010; Kamangar et al., 2006; Kamei et al., 2008; Wang et al., 2009; Zhou et al., 2008).

29 Aims

The overall aim of this thesis was to investigate the role of gene and genome duplications in the formation of peptide and receptor families.

Specific aims of the different papers

• Paper I. To elucidate the evolutionary history of the neuropeptide Y family of peptides in teleost fishes using whole genome data from five teleost fish species.

• Paper II. To investigate the endogenous peptide PYYb in zebrafish with regard to its interaction with the Y4, Y7, Y8a and Y8b receptors.

• Paper III. To characterize the neuropeptide Y system in the frog Silurana tropicalis with regard to number of receptors and peptides, pharmacological properties and expression profiles.

• Papers IV and V. To delineate the evolutionary history of the opioid receptor and peptide families, including repertoire in different species and duplication events.

• Papers VI, VII, VIII. To investigate the evolutionary history of several gene families on the Hox-bearing chromosomes in relation to 2R and 3R; including detailed studies of the SCN alpha and IGFBP gene families regarding repertoire in different species, exon-intron structures and duplication events.

30 Materials and Methods

Genomes examined During the past five years the explosion of the number of whole genome sequences from various species available in different databases has made it possible to conduct new types of studies. The main focus of the vertebrate sequencing projects has been sequencing of mammalian genomes, however non-mammalian species with key positions in vertebrate species phylogenies have also been sequenced.

Urochordates and Cephalochordates The relationship between the urochordates and cephalochordates and their relationship to vertebrates has been a widely discussed subject. Molecular data places the urochordates as more closely related to vertebrates than cephalochordates (Delsuc et al., 2006; Vienne and Pontarotti, 2006). Whole genome sequences are currently available for three different urochordates, Ciona intestinalis, Ciona savignyi and Oikopleura dioca. The genome sequence for Ciona intestinalis have been assembled into chromosomes while the sequences for Ciona savignyi and Oikopleura dioca are partially assembled. All three species have a split Hox-cluster (Ikuta and Saiga, 2005; Seo et al., 2004) which limits the use of this species regarding genome organization analyses in these regions. The cephalochordate amphioxus, Branchiostoma floridae is often used as a model for an ancestral chordate, both regarding morphology and molecular data. Many genes that are found in duplicates in vertebrates have a single ortholog in amphioxus, and the whole genome sequencing of this species seems to have settled the case regarding the occurrence of the two whole genome duplications in early vertebrates (Garcia-Fernandez and Benito- Gutierrez, 2009; Holland et al., 2008; Putnam et al., 2008). The genome assembly is unfortunately still very fragmented; however a map depicting conserved synteny between amphioxus scaffolds and human chromosome is available (Putnam et al., 2008).

31 Agnathans Two main lineages of agnathans are present today, the hagfishes and the lampreys. There has been some discussion regarding their monophyletic origin (Kuraku and Kuratani, 2006). Their phylogenetic position in respect to the two whole genome duplications has also been discussed (Escriva et al., 2002; Kuraku et al., 2009). One lamprey genome, that of the sea lamprey Petromyzon marinus, has been sequenced, however the preliminary genome assembly is highly fragmented which makes it difficult to identify the complete gene if several introns are present. For the same reason it is difficult to conduct analyses of conserved synteny. There is also a massive loss of DNA when comparing the genomic content of germline cells and somatic cells. Even functional genes transcribed in the germline are deleted from the genome in the somatic cells. Genomic resources based on germline cells are therefore on the way (Smith et al., 2009; Smith et al., 2010).

Amphibians One genome of a diploid frog is sequenced, that of Silurana tropicalis (Hellsten et al., 2010) also known as Xenopus tropicalis. Both molecular and morphological data shows that the sister taxa Xenopus and Silurana are monophyletic. The name Silurana tropicalis is used to emphasize the age of the divergence between the taxa which has been estimated to be 63.7 million of years (Evans et al., 2004). The genome is sequenced with high coverage but unfortunately not assembled into chromosomes, which limit the usefulness to some degree. De- spite its limitations it gives important information about events that took place in the divergence of amphibians and other tetrapods.

Birds and Lizards There are several bird genomes sequenced, chicken (Gallus gallus), zebra finch (Taeniopygia guttata), duck (Anas platyrhynchos), turkey (Meleagris gallopavo) together with one lizard (Anolis carolinensis) genome. The duck and lizard genomes are not assembled into chromosomes, while the genome sequences of the other three have been assigned to chromosomes. Both the turkey and duck genomes have just recently been available and therefore not used in this thesis. One special characteristic of bird genomes are microchromosomes. These chromosomes are small with a high gene density. Some of the microchromosomes might be retained ancestral chromosomes (Burt, 2002) and the chicken genome has been used in attempts to reconstruct the ancestral vertebrate karyotype (Nakatani et al., 2007).

32 Songbirds such as zebra finch have long been used as model species in neuroscience and an analysis of the neuropeptidome in this species identified orthologs to a majority of the prohormones in mammals (Xie et al., 2010).

