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The acquisition of movable jaws was a major event during vertebrate evolution. The role of NK3 homeobox 2 (Nkx3.2) transcription factor in patterning the primary jaw joint of gnathostomes (jawed vertebrates) is well known, however knowledge about its regulatory mechanism is lacking. In this study, we report a proximal enhancer element of Nkx3.2 that is deeply conserved in gnathostomes but undetectable in the jawless hagfish. This enhancer is active in the developing jaw joint region of the zebrafish Danio rerio, and was thus designated as jaw joint regulatory sequence 1 (JRS1). We further show that JRS1 enhancer sequences from a range of gnathostome species, including a chondrichthyan and mammals, have the same activity in the jaw joint as the native zebrafish enhancer, indicating a high degree of functional conservation despite the divergence of cartilaginous and bony fish lineages or the transition of the primary jaw joint into the middle ear of mammals. Finally, we show that deletion of JRS1 from the zebrafish genome using CRISPR/Cas9 leads to a transient jaw joint deformation and partial fusion. Emergence of this Nkx3.2 enhancer in early gnathostomes may have contributed to the origin and shaping of the articulating surfaces of vertebrate jaws.

The acquisition of movable jaws was a major event during vertebrate evolution. The role of NK3 homeobox 2 (Nkx3.2) transcription factor in patterning the primary jaw joint of gnathostomes (jawed vertebrates) is well known, however knowledge about its regulatory mechanism is lacking. In this study, we report a proximal enhancer element ofiNkx3.2/ithat is deeply conserved in most gnathostomes but undetectable in the jawless hagfish and lamprey. This enhancer is active in the developing jaw joint region of the zebrafishiDanio rerio/i, and was thus designated asijaw joint regulatory sequence 1/i(JRS1). We further show that JRS1 enhancer sequences from a range of gnathostome species, including a chondrichthyan and mammals, have the same activity in the jaw joint as the native zebrafish enhancer, indicating a high degree of functional conservation despite the divergence of cartilaginous and bony fish lineages or the transition of the primary jaw joint into the middle ear of mammals. Finally, we show that deletion of JRS1 from the zebrafish genome using CRISPR/Cas9 results in a significant reduction of early gene expression ofinkx3.2/iand leads to a transient jaw joint deformation and partial fusion. Emergence of thisiNkx3.2/ienhancer in early gnathostomes may have contributed to the origin and shaping of the articulating surfaces of vertebrate jaws.

The transcription factor Nkx3.2 (Bapx1) is an important chondrocyte maturation inhibitor. PreviousNkx3.2knock-down and overexpression studies in non-mammalian gnathostomes have focused on its role in primary jaw joint development, while little is known about the function of this gene in broader skeletal development. We generated CRISPR/Cas9 knockout ofnkx3.2in zebrafish and applied a range of techniques to characterize skeletal phenotypes at developmental stages from larva to adult, revealing fusions in bones of the occiput, the loss or deformation of bony elements derived from basiventral cartilages of the vertebrae, and an increased length of the proximal radials of the dorsal and anal fins. These phenotypes are reminiscent ofNkx3.2knockout phenotypes in mammals, suggesting that the function of this gene in axial skeletal development is ancestral to osteichthyans. Our results highlight the broad role ofnkx3.2in zebrafish skeletal development and its context-specific functions in different skeletal elements.

The transcription factor Nkx3.2 (Bapx1) is an important chondrocyte maturation inhibitor. Previous Nkx3.2 knockdown and overexpression studies in non-mammalian gnathostomes have focused on its role in primary jaw joint development, while the function of this gene in broader skeletal development is not fully described. We generated a mutant allele of nkx3.2 in zebrafish with CRISPR/Cas9 and applied a range of techniques to characterize skeletal phenotypes at developmental stages from larva to adult, revealing loss of the jaw joint, fusions in bones of the occiput, morphological changes in the Weberian apparatus, and the loss or deformation of bony elements derived from basiventral cartilages of the vertebrae. Axial phenotypes are reminiscent of Nkx3.2 knockout in mammals, suggesting that the function of this gene in axial skeletal development is ancestral to osteichthyans. Our results highlight the broad role of nkx3.2 in zebrafish skeletal development and its context-specific functions in different skeletal elements.

Glycosaminoglycans are sulfated polysaccharide molecules, essential for many biological processes. The 6-O sulfation of glycosaminoglycans is carried out by carbohydrate 6-O sulfotransferases (C6OST), previously named Gal/GalNAc/GlcNAc 6-O sulfotransferases. Here for the first time we present a detailed phylogenetic reconstruction, analysis of gene synteny conservation and propose an evolutionary scenario for the C6OST family in major vertebrate groups, including mammals, birds, non-avian reptiles, amphibians, lobe-finned fishes, ray-finned fishes, cartilaginous fishes and jawless vertebrates. The C6OST gene expansion likely occurred in the chordate lineage, after the divergence of tunicates and before the emergence of extant vertebrate lineages.The two rounds of whole genome duplication in early vertebrate evolution (1R/2R) only contributed two additional C6OST subtype genes, increasing the vertebrate repertoire from four genes to six, divided into two branches. The first branch includes CHST1 and CHST3 as well as a previously unrecognized subtype, CHST16, that was lost in amniotes. The second branch includes CHST2, CHST7 and CHST5. Subsequently, local duplications of CHST5 gave rise to CHST4 in the ancestor of tetrapods, and to CHST6 in the ancestor of primates.The teleost specific gene duplicates were identified for CHST1, CHST2 and CHST3 and are result of whole genome duplication (3R) in the teleost lineage. We could also detect multiple, more recent lineage-specific duplicates. Thus, the repertoire of C6OST genes in vertebrate species has been shaped by different events at several stages during vertebrate evolution, with implications for the evolution of the skeleton, nervous system and cell-cell interactions.

