key: cord-009969-ln91qfg4
authors: Bertolesi, Gabriel E.; Zhang, John Zhijia; McFarlane, Sarah
title: Plasticity for colour adaptation in vertebrates explained by the evolution of the genes pomc, pmch and pmchl
date: 2019-03-10
journal: Pigment Cell Melanoma Res
DOI: 10.1111/pcmr.12776
sha: 
doc_id: 9969
cord_uid: ln91qfg4

Different camouflages work best with some background matching colour. Our understanding of the evolution of skin colour is based mainly on the genetics of pigmentation (“background matching”), with little known about the evolution of the neuroendocrine systems that facilitate “background adaptation” through colour phenotypic plasticity. To address the latter, we studied the evolution in vertebrates of three genes, pomc, pmch and pmchl, that code for α‐MSH and two melanin‐concentrating hormones (MCH and MCHL). These hormones induce either dispersion/aggregation or the synthesis of pigments. We find that α‐MSH is highly conserved during evolution, as is its role in dispersing/synthesizing pigments. Also conserved is the three‐exon pmch gene that encodes MCH, which participates in feeding behaviours. In contrast, pmchl (known previously as pmch), is a teleost‐specific intron‐less gene. Our data indicate that in zebrafish, pmchl‐expressing neurons extend axons to the pituitary, supportive of an MCHL hormonal role, whereas zebrafish and Xenopus pmch+ neurons send axons dorsally in the brain. The evolution of these genes and acquisition of hormonal status for MCHL explain different mechanisms used by vertebrates to background‐adapt.

Different types of camouflage (i.e., masquerade, disruptive and distractive coloration, surface disruption and countershading) are generated by a combination of skin patterns and colours selected through evolution in many prey and predator species as a means to reduce the likelihood of detection (crypsis) (Skelhorn & Rowe, 2016) .

In general, they are not mutually exclusive and work best when several of their components match the background colour (Eterovick et al., 2018) . The matching of skin pigmentation to background colour can be genetically determined (defined here as "background matching"), or induced by physiological plasticity through differential secretion of hormones by neuroendocrine systems ("background adaptation") (Baker & Bird, 2002; Hoekstra, 2006 ). An example of genetically based background matching are mutations that produced a hyperactive melanocortin 1 receptor (Mc1r), the main receptor for α-melanocyte-stimulating hormone (α-MSH) (Robbins et al., 1993) . The emergence of genetic polymorphisms in Mc1r expressed by pigment cells, and the selection of standing genetic variations, resulted in adaptive evolution of background matching in populations of reptiles, birds and mammals (Linnen et al., 2013; Price & Bontrager, 2001; Rosenblum, Hoekstra, & Nachman, 2004) .

In contrast, background adaptation is plastic, with colour change mediated by pigment cell receptors activated by hormones secreted after eye photoperception of surface colour. Although background adaptation occurs in many species, differences exist in the mechanisms between species that are not well understood. Here, the evolutionary analysis of three critical genes involved in background increase in organisms with a black background de Rijk, Jenks, & Wendelaar Bonga, 1990; Jenks, Overbeeke, & McStay, 1977) .