Mammals The human genome has been available for almost a decade and there are now over 35 mammalian species with their whole genome sequenced in the Ensembl genome database. The majority of these are however low-coverage genomes and this might affect the usefulness of the data, depending on what analysis you want to perform (Milinkovitch et al., 2010). Not all of the genomes are assembled into chromosomes and it is not possible to study conserved synteny in these cases. Most mammalian genomes give the same information regarding the whole genome duplications and we have therefore used a few species, including human, to highlight differences and similarities in the mammalian lineage. Mouse (Mus musculus) is one of the classical laboratory animals and many functional studies of genes have been performed in this species. Many genes have also been cloned in mice, these sequences can be used as a complement to the sequences retrieved from the genome database. The mouse genome is however highly rearranged (Trifonov et al., 2010). The extent of rearrangement differs depending on chromosomal region, this is also true for the other mammalian genomes used in these studies. The dog (Canis familiaris) genome proved to be useful with good coverage and fewer rearrangements than mouse (Lindblad-Toh et al., 2005). The genome of grey short-tailed opossum (Monodelphis domestica) is in- teresting since it can provide information about eutherian specific event (Lindblad-Toh et al., 2005). Compared to human, mouse and dog the karyotype of opossum is different with a low number of chromosomes that seems to be the result of several fissions and fusions in the marsupial lineage (Rens et al., 2003; Svartman and Vianna-Morgante, 1998) and diminish the usefulness of this species in conserved synteny analysis.

Teleost fishes Teleost fishes constitute approximately half of all extant vertebrates (Meyer and Van de Peer, 2005). The whole genomes from five different teleost fishes are available in the Ensembl genome database, zebrafish (Danio rerio), fugu (Takifugu rubripes), tetraodon (Tetraodon nigriviridis), stickleback (Gasterosteus aculeatus) and medaka (Oryzia latipes). The accepted view is that zebrafish are the most basal of the five specie and that the two pufferfishes, fugu and tetraodon are more closely related to stickleback than to medaka. Phylogenetic analyses using several different datasets give in-

33 conclusive results regarding the relationship between medaka and stickleback (Negrisolo et al., 2010 and references therin). All of the teleost fish genomes have been assembled into chromosomes, the fugu genome most recently. However, all of them still have some regions which have not been assigned to any chromosomes. The degree of unas- signed genomic stretches varies over the genomes and from genome to genome. The teleost fishes have undergone a third round of genome duplication, 3R, resulting in duplicates of genes discovered in tetrapod genomes (Meyer and Van de Peer, 2005). Differential losses have occurred and the five genomes complement each other. The genomes have not only proved to be useful in detecting double conserved synteny (Jaillon et al., 2004) but also in order to construct an ancestral vertebrate karyotype (Kasahara et al., 2007; Woods et al., 2005).

Other investigated species The genome of the cartilaginous fish elephant shark, Callorhincus millii is important since it is the only genome to represent this lineage of vertebrates (Venkatesh et al., 2007; Venkatesh et al., 2005). It is a low-coverage sequence of a compact genome that is not assembled into chromosomes yet. This makes it hard to identify full-length sequences of genes with large introns. Investigations of conserved non-coding elements show more similarities between human and elephant shark than between human and teleost fishes (Venkatesh et al., 2006). Two genomes of the closest relatives to chordates are available, one from the hemichordate Saccoglossus kowalevskii (acorn worm) and one from the echinoderm Strongylocentrotus purpuratus (sea urchin). Their close relationship to chordates is useful when to define evolutionary event as chordate specific or not. Neither of these genomes is however assembled in to chromosomes.

Database searches and sequence identification The protein family predictions in the Ensembl database (http://www.ensembl.org) were used to retrieve the protein sequences for all families included in the papers. For detailed information regarding Ensembl release versions, species and selection criteria for the protein families included in the analyses, see material and methods in the respective papers. In general the longest transcript predictions were selected for analysis, if the predicted protein sequence was not too divergent. In such cases automatic GenScan predictions (Burge and Karlin, 1997) in the Ensembl database were used instead. To identify additional sequences not included in the En-

34 sembl protein family prediction, tblastn searches (Altschul et al., 1990) with standard settings were carried out in the Ensembl database and in the Na- tional Center for Biotechnology Information (NCBI) database. The protein family database Pfam (http://pfam.sanger.ac.uk/search) (Finn et al., 2010) was used to identify protein domains in the sequences. Short, incomplete or divergent protein predictions were extended by a search for missing exons in the sequences flanking the genes or inside introns, following consensus for splice donor and acceptor sites as well as sequence identities to other family members. To complement the manual curation, the Genscan gene prediction server (Burge and Karlin, 1997) was used.