In order to characterize the molecular bases of mineralizing cell evolution, we targeted type X collagen, a nonfibrillar network forming collagen encoded by the Col10a1 gene. It is involved in the process of endochondral ossification in ray-finned fishes and tetrapods (Osteichthyes), but until now unknown in cartilaginous fishes (Chondrichthyes). We show that holocephalans and elasmobranchs have respectively five and six tandemly duplicated Col10a1 gene copies that display conserved genomic synteny with osteichthyan Col10a1 genes. All Col10a1 genes in the catshark Scyliorhinus canicula are expressed in ameloblasts and/or odontoblasts of teeth and scales, during the stages of extracellular matrix protein secretion and mineralization. Only one duplicate is expressed in the endoskeletal (vertebral) mineralizing tissues. We also show that the expression of type X collagen is present in teeth of two osteichthyans, the zebrafish Danio rerio and the western clawed frog Xenopus tropicalis, indicating an ancestral jawed vertebrate involvement of type X collagen in odontode formation. Our findings push the origin of Col10a1 gene prior to the divergence of osteichthyans and chondrichthyans, and demonstrate its ancestral association with mineralization of both the odontode skeleton and the endoskeleton.

G protein-coupled receptors (GPCRs) are a class of integral membrane proteins mediating intercellular interactions of fundamental physiological importance for survival including regulation of food intake, blood pressure, and hormonal sensing signaling, among other roles. Homeostatic alterations in the physiological status of GPCRs are often associated with underlying causes of disease, and to date, several orphan GPCRs are still uncharacterized.Findings from our previous study demonstrate that the Rhodopsin family protein GPR162 is widely expressed in GABAergic as well as other neurons within the mouse hippocampus, whereas extensive expression is observed in hypothalamus, amygdala, and ventral tegmental area, regions strictly interconnected and involved in the regulation of energy homeostasis and hedonic feeding.In this study, we provide a further anatomical characterization of GPR162 in mouse brain via in situ hybridization as well as detailed mRNA expression in a panel of rat tissues complementing a specie-specific mapping of the receptor. We also provide an attempt to demonstrate a functional implication of GPR162 in food intake-related behavior via antisense knockdown studies. Furthermore, we performed human genetic studies in which for the first time, variants of the GPR162 gene were associated with impairments in glucose homeostasis. Fil: Caruso, Vanni. Uppsala University. Department Of Neuroscience. Functional Pharmacology; Suecia. University of Tasmania; Australia Fil: Sheedharan, Smitha. Uppsala University. Department Of Neuroscience. Functional Pharmacology; Suecia Fil: Carlini, Valeria Paola. Uppsala University. Department Of Neuroscience. Functional Pharmacology; Suecia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Jacobsson, Josefin A.. Uppsala University. Department Of Neuroscience. Functional Pharmacology; Suecia Fil: Haitina, Tatjana. Uppsala University. Department Of Neuroscience. Functional Pharmacology; Suecia Fil: Hammer, Joanna. Uppsala University. Department Of Neuroscience. Functional Pharmacology; Suecia Fil: Stephansson, Olga. Uppsala University. Department Of Neuroscience. Functional Pharmacology; Suecia Fil: Crona, Filip. Uppsala University. Department Of Neuroscience. Functional Pharmacology; Suecia Fil: Sommer, Wolfgang. Central Institute of Mental Health. Department of Psychopharmacology; Alemania Fil: Risérus, Ulf. Uppsala University. Clinical Nutrition and Metabolism. Department of Public Health and Caring Sciences; Suecia Fil: Lannfelt, Lars. Department Of Neuroscience, Functional Pharmacology; Suecia. Uppsala University. Department of Public Health and Caring Sciences, Geriatrics; Suecia Fil: Marcus, Claude. Department Of Neuroscience, Functional Pharmacology; Suecia. Karolinska Institute. National Childhood Obesity Centre. Department for Clinical Science, Intervention and Technology. Division of Pediatrics; Suecia Fil: Heiling, Markus. National Institutes of Health. National Institute on Alcohol Abuse and Alcoholism. Laboratory of Clinical and Translational Studies; Estados Unidos Fil: Rubiales, Susana Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Farmacología Experimental de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Farmacología Experimental de Córdoba; Argentina Fil: Fredriksson, Robert. Uppsala University. Unit of Functional Pharmacology. Department of Neuroscience ; Suecia Fil: Schiöth, Helgi B.. Uppsala University. Unit of Functional Pharmacology. Department of Neuroscience ; Suecia