The existence of a second hormone secreted from the pars nervosa of the pituitary, with opposite effects on background adaptation to α-MSH, was suggested initially by Hogben and Slome (1936) in their "dual-hormonal control" theory. MCH is processed from the C-terminal end of the preprohormone PMCH ( Figure 1 ). In fish, an "MCH" peptide is produced in the hypothalamus and transported down axons to be secreted from the neurohypophysis upon stimulation (Baker & Bird, 2002; Enami, 1955; Kawauchi, 2006; Kawauchi, Kawazoe, Tsubokawa, Kishida, & Baker, 1983) (Figure 1 ). The ability to adapt to a white background by aggregating melanosomes associates with the synthesis and secretion of an "MCH" in several teleost species (Gröneveld, Balm, Martens, & Wendelaar Bonga, 1995a; Kishida, Baker, & Eberle, 1989; Mizusawa et al., 2012; Sugimoto, F I G U R E 2 Zebrafish and Xenopus both background-adapt but only in zebrafish do MCHL+ neurons project axons to the pituitary. (a) Schematic of the genomic structure of the three-exon pmch and one-exon pmchl genes, the mRNAs, the prehormones and the aligned a.a. sequence of MCH and MCHL. The two cysteines involved in the disulphide bonds in MCH and MCHL are indicated in red, and a.a. identity in green, while the predicted dibasic peptide at the cleavage site (RR) is underlined. (b) Quantification of the pigmentation index (left) and dorsal pictures of the head (right) from zebrafish and frog larvae developed for 5 days with a white (WB) or a black (BB) background. Bars indicate average (normalized to WB) from two independent experiments (n = 10; N = 2; ±SE; *p < 0.05, ANOVA). Bottom right panels are enlargements of the red squares. (c) FISH against pmch and pmchl followed by immunohistochemistry against MCH/MCHL. Transverse brain sections (12 µm) processed for FISH (red; white arrows) and with a polyclonal anti-human MCH antibody (green) that recognizes both MCH and MCHL. Note MCH immunoreactivity surrounding the pituitary (P) in zebrafish but not Xenopus. pmchl+ neurons project their axons to the pituitary in fish (yellow arrows), while the axons of pmch+ neurons from both species project dorsally and are restricted to the brain (hypothalamus areas) Nagamori, Yasui, & Oshima, 1997) . Thus, a dual-hormonal control of regulation by α-MSH and an MCH with "hormonal status" may exist in fish. MCH can also act as a neurotransmitter or neuromodulator to regulate physiological processes associated with feeding and energy homoeostasis (Amano & Takahashi, 2009; Ludwig et al., 2001; Matsuda et al., 2007) . The fish MCH system differs from that in mammals with regard to the hormonal status of MCH and the presence of two genes, pmch and pmchl, with distinct genetic structures (Berman, Skariah, Maro, Mignot, & Mourrain, 2009 ). The single PMCH gene of mammals and birds contains three exons and encodes a prohormone that generates an MCH that acts as a neurotransmitter (Breton, Presse, Hervieu, & Nahon, 1993; Cui et al., 2017; Nahon, Presse, Bittencourt, Sawchenko, & Vale, 1989) . Zebrafish have this gene, named originally pmch2 (renamed here pcmh), but also possess a one-exon gene that ultimately produces a peptide with "melaninconcentrating activity" (Berman et al., 2009; Ono, Wada, Oikawa, Kawazoe, & Kawauchi, 1988; Takayama, Wada, Kawauchi, & Ono, 1989) (Figure 1 ). The mammalian MCH, encoded by the three-exon gene, is almost identical to the 17-a.a. fish "MCH," encoded by the single-exon gene (Berman et al., 2009; Kawauchi, 2006; Kawauchi et al., 1983; Nahon et al., 1989; Ono et al., 1988) . Because rat MCH was characterized after the fish peptide, both received the same name (Nahon et al., 1989) . Synteny analysis indicates, however, that the mammalian and zebrafish three-exon genes are linked evolutionarily, and higher similarity exists between the MCH peptides encoded by the three-exon genes than that by the one-exon gene (named hereafter pmchl) (Figure 2a ) (Berman et al., 2009) . A few studies have compared the expression of the pmch genes in fish adapted to different backgrounds (Berman et al., 2009; Kang & Kim, 2013; Mizusawa et al., 2015; Zhang et al., 2010) , but it is unclear whether it is MCH or MCHL that mediates teleost background adaptation. Moreover, an evolutionary analysis of pmch and pmchl genes is lacking.

Our data indicate that pmchl-expressing cells extend "MCHL"positive axons to the zebrafish pituitary, while the pmch-expressing neurons extend "MCH"-positive axons dorsally in the brain, suggesting that MCHL rather than MCH has the hormonal role. We used the phylogeny of chordates to analyse the evolution of pomc, pmch and pmchl genes. We suggest an explanation for the differences in the mechanisms that evolved to regulate background adaptation by inferring ancestral relationships, and assessing associations with plastic colour change, the ability of α-MSH, MCH and MCHL peptides to be synthesized and secreted and to work as neuroendocrine hormones, as well as their effects on skin pigmentation. We propose that the evolution of background adaptation relates mainly to key changes in the pmch genes, with the topological structure of the pomc gene and the α-MSH a.a sequence both conserved during evolution. We show that the intron-less pmchl gene appears only in the Teleostei lineage, possibly as a result of an ancestral retroposition or during the teleost-specific genome duplication (TSD). Based on our analyses, we suggest renaming the intron-less gene, originally named pmch in several fish (Kawauchi, 2006; Ono et al., 1988; Takayama et al., 1989) , as pmchl, which is in concordance with the current zebrafish nomenclature convention and reflects its evolutionary origin. PMCHL gained "hormonal status" and is found only in teleosts, while the three-exon pmch peptide product was conserved as a brain neurotransmitter that regulates feeding behaviour. To be consistent with the evolutionary origin of both genes, in this paper we call the mammalian and fish three-exon gene, and the prehormone and the peptide generated, as pmch, PMCH and MCH, respectively, and the fish intron-less gene, pmchl, and its corresponding peptides PCMHL and MCHL. Of note, special care should be taken with the literature, as a different nomenclature was used. 