Sequence alignments and phylogenetic analysis Different software packages were used for the alignment of sequences and the phylogenetic analyses. The alignments were manually inspected to im- prove incorrectly annotated sequences and remove incomplete sequences. The sequences were aligned using the Windows version of Clustal X 1.81 (Jeanmougin et al., 1998; Thompson et al., 1994) in Paper III, IV, V and VI. In Paper VII and VIII sequence alignments were made using Jalview2.4 (Waterhouse et al., 2009) applying the ClustalW (Thompson et al., 1994) tool with standard settings. Phylogenetic trees were constructed using the neighbor-joining (NJ) method with standard settings and 1000 bootstrap replicates in Clustal X 1.81 (Jeanmougin et al., 1998; Thompson et al., 1994) for Paper III, IV, V and VI and in ClustalX 2.0 (Larkin et al., 2007) for Paper VII and VIII. Quartet- puzzling maximum likelihood (QP) trees were constructed in paper IV, V, VI and VII using Treepuzzle 5.2 (Schmidt et al., 2002). Phylogenetic maximum likelihood (PhyML) trees were constructed in Paper III and VIII using the online execution of the PhyML 3.0 algorithm available at http://www.atgc-montpellier.fr/phyml/ (Guindon and Gascuel, 2003).

Cloning and Sequencing PCR primers for the NPY receptors in Silurana tropicalis (including restric- tion sites for Xho and Hind) were constructed based on the sequences in the genome database. PCR products were ligated into a pcDNA3 vector and transformed into chemically competent DH5a E.coli cells (Invitrogen). The insert was sequenced using T7 vector specific primer and BigDye V3 terminator sequencing kit (Applied Biosystems, USA). The product was analyzed on an ABI PRISM 310 GeneticAnalyzer (PE Applied Biosystems) automatic

35 sequencer to confirm sequence. Sequences were analyzed using the laser- gene software (DNASTAR, USA)

Receptor binding studies Plasmid DNA was transfected to 293-HEK cells. The zebrafish receptors had previously been cloned and expressed in cell lines by our laboratory. Trans- fected cells were grown for 24 hours, harvested and frozen at −80ºC. Thawed aliquots of transfected cells were used for competition binding experiments which were performed with serial dilutions of the endogenous peptides (NPY, PYY and PP for the frog and NPYa, PYYa and PYYb for zebrafish). All binding experiments were performed with a total volume of 100µl, with 125I-labeled pPYY (porcine PYY) as radioligand and an incuba- tion time of 3 hours. Saturation experiments for the frog receptors were performed with 12 different concentrations of radioligand ranged from 8 to 1000 pM. Results were analyzed using Prism 4.0 software package (Graph- Pad, San Diego, CA, USA). The pKi values were compared to each other using one-way ANOVA followed by Tukey-Kramer Multiple Comparison Test (GraphPad, San Diego, CA, USA).

RT-PCR Reverse transcriptase PCR was performed on total RNA (isolated using TRIzol reagent, Life Technologies, USA) in eleven tissues from Takifugu rubripes. cDNA was synthesized using AMV transcriptase first strand cDNA synthesis kit (Gibco, BRL, USA). For PCR conditions and primer sequences see Paper I. RT-PCR products were sequenced to confirm their identity using dye terminator chemistry on an Applied Biosystems 3700 DNA Analyzer.

RNA extraction and quantitative real time PCR Four adult zebrafish were purchased from Akvarie Hobby (Uppsala, Swe- den) and all procedures involving animals were conducted in accordance with approval #C264/6 from the local ethics committee. Adult S. tropicalis frogs were purchased from NASCO and maintained in the laboratory in well water (25-26 oC) under a 12L:12D photoperiod. Frogs were fed frog brittle (NASCO). All procedures involving animals were conducted in accordance with the guidelines of the University Committee on the Care and Use of Animals of the University of Michigan (animal care and use protocol # 09860).

36 Total RNA was prepared using the Trizol Reagent (Invitrogen, Sweden) following the manufacturer’s instructions. cDNA was synthesized using M- MLV reverse transcriptase (Invitrogen, Sweden) and random hexamers as primers following the manufacturer’s recommendations. Absence of genomic DNA in the cDNA was controlled for by PCR. Primers for the quantitative PCR were designed using the Beacon De- signer v4.0 (Premier Biosoft, USA). The optimal annealing temperatures for each primer combination were determined with PCR on genomic DNA. The real-time PCR reactions were preformed in a MyIQ thermal cycler (Bio-Rad Laboratories, Sweden). To confirm that only one product was formed, melt curve analyses were conducted. For PCR conditions and primer sequences see Paper II and III. The data were analyzed and threshold cycle (Ct) values derived using MyIQ software v 1.04 (Bio-Rad Laboratories, Sweden). Differences in the primer efficiency was corrected for using LinRegPCR (Ramakers et al., 2003), Grubbs test for outliers (GraphPad, USA) was used to calculate the mean primer efficiency for each primer pair and detection and exclusion of outliers. The Ct values were transformed into quantities using the delta Ct method (Livak and Schmittgen, 2001) and the highest expression was set to 1. geNorm (Vandesompele et al., 2002) was used to identify the two most stable housekeeping genes which thereafter were used to normalize expression due to differences in cDNA concentration between samples.