International audience; There are two types of dermal skeletons in jawed vertebrates: placoderms and osteichthyans carry large bony plates (macromery), whereas chondrichthyans and acanthodians are covered by small scales (micromery). Fin spines are one of the last large dermal structures found on micromeric taxa and offer a potential source of histology and morphology that can be compared to those found on macromeric groups. Dermal fin spines offer a variety of morphology but aspects of their growth modes and homology are unclear. Here, we provide detailed descriptions of the microstructure and growth of a dorsal ridge spine from the acanthothoracid placoderm, Romundina stellina, using virtual three-dimensional paleohistological datasets. From these data we identify several layers of dentine ornamentation covering the lateral surfaces of the spine and reconstructed their growth pattern. We show that this spine likely grew posteriorly and proximally from a narrow portion of bone located along the leading edge of the spine. The spine is similarly constructed to the scales with a few exceptions, including the absence of polarized fibers distributed throughout the bone and the presence of a thin layer of perichondral bone. The composition of the spine (semidentine odontodes, dermal bone, perichondral bone) is identical to that of the Romundina dermal plates. These results illustrate the similarities and differences between the dermal tissues in Romundina and indicate that the spine grew differently from the dentinous fin spines from extant and fossil chondrichthyans. The morphology and histology of Romundina is most similar to the fin spine of the probable stem osteichthyan Lophosteus, with a well-developed inner cellular bony base and star-shaped odontodes on the surface. Results from these studies will undoubtedly have impact on our understanding of fossil fin spine histology and evolution, contributing to the on-going revision of early gnathostome phylogeny.

Background: Chondroitin/dermatan sulfate (CS/DS) proteoglycans present in the extracellular matrix have important structural and regulatory functions. Results: Six human genes have previously been shown to catalyze CS/DS polymerization. Here we show that one of these genes, chpf, is represented by two copies in the zebrafish genome, chpfa and chpfb, while the other five human CS/DS glycosyltransferases csgalnact1, csgalnact2, chpf2, chsy1, and chsy3 all have single zebrafish orthologues. The putative zebrafish CS/DS glycosyltransferases are spatially and temporally expressed. Interestingly, overlapping expression of multiple glycosyltransferases coincides with high CS/DS deposition. Finally, whereas the relative levels of the related polysaccharide HS reach steady-state at around 2 days post fertilization, there is a continued relative increase of the CS amounts per larvae during the first 6 days of development, matching the increased cartilage formation. Conclusions: There are 7 CS/DS glycosyltransferases in zebrafish, which, based on homology, can be divided into the CSGALNACT, CHSY, and CHPF families. The overlap between intense CS/DS production and the expression of multiple CS/DS glycosyltransferases suggests that efficient CS/DS biosynthesis requires a combination of several glycosyltransferases. Developmental Dynamics 242:964–975, 2013. © 2013 Wiley Periodicals, Inc.

Transmembrane proteins are gatekeepers of the cells; controlling the transport of substrates as well as communicating signals among cells and between the organelles and cytosol. Solute carriers (SLC) and G protein-coupled receptors (GPCR) are the largest family of membrane transporters and membrane receptors respectively. The overall aim of this thesis was to provide a basic understanding of some of the novel SLCs and GPCRs with emphasis on expression, transport property, evolution and probable function. The first part of the thesis directs towards the study of some novel solute carriers. In an initial study, we provided an overall picture of the sequence relationship and tissue expression of 14 diverse atypical SLCs confirming some of their evolutionary conservation and highly specific expression pattern. The focus then was on the SLC17 family (mainly vesicular proteins) and a novel member named Slc17a9. This study revealed that SLC17 family could be divided into four main phylogenetic clades which were all present before the divergence of the insect lineage with Slc17a9 having the most restricted evolutionary history. Detailed expression study of Slc17a9 in the mouse brain suggests that it is also expressed in some regions important for purinergic neurotransmission. Further, we deorphanised an aminoacid transporter Slc38a7 which was expressed in a majority of neurons in the CNS and showed that it preferably mediate transport of L–glutamine and L–histidine. The second part of the thesis focuses on the study of two GPCRs belonging to the Rhodopsin superfamily, Gpr162 and Gpr153. A phylogenetic analysis revealed that both Gpr153 and Gpr162 originated from a common ancestor before the radiation of the mammalian lineage. Expression study revealed that Gpr162 had a predominant expression in the CNS and relatively lower expression in the other tissue tested whereas Gpr153 had a more widespread and similar expression pattern in both CNS and peripheral tissues. The functional studies of the two GPCRs were done using the antisense oligodeoxynucleotide knockdown rat model. These studies provided evidence linking the orphan Gpr162 gene with the regulation of food intake– related behaviour whereas Gpr153 gene caused only a slight reduction in food intake.

We have characterized the biochemical function of the melanocortin 1 receptor (MC1R), a critical regulator of melanin synthesis, from 9 phylogenetically diverse primate species with varying coat colors. There is substantial diversity in melanocyte-stimulating hormone (MSH) binding affinity and basal levels of activity in the cloned MC1Rs. MSH binding was lost independently in lemur and New World monkey lineages, whereas high basal levels of MC1R activity occur in lemurs and some New World monkeys and Old World monkeys. Highest levels of basal activity were found in the MC1R of ruffed lemurs, which have the E94K mutation that leads to constitutive activation in other species. In 3 species (2 lemurs and the howler monkey), we report the novel finding that binding and inhibition of MC1R by agouti signaling protein (ASIP) can occur when MSH binding has been lost, thus enabling continuing regulation of the melanin type via ASIP expression. Together, these findings can explain the previous paradox of a predominantly pheomelanic coat in the red ruffed lemur (Varecia rubra). The presence of a functional, MSH-responsive MC1R in orangutan demonstrates that the mechanism of red hair generation in this ape is different from the prevalent mechanism in European human populations. Overall, we have found unexpected diversity in MC1R function among primates and show that the evolution of the regulatory control of MC1R activity occurs by independent variation of 3 distinct mechanisms: basal MC1R activity, MSH binding and activation, and ASIP binding and inhibition. This diversity of function is broadly associated with primate phylogeny and does not have a simple relation to coat color phenotype within primate clades.