Physiological skin pigmentation indices were measured on 8-dayold embryos at the middle of the light cycle period (7 hr ON), as described previously (Bertolesi, Hehr, & Mcfarlane, 2015) . Briefly, pictures of the dorsal head of tadpoles were taken with identical conditions of light, time exposure and diaphragm aperture, and converted to binary white/black images using NIH ImageJ (U. S. National

Institutes of Health, Bethesda, MD) public domain software. The areas of ten dorsal head melanophores/fish were measured (n = 10 fish; N = 2 experiments), while in Xenopus (n = 10; N = 2) the pigmentation present within an area of the dorsal head between the eyes was used for quantification, since melanophore processes overlap.

Statistical differences were determined by ANOVA (*p < 0.05).

pmch and pmchl from zebrafish were amplified by reverse transcriptase PCR (RT-PCR) from single-strand cDNA generated from zebrafish embryos at 8 days post-fertilization (dpf) using SuperScript™ II RNase H reverse transcriptase (Invitrogen) according to the manufacturer's instructions. PCR amplifications were carried out in a total volume of 20 μl using Fermentas PCR mix (Thermo Fisher Scientific Inc., Ottawa, Ontario). PCR amplification products were cloned into the pCRII-Topo vector and sequenced at the DNA Services Facility, U. Calgary. pCRII-TOPO-pmch-ZF and pCRII-TOPO-pmchl-ZF amplified by PCR produced sequences with 100% identity to those deposited previously in the GenBank database [FJ392644.1 between nucleotides 1 and 481 (pmch) and FJ204827.1 between nucleotides 15 and 452 (pmchl), respectively). A pCMV sport6 plasmid, containing the full-length cDNA encoding X. laevis pmch obtained from an image clone (Open Biosystems Image Clone number IC6947676;

pCMV-Sport6-pmch-Xl), was used to generate pmch probes.

Antisense and sense riboprobes were synthesized from linearized plasmid using SP6 or T7 RNA polymerases (Roche) and digoxigenin (DIG) (Roche, Montreal, QC, Canada)-labelled nucleotides and stored at −80°C. FISH was performed on 12-µm cryostat sections obtained from embryos at 8 dpf. Hybridized probes were detected using anti-DIG antibody (1:500; Roche) conjugated to horseradish peroxidase. The staining process was carried out with a TSA Cyanine 3 (red) system kit (Perkin Elmer, USA), according to the manufacturer's instructions.

Immunohistochemistry was performed on sections after FISH using standard procedures. Briefly, the FISH reaction was stopped with several washes of TN-T (100 mM Tris pH 7.5, 150 mM NaCl, 0.1% bovine serum albumin (Sigma), and 0.5% Tween-20 (BDH)) before addition of primary antibody (anti-MCH rabbit polyclonal M8440; 1/100 dilution; Sigma-Aldrich) in blocking buffer (0.5% blocking F I G U R E 3 Schematic diagram of related taxa (classes or orders) of vertebrates with a similar state of evolution and/or mechanism for background adaptation. Extant species divided (see Materials and Methods) into 16 groups corresponding with taxonomic classes (top rectangles) or taxonomic orders. Evolutionary years (millions) dividing different era are indicated on the left. Whole-genome duplication events (2R; teleost-specific duplication [TSD 3R] and salmonid-specific duplication [SS 4R]) are indicated in yellow, while blue circles denote the age of the oldest preserved fossils with fur or feathers with insulation capacity. Homoeotherms (birds and mammals) are in grey boxes. Background adaptation is indicated with a white/black circle. Group 1: Cyclostome (agnathans: hagfish and lamprey); Group 2: Chondrichthyes (cartilaginous fish; sharks); Group 3: Cladistei (Polypteriformes; bichir); Group 4: Chondrostei (sturgeon); Group 5: Holostei (bowfin; gar); Group 6: Teleostei; Group 7: Euteleostei and Neoteleostei; Group 8: Acanthomorpha (See taxonomic orders for Groups 6, 7 and 8 in Table S2 ); Group 9: Coelacanthi and Dipnoi (lungfish); Group 10: Anura (frogs); Group 11: Caudata (salamanders); Group 12: Squamata (lizard and snakes); Group 13: Testudine (turtles); Group 14: Crocodilia (crocodiles, alligators); Group 15: Aves (birds); and Group 16: Mammalian (mammals). Not included in the analysis are the taxonomic orders from amphibian Gymnophiona (caecilians) and from reptile Rhynchocephalia (tuatara) (grey circle) reagent FP1020 in TN-T; Perkin Elmer). Alexa Fluor-tagged secondary antibodies (green; 488 nm emission, anti-rabbit) diluted 1:1,000 were used to detect the primary antibody.