37 Results and discussion

Paper I The NPY peptide family in tetrapods consists of three peptides, NPY, PYY and PP, but the situation in teleost fishes is more complex. Three peptides were previously known in zebrafish, designated NPY, PYY and PY. Investi- gation in the zebrafish genome confirmed this, while it was possible to identify a fourth peptide in the genomes of tetraodon, fugu and stickleback. The fifth teleost species with a whole genome sequence available, medaka, has the same number of peptides as zebrafish. However, zebrafish have NPY, PYYa and PYYb (previously known as PY) while medaka have NPYa, NPYb and PYYa. The identification of four different peptides in teleosts also made it possible to rename peptide sequences from the sea bass (Dicentrarchus labrax) and the Japanese flounder (Paralichthys olivaceus) (Cerdá-Reverter et al., 2000; Kurokawa & Suzuki, 2002) that previously had been misidentified. The expression of the mRNA of the four peptides in fugu was studied in eleven different tissues using RT-PCR. Both of the NPY copies displayed a similar expression pattern as did the two PYY copies. The NPY peptides had a predominantly neural expression compared to PYY peptides. The prepropeptides themselves are too short and too conserved to use for phylogenetic analyses. Because of this lack of phylogentic signal we chose to investigate a set of neighboring genes in order to deduce their evolutionary history. Our lab has previously proposed that NPY and PYY were the product of a chromosomal duplication together with the Hox-clusters. In this study we show that the expanded peptide family in teleost fishes is the result of chromosomal duplications in the teleost lineage. The five neighboring gene-families we studied all showed a similar evolutionary history with expansion and duplication of genes after the teleost diverged from tetrapods. The same is true for the Hox-clusters and the duplication ot these clusters have been examples of the teleost specific whole genome duplication. The evolution of the NPY family is therefore the result of the two early vertebrate whole genome duplications (2R) generating NPY and PYY and the third teleost specific duplication (3R) resulting in NPYa, NPYb, PYYa, PYYb. Differential losses have occurred among the fishes and PYY in tetrapods have been locally duplicated generating PP.

38 Paper II The NPY system in zebrafish consists of at least six receptors, Y1, Y2, Y4, Y7, Y8a and Y8b and three peptides; NPY, PYYa and PYYb. One of the peptides and one of the receptors were probably formed as a result of the teleost-specific tetraploidization (Larsson et al., 2008 and Paper I). All of the receptors, except the recently discovered Y1 receptor, have previously been pharmacologically characterised with regard to binding affinity for the endogenous peptides NPY and PYYa and also to truncated fragment of mammalian peptides. One of the receptors, Y2, has also been characterized using the third endogenous peptide, PYYb and these studies concluded that PYYb has a high affinity for the Y2 receptor. In this paper we have investigated the endogenous peptide PYYb in zebrafish with regard to its binding to four previously cloned receptors, the Y7 receptor from the Y2 subfamily and three receptors from the Y1 subfamily, the Y4 receptor (formerly called Ya) and the two receptors duplicated in the teleost tetraploidization, Y8a and Y8b (formerly Yc and Yb). The affinity of NPY and PYYb for the Y7 receptor was equal and could be distinguished from PYYa that showed lower affinity for the receptor. No difference in affinity was detected between the three peptides tested on the Y4, Y8a and Y8b receptor. This indicates that Y2 may have a unique ligand preference for PYYb in zebrafish, just as the case for the Y4 and PP in mammals. We investigated the mRNA expression of both the peptides and receptors in a panel of eight tissues using quantitative PCR. All investigated genes showed expression in heart, kidney and brain. PYYb was the only peptide with expression in the gastrointestinal tract. The broadest expression profile was found for the Y7 receptor with expression in the tissues mentioned above and in muscle, spleen and with high expression in the liver.

Paper III The neuropeptide Y system consist of 3-4 peptides and 4-7 receptors depending of species examined. We have previously suggested that the ancestral gnathostome had seven receptors and two peptides, this repertoire is intact today in the cartilaginous fish elephant shark, Callorhincus millii. All tetrapods have three peptides but the number of receptors varies. The whole genome of the diploid frog Silurana tropicalis has recently been sequenced and we used BLAST to search for the complete NPY receptor and peptide repertoires followed by phylogenetic analyses to identify the different subtypes of receptors. Six members of the NPY receptor family were identified in the genome. We have previously cloned a seventh receptor in another frog species, the marsh frog Pelophylax ridibundus (previously named Rana

39 ridibunda). Thus, all seven proposed ancestral NPY receptors have been found among amphibians. All six frog receptor genes were inserted into expression vectors for functional expression. Although all six receptors were transfected, no binding to the Y1, Y2 and Y4 receptors was detected in either human HEK-293 cells or the frog cell-lines XTC-2 and XL-58. PYY had the highest affinity for the three receptors investigated, the Y5, Y7 and Y8 receptors. All three peptides had their highest affinity to the Y8 receptor. This is also the only receptor where PP seems to have a physiological relevant affinity. The six receptors have a fairly similar tissue distribution, analyzed with quantitative real time PCR in a panel of 19 tissues, with the highest expression in skin, blood and heart. Also the peptides were expressed to a high degree in skin and blood. The local duplicates PYY and PP showed an almost identical expression profile. It was only possible to detect expression in neural tissues for NPY, the Y1 and Y2 receptors. However, all peptides and receptors showed expression in at least two of the three tissues representing the gastrointestinal system: stomach, large intestine and small intestine.