The Adhesion family of G protein-coupled receptors (GPCRs) includes 33 receptors and is the second largest GPCR family. Most of these proteins are still orphans and fairly little is known of their tissue distribution and evolutionary context. We report the evolutionary history of the Adhesion family protein GPR123 as well as mapping of GPR123 mRNA expression in mouse and rat using in situ hybridization and real-time PCR, respectively. GPR123 was found to be well conserved within the vertebrate lineage, especially within the transmembrane regions and in the distal part of the cytoplasmic tail, containing a potential PDZ binding domain. The real-time PCR data indicates that GPR123 is predominantly expressed in CNS. The in situ data show high expression in thalamic nuclei and regions containing large pyramidal cells like cortex layers 5 and 6 and subiculum. Moreover, we found distinct expression in amygdala, hypothalamus, inferior olive and spinal cord. The CNS specific expression, together with the high sequence conservation between the vertebrate sequences investigated, indicate that GPR123 may have an important role in the regulation of neuronal signal transduction.

Enamel, the hardest vertebrate tissue, covers the teeth of almost all sarcopterygians (lobe-finned bony fishes and tetrapods) as well as the scales and dermal bones of many fossil lobe-fins. Enamel deposition requires an organic matrix containing the unique enamel matrix proteins (EMPs) amelogenin (AMEL), enamelin (ENAM) and ameloblastin (AMBN). Chondrichthyans (cartilaginous fishes) lack both enamel and EMP genes. Many fossil and a few living non-teleost actinopterygians (ray-finned bony fishes) such as the gar, Lepisosteus, have scales and dermal bones covered with a proposed enamel homologue called ganoine. However, no gene or transcript data for EMPs have been described from actinopterygians. Here we show that Psarolepis romeri, a bony fish from the the Early Devonian period, combines enamel-covered dermal odontodes on scales and skull bones with teeth of naked dentine, and that Lepisosteus oculatus (the spotted gar) has enam and ambn genes that are expressed in the skin, probably associated with ganoine formation. The genetic evidence strengthens the hypothesis that ganoine is homologous with enamel. The fossil evidence, further supported by the Silurian bony fish Andreolepis, which has enamel-covered scales but teeth and odontodes on its dermal bones made of naked dentine, indicates that this tissue originated on the dermal skeleton, probably on the scales. It subsequently underwent heterotopic expansion across two highly conserved patterning boundaries (scales/head-shoulder and dermal/oral) within the odontode skeleton.

Chondroitin/dermatan sulfate (CS/DS) proteoglycans consist of unbranched sulfated polysaccharide chains of repeating GalNAc-GlcA/IdoA disaccharide units, attached to serine residues on specific proteins. The CS/DS proteoglycans are abundant in the extracellular matrix where they have essential functions in tissue development and homeostasis. In this report a phylogenetic analysis of vertebrate genes coding for the enzymes that modify CS/DS is presented. We identify single orthologous genes in the zebrafish genome for the sulfotransferases chst7, chst11, chst13, chst14, chst15 and ust and the epimerase dse. In contrast, two copies were found for mammalian sulfotransferases CHST3 and CHST12 and the epimerase DSEL, named chst3a and chst3b, chst12a and chst12b, dsela and dselb, respectively. Expression of CS/DS modification enzymes is spatially and temporally regulated with a large variation between different genes. We found that CS/DS 4-O-sulfotransferases and 6-O-sulfotransferases as well as CS/DS epimerases show a strong and partly overlapping expression, whereas the expression is restricted for enzymes with ability to synthesize di-sulfated disaccharides. A structural analysis further showed that CS/DS sulfation increases during embryonic development mainly due to synthesis of 4-O-sulfated GalNAc while the proportion of 6-O-sulfated GalNAc increases in later developmental stages. Di-sulfated GalNAc synthesized by Chst15 and 2-O-sulfated GlcA/IdoA synthesized by Ust are rare, in accordance with the restricted expression of these enzymes. We also compared CS/DS composition with that of heparan sulfate (HS). Notably, CS/DS biosynthesis in early zebrafish development is more dynamic than HS biosynthesis. Furthermore, HS contains disaccharides with more than one sulfate group, which are virtually absent in CS/DS.

Heparan sulfate (HS) proteoglycans are ubiquitous components of the extracellular matrix and plasma membrane of metazoans. The sulfation pattern of the HS glycosaminoglycan chain is characteristic for each tissue and changes during development. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes catalyze N-deacetylation and N-sulfation during HS biosynthesis and have a key role in designing the sulfation pattern. We here report on the presence of five NDST genes in zebrafish. Zebrafish ndst1a, ndst1b, ndst2a and ndst2b represent duplicated mammalian orthologues of NDST1 and NDST2 that arose through teleost specific genome duplication. Interestingly, the single zebrafish orthologue ndst3, is equally similar to tetrapod Ndst3 and Ndst4. It is likely that a local duplication in the common ancestor of lobe-finned fish and tetrapods gave rise to these two genes. All zebrafish Ndst genes showed distinct but partially overlapping expression patterns during embryonic development. Morpholino knockdown of ndst1b resulted in delayed development, craniofacial cartilage abnormalities, shortened body and pectoral fin length, resembling some of the features of the Ndst1 mouse knockout.