Paper IV and V The opioid system is involved in reward and pain mechanisms and consists of four receptors and several peptides in mammals. We have investigated species representing a broad range of vertebrates and found that the four opioid receptor types, called delta, kappa, mu and orphanin receptors, are present in most of the species. The opioid peptides are derived from four prepropeptide genes, PENK, PDYN, PNOC and POMC, encoding enkephalins, dynorphins, orphanin/nociceptin and beta-endorphin, respectively. We have analyzed the evolution of both the peptide and receptor families using a combination of sequence-based phylogeny and chromosomal locations of the genes and their neighboring gene families. The results show that the vertebrate opioid receptor gene family was largely formed by the genome doublings that took place early in vertebrate evolution. Additional duplicates have been retained after the teleost specific tetraploidization. It was possible to identify duplicates for the delta receptor in the zebrafish and stickleback genomes. Medaka, stickleback and tetraodon have duplicates of the kappa receptor, whereas the orphanin gene is missing in the medaka genome and the mu receptor gene is missing in the genome of tetraodon. We propose that the ancestral peptide gene gave rise to two additional copies in the genome doublings. The genes encoding POMC and PNOC are located on the same chromosome in the chicken genome and all three teleost genomes that we have studied, zebrafish, medaka and stickleback. This implies that the fourth member of the peptide family was generated by a local

40 gene duplication. A translocation has disrupted this synteny in mammals. Our results show that chromosomal rearrangements in these regions have been frequent. As an example, genes on chicken chromosome 3, which har- bors the gene for the mu receptor, POMC precursor and PNOC precursor, are represented on three different human chromosomes (chromosomes 6, 2 and 8). The PDYN gene seems to have been lost in chicken, but not in zebra finch, duck or turkey. Duplicates of some peptide genes have arisen in the teleost fishes through 3R, both duplicates have been retained for the POMC and PNOC genes in all three species, while the PENK gene exists in duplicates only in the zebrafish genome. Interestingly, the ancestral peptide and receptor genes were located on the same chromosome and were duplicated concomitantly. This suggests that there may once have been a functional reason for the genetic linkage, such as coexpression and/or coevolution. However, subsequently genetic linkage has been lost and receptor-ligand preferences have evolved. Thus the system of opioid peptides and receptors reached it present complexity more than 420/450 million years ago.

Paper VI, VII and VIII There are four Hox-clusters in tetrapods, seven to eight of them in teleost fishes and only one in cephalochordates. This phenomenon was one of the first arguments for the whole genome doublings early in vertebrate evolution and among teleost fishes. We phylogenetically analyzed gene 14 families (Paper VI) in the vicinity of the Hox genes in order to further characterize the chromosomal regions harboring Hox clusters in human, mouse, zebrafish, fugu and tetraodon. All except one displayed a tree topology that shows expansion in the vertebrate lineage after the split from urochordates and cephalochordates. The chromosomal location of the genes in the human and mouse genomes shows conserved synteny and fourfold paralogy. The three teleost fishes studied had duplicated genes in several of the gene families investigated, and the three topologies suggest that these originated in the teleost whole genome duplication. In humans a part of chromosome 3 reflects an ancient mammalian translocation from what is now human HoxA-bearing chromosome 7. A com- bined analysis using phylogenetic and chromosomal information for the sodium channel (SCN) alpha subunit gene family and five of the neighboring gene families supports this translocation (PaperVII). In-depth studies of two gene families located near the Hox-clusters, SCN alpha family and the insulin-like growth factor binding protein (IGFBP) family (Paper VIII) supported genome duplication as one of the mechanisms responsible for forming the Hox-paralogon. The studies also highlight the role of local duplications in the generation of gene families. A pair of genes

41 in the IGFBP family was present in the ancestral chordate before the whole genome duplications. Two losses have then occurred on different chromosomes, resulting in two chromosomes harboring a pair of IGFBP genes next to each other and two chromosomes with single IGFBP genes. These genes have then been duplicated in the teleost fish whole genome duplication followed by differential losses in the different teleost species investigated. Different types of duplication mechanisms have contributed in the expansion of the SCN alpha gene family in tetrapods and teleost fishes. The two whole genome duplications generated four genes in the ancestral gnathostome, these were then doubled in the teleost whole genome duplication, resulting in the present set up of eight genes. In the tetrapod lineage local duplications have played the major part in generating SCN alpha genes. A quartet was formed after local duplications of one gene, followed by one additional duplication in eutherian mammals, resulting in a quintet. Among amniotes, another one of the genes was locally duplicated twice, resulting in a triplet. All these duplications resulted in a set up of ten genes in eutherian mammals and nine genes in chicken. The analyses in both Paper VII and Paper VIII have shown that with care- ful inspection of sequence alignments and inclusion of wide selection of species, representing many branches in the vertebrate tree, it is possible to reconcile topologies of phylogenetic trees and chromosomal location of genes. This helps to delineate the evolution of gene families, including identification of true orthologs.