We have cloned melanocortin receptors (MCRs) from several species of fish. The MC4R and MC5R subtypes arose early in vertebrate evolution and their primary structure is remarkably conserved. Expression and pharmacological characterization of the MCRs in fish has revealed that they bind and respond to melanocortin peptides with high potency. Detailed characterization of the binding properties of the different subtypes suggests that MCRs in early vertebrates had preference for adrenocorticotropic hormone (ACTH) peptides, while the high sensitivity for the shorter proopiomelanocortin (POMC) products, such as the alpha-, beta-, and gamma-melanocyte-stimulating hormone (MSH), has appeared later, perhaps as the MCR subtypes gained more specialized functions. The MCR repertoire shows in general high similarities in their primary structures, while they are however not similar in terms of functional roles. The MCRs serve therefore as an interesting model family to understand the molecular mechanisms of how functions of the genes can diverge during evolution. In this review, we provide an overview of our recent studies on the cloning, expression, pharmacology, 3D modeling, and genomic studies of the MCRs in non-mammalian species.

Food restriction is associated with a number of endocrine disturbances. We validated the experimental conditions for several house-keeping genes and determined the effects of 12 day 50% food restriction on hypothalamic and pituitary transcription of genes involved in different neuroendocrine systems, using real-time quantitative polymerase chain reaction (PCR). A total of 7 nuclear receptors and 12 neuropeptides and peptide hormones were investigated in the dorsal and ventral hypothalamus and the pituitary gland in rats. In the hypothalamus, food restriction reduced mRNA levels of estrogen receptor alpha (ERalpha), progesterone receptor, glucocorticoid receptor, thyroid hormone receptor alpha and beta, pro-opiomelanocortin (POMC), growth hormone-releasing factor (GHRF), corticotropin-releasing factor (CRF), thyrotropin-releasing factor (TRF), somatostatin, and increased that of neuropeptide Y (NPY). In the pituitary, the treatment reduced growth hormone (GH), luteinizing hormone beta (LHbeta) and thyrotropin beta, but increased ERalpha mRNA levels. The study provides a map of how food restriction affects the regulation of a number of transcripts involved in neuroendocrine control.

1 Melanocortin (MC) receptors are widely distributed throughout the body of chicken, like in mammals, and participate in a wide range of physiological functions. 2 To clarify the pharmacological impact of ligands acting in the MC system, we expressed the chicken MC1, MC2, MC3, MC4 and MC5 (cMC1-5) receptors in eukaryotic cells and performed comprehensive pharmacological characterization of the potency of endogenous and synthetic melanocortin peptides. 3 Remarkably, the cMC receptors displayed high affinity for ACTH-derived peptides and in general low affinity for alpha-MSH. It is evident that not only the cMC2 receptor but also the other cMC receptors interact with ACTH-derived peptide through an epitope beyond the sequence of alpha-MSH. 4 The synthetic ligand MTII was found to be a potent agonist whereas HS024 was a potent antagonist at the cMC4 receptor, indicating that these ligands are suitable for physiological studies in chicken. 5 We also show the presence of prohormone convertase 1 (PC1) and PC2 genes in chicken, and that these peptides are coexpressed with proopiomelanocortin (POMC) in various tissues.

We report the cloning and characterization of two melanocortin receptors (MCRs) from the spiny dogfish (Squalus acanthias) (Sac). Phylogenetic analysis shows that these shark receptors are orthologues of the MC3R and MC5R subtypes, sharing 65% and 70% overall amino acid identity with the human counterparts, respectively. The SacMC3R was expressed and pharmacologically characterized in HEK293 cells. The radioligand binding results show that this receptor has high affinity for adrenocorticotropic hormone (ACTH)-derived peptides while it has comparable affinity for alpha- and beta-melanocyte stimulating hormone (MSH), and slightly lower affinity for gamma-MSH when compared with the human orthologue. ACTH(1-24) has high potency in a second-messenger cAMP assay while alpha- and gamma-MSH had slightly lower potency in cells expressing the SacMC3R. We used receptor-enhanced green fluorescence protein (EGFP) fusion to show the presence of SacMC3R in plasma membrane of Chinese hamster ovary and HEK293 cells but the SacMC5R was retained in intracellular compartments of these cells hindering pharmacological characterization. The anatomical distribution of the receptors were determined using reverse transcription PCR. The results showed that the SacMC3R is expressed in the hypothalamus, brain stem and telencephalon, optic tectum and olfactory bulbs, but not in the cerebellum of the spiny dogfish while the SacMC5R was found only in the same central regions. This report describes the first molecular characterization of a MC3R in fish. The study indicates that many of the important elements of the MC system existed before radiation of gnathostomes, early in vertebrate evolution, at least 450 million years ago.