42 Conclusions and perspectives

The investigations that this thesis is based on started in 2004 and 2005. At that time the proposition that two whole genome duplications had occurred early in vertebrate evolution was not firmly established within the field. Fol- lowing the sequencing of a broad range of vertebrate genomes, large scale studies using whole genome data from these vertebrates were conducted (Dehal and Boore, 2005; Kasahara et al., 2007; Nakatani et al., 2007) and provided strong argumentation in favor of this theory. After the publication of the whole genome sequence of the cephalochordate amphioxus it is now proven that two whole genome duplications (2R) occurred in vertebrate evolution (Putnam et al., 2008). This thesis highlights the impact of these genome duplications in vertebrate evolution through the expansion of many gene families, including both the peptides and receptors for neuropeptide Y- and opioid systems. Paper I and Paper VI were written before the discussion about 2R was settled and the results of Paper VI (and to some extent also Paper I) are therefore part of the argument for the 2R and 3R theory. In Papers IV, V, VII and VIII these large-scale events provide the framework for the elucidation of the evolutionary history of the opioid receptor and peptide families, the insulin-like growth factor binding protein (IGFBP) family and the sodium channel alpha subunit (SCN) family. Within this framework, information on the chromosomal location of genes makes it possible to delineate the evolutionary history of gene families with inconclusive phylogenetic tree topologies. Both the IGFBP and SCN alpha gene families are such families where the evolutionary history has been difficult to resolve and conflicting hypothesis have been proposed, even though they are located within the same chromosomal regions as the Hox-clusters in many cases. The discovery that lineage-specific losses and duplications have contributed greatly to gene family diversity, highlights the need for a wide tax- onomic representation in the studies. The SCN alpha family is a striking example of how a similar number of genes in different lineages can have originated through different mechanisms, obscuring orthology relationships. The eight genes in teleost fishes are the result of in total three whole genome duplications, while local duplications have had a more prominent role in the expansion of the gene family in the tetrapod lineage.

43 Even if the chromosomal location is sometimes able to resolve the ambi- guities seen in the tree topologies, it is important to remember that translocations have been frequent and differ between species. The usefulness of non- mammalian genomes is shown several times in this thesis, not least in trac- ing translocations and to reconstruct ancestral diversities and arrangements of genes. The proorphanin and proopiomelanocortin genes have been cloned in a broad range of species, but before information was available about the chromosomal location of these genes in the chicken and teleost fish genomes, there was no indication that they were the result of a local gene duplication. Even if these genes belong to the same gene family it is difficult to make informative phylogenetic analyses due to the insertion of melanocortin sequences in the POMC precursor. It has also been difficult to produce reliable phylogenetic trees for the NPY peptide family because the precursor sequences have too few informative sites. Fortunately, by using information on chromosomal location and by making phylogenetic trees for neighboring gene families it was possible to identify the different peptides as either NPY or PYY. The genome duplications concomitantly increased the receptor and peptide repertoire and thereby provided prerequisites for coevolution. The kind of innovations that new genes can give rise to is exemplified in the functional studies of the NPY system in zebrafish and the frog Silurana tropicalis in Papers II and III. The 3R duplicates of PYY in zebrafish, PYYa and PYYb differ only in a few amino acids and the majority of these are located in a region that has not been shown to be important for binding to the receptor. Nevertheless, it has been possible to identify differences in affinity to the receptors, especially the Y2 receptor, for the peptide ligands. Comparative sequence information from receptors and peptides that shows great differences in affinity will be helpful for mutagenesis studies of the human NPY receptor subtypes and in computer modeling of the interaction between receptors and ligands. The differences between genes belonging to the same family can also be illustrated by analyses of the tissue distribution. One example from the frog Silurana tropicalis is NPY. It is the only peptide with detectable neuronal mRNA expression in our analysis. The whole genome duplications have not only contributed to the complexity of the neuropeptide Y and opioid systems, it has been reported that several other receptor-ligand systems have expanded in this time period such as endothelin-, secretin-, and tachykinin systems (Braasch et al., 2009; Conlon and Larhammar, 2005; Hyndman et al., 2009; Roch et al., 2009; Vaudry et al., 2009). The genome duplications also seem to have been important in the formation of components in signal cascades for specific neuro- logical systems, such as the visual signal transduction (Larhammar et al., 2009; Nordstrom et al., 2004). This works shows that sequencing of whole genomes from a broad range of vertebrates makes it possible to use a comparative approach and analyze

44 several lineages among chordates both regarding evolution and function. This approach is helpful to elucidate co-evolution (in time) of receptors and their ligands. Studies of how the binding properties differ in various species help determine important amino acids that are involved in the interactions between receptors and peptides. It becomes possible to delineate the evolution of gene families in different lineages, with losses and duplications, and also to see traces of more global genomic events such as whole genome duplications.

45 Svensk sammanfattning (Swedish summary)

Alla celler i en organism innehåller samma uppsättning DNA, denna upp- sättning kallas för ett genom och i genomet finns alla gener. Man har se- kvenserat, dvs läst av, hela genomet för många ryggradsdjur, inklusive män- niskan. När man jämför genom mellan olika arter kan man se hur mycket som är lika och hur mycket som skiljer mellan dem. Att mutationer är grundläggande i evolutionen är välkänt. När en mutation inträffar i en gen, leder det ibland till förändring i proteinet som genen kodar för och detta kan leda till att proteinet får en ny funktion. Beroende på var och hur förändringen sker så kan den gamla funktionen försvinna. Man vinner alltså en ny funktion till priset av den gamla. Detta behöver dock inte vara fallet om genen duplicerats först. Om en gen dupliceras och sedan en av kopiorna muteras och förändrar proteinet så kan man både äta kakan och ha den kvar. En av kopiorna behåller den gamla funktionen och den andra kan få en ny. Den nya funktionen kan till exempel vara att proteinet bildas vid en annan tidpunkt eller i en annan vävnad. Det kan också innebära att en helt ny funktion tillkommer. Genom duplikationer formas genfamiljer, familjer av gener som kodar för protein som liknar varandra till egenskaper och funktion. Hur lika de är beror på hur länge sedan duplikationerna skedde. Gener kan dupliceras på olika sätt, antingen en i taget eller alla gener i en organism på samma gång. Det sistnämnda kallas för genomduplikation. Ge- nomduplikationer är t ex vanliga hos många växter. Man har länge haft en hypotes att två genomduplikationer skedde tidigt i ryggradsdjurens evolution. Det har bland annat uppmärksammats att om en gen finns i en kopia i ett ryggradslöst djur så finns den ofta i flera kopior hos ett ryggradsdjur. En gen har då genom duplikationer blivit en genfamilj. Flera genfamiljer är undersökta i denna avhandling där man just ser detta mönster. Det är argument för att genomduplikationer har skett hos ryggradsdjuren. Andra forskare har gjort storskaliga undersökningar av hela genom och sett samma sak, hela genomet har duplicerats två gånger hos ryggradsdjur. Äkta benfiskar har dessutom genomgått ytterligare en genomduplikation. Efter duplikationen har skett är det vanligaste att det sker så mycket mutationer att en av kopiorna förstörs, det går inte att få fram något användbart protein från genen. Det är det huvudsakliga skälet till att vi som människor inte har fyra gånger fler gener än insekter och att en zebrafisk inte har dub-

46 belt så många gener som en människa trots ovan nämnda duplikationer. Men vid vissa tillfällen bevaras kopian, den får nya funktioner och nya medlemmar i en genfamilj skapas. Många signalsystem i vår kropp består av flera mottagare (receptorer) och flera signalmolekyler (ligander). Liganderna kan vara små molekyler eller proteiner, peptider. Liganderna binder till en receptor som sedan sätter igång en händelskedja som leder till att signalen förs vidare. Receptorerna ingår i en genfamilj och peptiderna i en annan. Ett exempel på dessa system är neu- ropeptid Y (NPY) -systemet och ett annat är det opioida systemet. I den här avhandlingen beskrivs evolutionen av både receptorer och peptider i NPY-systemet och det opioida systemet. Genom att undersöka generna för receptorerna, peptiderna och de gener som ligger intill dessa på samma kromosomer kan man se att de alla har fått fler medlemmar vid samma tidpunkt. Att händelserna verkar ha skett samtidigt är ett starkt argument för att duplikationerna som gav de nya medlemmarna skedde i hela genomet samtidigt. Både NPY och opioidsystemet har alltså fått fler medlemmar på grund av genomduplikationerna. Med många medlemmar i familjerna finns det fler sätt som de kan interagera på än om det bara fanns en receptor och en peptid. NPY-systemet är bland annat inblandat i födointag. Vissa receptorer sig- nalerar hunger och vissa mättnad. Både receptorerna och peptiderna finns i alla ryggradsdjur. Även om det stora mönstret är detsamma finns det varia- tioner beroende på vilken art man studerar. NPY-systemet består av fyra olika sorters receptorer och tre typer av peptider i människa. I en av artik- larna i denna avhandling studerades vilka receptorer som finns i groda. Sex receptortyper kunde identifieras tillsammans med tre olika sorters peptider. För att se om det var någon skillnad i funktion mellan grodan och andra djur undersökte vi också vilka peptider som binder bäst till vilka receptorer. Det var bara möjligt att undersöka tre av de sex receptorerna och där kom vi fram till att en av peptiderna, PP, i stort sett bara binder till en av receptorerna, om man studerar koncentrationer av liganden som liknar de som finns i kroppen. Vi har också studerat systemet i zebrafisk, som har sex receptorer och tre peptider. De sex receptorerna och tre peptiderna motsvarar inte rakt av de som finns i groda då familjerna har utökats vid olika tillfällen och på olika sätt efter de tidiga genomduplikationerna, både genom ytterligare genomduplikationer och genom duplicering av enskilda gener. Vi studerade vilken receptor som en av peptiderna, PYYb, band bäst till. Denna peptid visade sig ha en favoritreceptor, kallad Y2. För både groda och zebrafisk studerade vi i vilka vävnader i kroppen som generna för receptorer och peptider uttrycks. Det fanns några skillnader: i groda kunde vi bara se uttryck i nervsystemet för en av de tre peptiderna, och för två av de sex receptorerna. För zebrafisk var det bara en av peptiderna som uttrycks i mag-tarmsystemet.