The rainbow trout (Oncorhynchus mykiss) is one of the most widely used fish species in aquaculture and physiological research. In the present paper, we report the first cloning, 3D (three-dimensional) modelling, pharmacological characterization and tissue distribution of two melanocortin (MC) receptors in rainbow trout. Phylogenetic analysis indicates that these receptors are orthologues of the human MC4 and MC5 receptors. We created 3D molecular models of these rainbow trout receptors and their human counterparts. These models suggest greater divergence between the two human receptors than between their rainbow trout counterparts. The pharmacological analyses demonstrated that ACTH (adrenocorticotropic hormone) had surprisingly high affinity for the rainbow trout MC4 and MC5 receptors, whereas α-, β- and γ-MSH (melanocyte-stimulating hormone) had lower affinity. In second-messenger studies, the cyclic MSH analogues MTII and SHU9119 acted as potent agonist and antagonist respectively at the rainbow trout MC4 receptor, indicating that these ligands are suitable for physiological studies in rainbow trout. Interestingly, we found that the rainbow trout MC4 receptor has a natural high-affinity binding site for zinc ions (0.5 µM) indicating that zinc may play an evolutionary conserved role at this receptor. Reverse transcription PCR indicates that the rainbow trout receptors are expressed both in peripheral tissues and in the central nervous system, including the telencephalon, optic tectum and hypothalamus. Overall, this analysis indicates that the rainbow trout MC4 and MC5 receptors have more in common than their mammalian counterparts, which may suggest that these two receptors have a closer evolutionary relationship than the other MC receptor subtypes.

The melanocortin (MC) receptor subtypes have distinctive characteristic binding profiles. We found that the trout and Fugu MC4 receptors have similar affinity for alpha-MSH and beta-MSH and a much higher affinity for ACTH than does the human MC4 receptor. The Fugu MC1 and the trout and Fugu MC5 receptors also have higher affinity for ACTH-derived peptides than alpha-, beta-, or gamma-MSH. It is tempting to speculate that ACTH-derived peptides may have played an important role as "original" ligands at the MC receptors, while the specificity of the different subtypes for the alpha-, beta-, and gamma-MSH peptides may have appeared at later stages during vertebrate evolution.

The Rhodopsin family of G protein coupled receptors (GPCRs) includes the phylogenetic α-group consisting of about 100 human members. The α-group is the only group of GPCRs that has many receptors for biogenic amines which are major drug targets. Several members of this group are orphan receptors and their functions are elusive. In this study we present a detailed phylogenetic and anatomical characterization of the Gpr153 receptor and also attempt to study its functional role. We identified the homologue of Gpr153 in the elephant shark genome and phylogenetic and synteny analyses revealed that Gpr162 and Gpr153 share a common ancestor that split most likely through a duplication event before the divergence of the tetrapods and the teleost lineage. A quantitative real-time PCR study reveals widespread expression of Gpr153 in the central nervous system and all the peripheral tissues investigated. Detailed in situ hybridization on mouse brain showed specifically high expression in the thalamus, cerebellum and the arcuate nucleus. The antisense oligodeoxynucleotide knockdown of Gpr153 caused a slight reduction in food intake and the elevated plus maze test showed significant reduction in the percentage of time spent in the centre square, which points towards a probable role in decision making. This report provides the first detailed characterization of the evolution, expression and primary functional properties of the Gpr153 gene.

The genome of Callorhinchus and the fossil record : a new perspective on SCPP gene evolution in gnathostomes

Background The dog is an important model organism and it is considered to be closer to humans than rodents regarding metabolism and responses to drugs. The close relationship between humans and dogs over many centuries has lead to the diversity of the canine species, important genetic discoveries and an appreciation of the effects of old age in another species. The superfamily of G protein-coupled receptors (GPCRs) is one of the largest gene families in most mammals and the most exploited in terms of drug discovery. An accurate comparison of the GPCR repertoires in dog and human is valuable for the prediction of functional similarities and differences between the species. Results We searched the dog genome for non-olfactory GPCRs and obtained 353 full-length GPCR gene sequences, 18 incomplete sequences and 13 pseudogenes. We established relationships between human, dog, rat and mouse GPCRs resolving orthologous pairs and species-specific duplicates. We found that 12 dog GPCR genes are missing in humans while 24 human GPCR genes are not part of the dog GPCR repertoire. There is a higher number of orthologous pairs between dog and human that are conserved as compared with either mouse or rat. In almost all cases the differences observed between the dog and human genomes coincide with other variations in the rodent species. Several GPCR gene expansions characteristic for rodents are not found in dog. Conclusion The repertoire of dog non-olfactory GPCRs is more similar to the repertoire in humans as compared with the one in rodents. The comparison of the dog, human and rodent repertoires revealed several examples of species-specific gene duplications and deletions. This information is useful in the selection of model organisms for pharmacological experiments.

Background The Adhesion G protein-coupled receptors (GPCRs) are membrane-bound receptors with long N termini. This family has 33 members in humans. Several Adhesion GPCRs are known to have important physiological functions in CNS development and immune system response mediated by large cell surface ligands. However, the majority of Adhesion GPCRs are still poorly studied orphans with unknown functions. Results In this study we performed the extensive tissue localization analysis of the entire Adhesion GPCR family in rat and mouse. By applying the quantitative real-time PCR technique we have produced comparable expression profile for each of the members in the Adhesion family. The results are compared with literature data and data from the Allen Brain Atlas project. Our results suggest that the majority of the Adhesion GPCRs are either expressed in the CNS or ubiquitously. In addition the Adhesion GPCRs from the same phylogenetic group have either predominant CNS or peripheral expression, although each of their expression profile is unique. Conclusion Our findings indicate that many of Adhesion GPCRs are expressed, and most probably, have function in CNS. The related Adhesion GPCRs are well conserved in their structure and interestingly have considerable overlap in their expression profiles, suggesting similarities among the physiological roles for members within many of the phylogenetically related clusters.