47 På samma kromosomer som generna för NPY-peptiderna finns en grupp av gener som kallas Hox-gener. Dessa gener är viktiga för organismernas fosterutveckling och har studerats i många år. Äkta benfiskar har sju till åtta olika grupper av Hox-gener på olika kromosomer och de övriga ryggradsdjuren har fyra grupper. Det är precis som det borde vara om genomet har duplicerats två gånger tidigt i ryggradsdjurens evolution och ytterligare en gång hos äkta benfiskar. Man har studerat hur Hox-generna är organiserade i lan- cettfiskar (ett ryggsträngsdjur som är nära släkt med ryggradsdjuren). Lan- cettfiskar har bara en grupp av Hox-gener och man kan därför datera genomduplikationerna till att ha skett efter lancettfiskarna skildes från de rygg- strängsdjur som utvecklades till ryggradsdjur. Förutom generna för NPY-peptiderna ligger det många andra genfamiljer på samma kromosomer som Hox-generna. Två av dessa är studerade i detalj i denna avhandling. En av familjerna kallas IGFBP och den andra SCN. Dessa familjer har fått nya medlemmar genom både lokala duplikationer och genomduplikationer. Det finns åtta medlemmar i SCN-familjen hos äkta benfiskar och tio medlemmar i samma familj hos däggdjur. I de äkta benfis- karna har medlemmarna uppkommit genom genomduplikationerna. Hos däggdjur är fyra av de tio medlemmarna resultatet av genomduplikationerna och de övriga sex kommer från enskilda duplikationer. Detta är ett bra exempel på att evolutionen av genfamiljer ser olika ut i olika arter. Sammanfattningsvis visar denna avhandling hur viktiga genomduplikationerna var för uppkomsten av de genfamiljer som vi har idag. Den visar också att olika gener bevaras och förloras i olika arter vilket leder till funktionsskillnader. Om man jämför många arter som är mer och mindre släkt med varandra kan man få en mer fullständig bild av vad som har skett och hur det har skett.

48 Acknowledgements

Tack till alla som medvetet och omedvetet har hjälpt till med denna avhandling.

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Dan, det är skönt att som doktorand veta att om man träffar på en konstig gen någonstans så är det nästan säkert att du vet vad den gör och om den ligger i ett paralogon (såklart). Tomas, för allt du kan, alla ”det borde jag hinna kolla på” och ”det tar bara 45 minuter”. Daniel, för den moderna re- nässansmänniska du är – en outsinlig kunskapsbas. Susanne, för att du ald- rig blev nedslagen när du var tvungen att göra om analyserna för femtioelfte gången. Jenny, för att din petighet lönar sig och för att du liksom jag vet nyttan av ett par raggsockor när man håller på med bioinformatik. Finn, Ludde och Robert O för all paralogonkunskap. Ingrid, för allt du vet om cellodling och bindning (och trerätters picknickar). Christina, för att du vet så mycket mer än att saker ligger på andra hyllan till vänster. Bo and Johan H for all the binding experiments and good company. Hille, för din osed(van)liga associationsförmåga. Helena, för att jobbet blir roligt när du säger vad du tänker. Hanna-Linn, för gott sällskap och alkoholprojektet. Frida, för första sommaren med ett fragmenterat fugugenom. Chus och David, för kunskap om och intresse för andra vertebrater än däggdjur. Lisa, Marie och Maddis för trevliga diskussioner om tokiga katter och annat. Marita och Birgitta, för all hjälp med praktiska saker och allt korsordslö- sande. Ulla, Lena, Emma och Maria L som har koll på viktiga saker som inte är forskning. Alla de som förgyllt både undervisning, forskning och luncher; Lars O, Robert F, Karin, Svante, Jossan, Hanna, Maria H, To- bias, Kalle, Malin, Josefin, Tatjana, Markus, Johan A, Bengt, Ola, To- run, Paula, Camilla och Niklas. Alla på BMC neuro och de projektstuden- ter som passerat i gruppen, tack för hjälp och trevligt sällskap.

Maria, Elisabeth och Stina, för ovärdeliga diskussioner om livet som doktorand. Jörgen, Karin och Viktoria för trevligt sällskap, inte minst i väntan på nationshamburgare. Maria B för bokcirkeln och teaterbesök.

Mamma och Pappa

Systrar med familjer; Gudrun, Gerd, Lars, Axel, Pelle, Magnus och Tove.

Anders och Sverker som gör att livet är så jädra roligt.

50 References

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