The solute carrier family 22 (SLC22) is a large family of organic cation and anion transporters. These are transmembrane proteins expressed predominantly in kidneys and liver and mediate the uptake and excretion of environmental toxins, endogenous substances, and drugs from the body. Through a comprehensive database search we identified six human proteins not yet cloned or annotated in the reference sequence databases. Five of these belong to the SLC22 family, SLC22A20, SLC22A23, SLC22A24, SLC22A25, and SPNS3, and the sixth gene, SVOPL, is a paralog to the synaptic vesicle protein SVOP. We identified the orthologs for these genes in mouse and rat and additional homologous proteins and performed the first phylogenetic analysis on the entire SLC22 family in human, mouse, and rat. In addition, we performed a phylogenetic analysis which showed that SVOP and SV2A-C are, in a comparison with all vertebrate proteins, most similar to the SLC22 family. Finally, we performed a tissue localization study on 15 genes on a panel of 30 rat tissues using quantitative real-time polymerase chain reaction.

G protein-coupled receptors (GPCRs) are a class of integral membrane proteins mediating physiological functions fundamental for survival, including energy homeostasis. A few years ago, an amino acid sequence of a novel GPCR gene was identified and named GPR178. In this study, we provide new insights regarding the biological significance of Gpr178 protein, investigating its evolutionary history and tissue distribution as well as examining the relationship between its expression level and feeding status. Our phylogenetic analysis indicated that GPR178 is highly conserved among all animal species investigated, and that GPR178 is not a member of a protein family. Real-time PCR and in situ hybridization revealed wide expression of Gpr178 mRNA in both the brain and periphery, with high expression density in the hypothalamus and brainstem, areas involved in the regulation of food intake. Hence, changes in receptor expression were assessed following several feeding paradigms including starvation and overfeeding. Short-term starvation (12-48h) or food restriction resulted in upregulation of Gpr178 mRNA expression in the brainstem, hypothalamus and prefrontal cortex. Conversely, short-term (48h) exposure to sucrose or Intralipid solutions downregulated Gpr178 mRNA in the brainstem; long-term exposure (10 days) to a palatable high-fat and high-sugar diet resulted in a downregulation of Gpr178 in the amygdala but not in the hypothalamus. Our results indicate that hypothalamic Gpr178 gene expression is altered during acute exposure to starvation or acute exposure to palatable food. Changes in gene expression following palatable diet consumption suggest a possible involvement of Gpr178 in the complex mechanisms of feeding reward.

The cloning of melanocortin (MC) receptors in distant species has provided us tools to get insight in how the ligand-receptors interactions in the MC system have evolved. We have however lacked studies on pharmacology of native ancient melanocortin peptides at the ancient MC receptors. In this paper we synthesized melanocortin peptides from both the sea lamprey (Petromyzon marinus) and spiny dogfish (Squalus acanthias) and tested them on the MC3 and MC4 receptors from spiny dogfish. The results show that both the dogfish and lamprey ACTH peptides have similar or higher affinity than the dogfish alpha-, beta- and gamma-MSH peptides to the dogfish MC3 and MC4 receptors. Moreover, both the dogfish and lamprey ACTH peptides have more than 10-fold higher affinity than alpha-MSH to the dogfish MC4 receptor. We also show that dogfish delta-MSH is able to bind to MC receptors and its potency is higher than of dogfish beta-MSH, which is considered to be its precursor. Our results provide the first evidence that native ACTH ligands from dogfish and lamprey have a preference above native MSH peptides to ancient version of the MC3 and MC4 receptors. This further strengthens the hypotheses that the ligand contributing to the first version of the melanocortin ligand-receptor system resembled ACTH.

The melanocortin (MC) receptors are found in five subtypes in mammals and chicken, while recent studies have shown that the Fugu (Takifugu rubripes) genome has only four MC receptors and the zebrafish genome has six subtypes. The MC3 receptor seems to be missing from the two closely related pufferfishes, Fugu and Tetraodon (Tetraodon nigroviridis). The MC2 and MC5 receptors in the pufferfish have introns. Moreover, these two receptors are found in a tandem that is remarkably conserved in several vertebrate species. Here, we speculate about the genomic origin of the MC receptors.

One of the most successful chromatic adaptations in vertebrates is the dorsal-ventral pigment pattern in which the dorsal skin is darkly colored, whereas the ventrum is light. In fish, the latter pattern is achieved because a melanization inhibition factor inhibits melanoblast differentiation and supports iridophore proliferation in the ventrum. In rodents, the patterned pigmentation results from regional production of the agouti-signaling protein (ASP). This peptide controls the switch between production of eumelanin and pheomelanin by antagonizing alphaMSH effects on melanocortin receptor (MCR) 1 in the melanocytes. In addition, ASP inhibits the differentiation and proliferation of melanoblast. Thus, the mammalian ASP may be homologous to the poikilotherm melanization inhibition factor. By screening of a genomic library, we deduced the amino acid sequence of goldfish ASP. The ASP gene is a four-exon gene spanning 3097 bp that encodes a 125-amino acid precursor. Northern blot analysis identified two different ASP mRNAs in ventral skin of red- and black-pigmented and albino fish, but no expression levels were observed in the dorsal skin of the same fish. The dorsal-ventral expression polarity was also detected in both black dorsally pigmented fish and albino fish. Pharmacological studies demonstrate that goldfish ASP acts as a melanocortin antagonist at Fugu MC1R and goldfish MC4R. In addition, goldfish ASP inhibited Nle4, D-Phe7-MSH-stimulated pigment dispersion in medaka melanophores. Our studies support agouti signaling protein as the melanization inhibition factor, a key factor in the development of the dorsal-ventral pigment pattern in fish.

The G-protein-coupled melanocortin receptors (MCRs) play an important role in a variety of essential functions such as the regulation of pigmentation, energy homeostasis, and steroid production. We performed a comprehensive characterization of the MC system in Fugu (Takifugu rubripes). We show that Fugu has an AGRP gene with high degree of conservation in the C-terminal region in addition to a POMC gene lacking gamma-MSH. The Fugu genome contains single copies of four MCRs, whereas the MC3R is missing. The MC2R and MC5R are found in tandem and remarkably contain one and two introns, respectively. We suggest that these introns were inserted through a reverse splicing mechanism into the DRY motif that is widely conserved through GPCRs. We were able to assemble large blocks around the MCRs in Fugu, showing remarkable synteny with human chromosomes 16 and 18. Detailed pharmacological characterization showed that ACTH had surprisingly high affinity for the Fugu MC1R and MC4R, whereas alpha-MSH had lower affinity. We also showed that the MC2R gene in Fugu codes for an ACTH receptor, which did not respond to alpha-MSH. All the Fugu receptors were able to couple functionally to cAMP production in line with the mammalian orthologs. The anatomical characterization shows that the MC2R is expressed in the brain in addition to the head-kidney, whereas the MC4R and MC5R are found in both brain regions and peripheral tissues. This is the first comprehensive genomic and functional characterization of a GPCR family within the Fugu genome. The study shows that some parts of the MC system are highly conserved through vertebrate evolution, such as regions in POMC coding for ACTH, alpha-MSH, and beta-MSH, the C-terminal region of AGRP, key binding units within the MC1R, MC2R, MC4R, and MC5R, synteny blocks around the MCRs, pharmacological properties of the MC2R, whereas other parts in the system are either missing, such as the MC3R and gamma-MSH, or different as compared to mammals, such as the affinity of ACTH and MSH peptides to MC1R and MC4R and the anatomical expression pattern of the MCRs.

We created a molecular model of the human melanocortin 4 receptor (MC4R) and introduced a series of His residues into the receptor protein to form metal ion binding sites. We were able to insert micromolar affinity binding sites for zinc between transmembrane region (TM) 2 and TM3 where the metal ion alone was able to activate this peptide binding G-protein-coupled receptor. The exact conformation of the metal ion interactions allowed us to predict the orientation of the helices, and remodeling of the receptor protein indicated that Glu100 and Ile104 in TM2 and Asp122 and Ile125 in TM3 are directed toward a putative area of activation of the receptor. The molecular model suggests that a rotation of TM3 may be important for activation of the MC4R. Previous models of G-protein-coupled receptors have suggested that unlocking of a stabilizing interaction between the DRY motif, in the cytosolic part of TM3, and TM6 is important for the activation process. We suggest that this unlocking process may be facilitated through creation of a new interaction between TM3 and TM2 in the MC4R.

Members of the solute carrier family 25 (SLC25) are known to transport molecules over the mitochondrial membrane. In this paper we present 14 novel members of SLC25 family in human. These were provided with following gene symbols by the HGNC: SLC25A32, SLC25A33, SLC25A34, SLC25A35, SLC25A37, SLC25A38, SLC25A39, SLC25A40, SLC25A41, SLC25A42, SLC25A43, SLC25A44, SLC25A45, and SLC25A46. We also identified the orthologues for these genes in rat and mouse. Moreover, we found yeast orthologues for 9 of these genes and show that the predicted substrate binding residues are highly conserved in the human and yeast proteins. We performed a comprehensive tissue localization study for 9 of these genes on a panel of 30 rat tissues with quantitative real-time polymerse chain reaction. We detected their mRNA in a wide number of tissues, both in brain and in periphery. This study provides an overall roadmap of the repertoire of the SLC25 family in mammals, showing that there are at least 46 genes in the human genome coding for mitochondrial transporters.

Background The melanocortin (MC) receptors have a key role in regulating body weight and pigmentation. They belong to the rhodopsin family of G protein-coupled receptors (GPCRs). The purpose of this study was to identify ancestral MC receptors in agnathan, river lamprey. Results We report cloning of two MC receptors from river lamprey. The lamprey receptors, designated MCa and MCb, showed orthology to the MC1 and MC4 receptor subtypes, respectively. The molecular clock analysis suggested that lamprey MC receptor genes were not duplicated recently and diverged from each other more than 400 MYR ago. Expression and pharmacological characterization showed that the lamprey MCa receptor was able to bind and be activated by both lamprey and human MSH peptides. The lamprey MCa receptor had relatively high affinity for ACTH derived peptides similarly to the fish MC receptors. We found that both of the lamprey MC receptors were expressed in skin, while the MCb receptor was also found in liver, heart and skeletal muscle. Conclusion This study shows presence of MC receptors in agnathans indicating early signs of specific functions of melanocortin receptor subtypes.