A variety of organisms from agnathans and gnathostomes with readily available protein reference sequence were used to probe for PMCH, PMCHL and POMC. Homologues were identified using zebrafish PMCH, PMCHL and POMC as queries with a cut-off expect (E) value for positive candidates set at 0.05. The organisms selected are listed in Tables S3 and S4 (pmch and pmchl) and 4 (pomc). The genetic-structural analysis of pmch and pmchl was performed from organisms listed in the NCBI gene database that contained the exonintron structure and gene identification number, as referred to in the organism list. Although organisms with fully sequenced genomes were chosen wherever possible, some organisms in key lineages with unpublished or incomplete genomes were also included. In a few cases, for example, for organisms that are used as model systems for background adaptation (i.e., Oncorhynchus keta and Verasper moseri), we included nucleotide sequence obtained from cDNA rather than derived from genomic sequences. Different species were chosen to represent a broad range of organisms in sixteen clades (taxonomic classes or orders) as referred to in Figure 3 .

Vertebrates were divided into sixteen clades. Group 1 contains species in the infraphylum agnathan (cyclostomes, represented by two taxonomic classes, Myxini and Cephalaspidomorphi, which includes the hagfish and lamprey, respectively). Groups 2-16 correspond to extant gnathostomes. Group 2 contains the cartilaginous fish (sharks; taxonomic class, Chondrichthyes), while the ray-fin fish (Actinopterygii) were divided into six groups representing taxonomic classes or orders (Groups 3-8). The taxonomic classes Cladistei, Chondrostei and Holostei were maintained as independent groups (Groups 3, 4 and 5, respectively), while the clade Teleostei was divided into three groups based on the taxonomic orders and differences in their background adaptation response (see Results4 and Table S2 ). The 38 taxonomic orders in Teleostei were divided as follows: Group 6 (Teleostei) contains 11 taxa, Group 7 (Euteleostei plus Neoteleostei) contains 7 taxa, and Group 8 (Acanthomorpha plus Percomorpha) contains 20 taxa (Table S2) .

Lobe-fin fish (Sarcopterygii) were Group 9. The amphibians were grouped into their three taxonomic orders: Anura (frogs; Group 10), Caudata (salamanders; Group 11) and Gymnophiona (caecilians, not analysed because of lack of information on pomc, pmch and pmchl).

Reptiles were also grouped according to their taxonomic order: Squamata (lizards and snakes; Group 12); Testudine (turtles; Group 13); Crocodilia (crocodiles; Group 14); and Rhynchocephalia (tuataras), with the latter not considered because only one living species (Sphenodon punctatus) exists and genetic information is lacking.

Finally, Groups 15 and 16 correspond to the clade Aves (birds) and

Mammals, respectively.

Validated sequences were aligned using MUSCLE (multiple sequence alignment) and used to build a hidden Markov model (HMM) by using a maximum-likelihood architecture construction algorithm. All phylogenetic analysis and alignments were performed by using the public domain MEGA X software (www.megasoftware.net) (Kumar, Stecher, Li, Knyaz, & Tamura, 2018) . Detailed descriptions and statistics are indicated in the figure legends.

MCH and MCHL show a similar a.a. sequence (Figure 2a ), and both bind cultured mammalian HEK293 cells that overexpress MCH1R and MCH2R (Mizusawa et al., 2015) . The data are more convincing, however, for MCHL having "hormonal status." At physiological concentrations (<1 nM), MCHL induces greater melanosome aggregation in cultured skin melanophores and xanthophores than MCH (Mizusawa et al., 2015) . Further, MCHL is the "factor" with melanin-concentrating capacity that was shown initially to be secreted by the pituitary of catfish (Parasilurus asotus) (Enami, 1955) , and was then purified from the pituitary of Chum salmon (O. keta) ( Figure 2a ) (Enami, 1955; Kawauchi et al., 1983) . Finally, pmchl mRNA is upregulated in organisms with a white background (Berman et al., 2009; Kang & Kim, 2013; Mizusawa et al., 2015; Zhang et al., 2010) , and pmchl overexpression in medaka (Oryzias latipes) increases MCHL plasma levels and lightens the skin, but leaves feeding behaviours unchanged (Kinoshita et al., 2001) . These data argue that MCHL has hormonal status.

Whether the related MCH is also secreted into the circulation blood to induce melanosome aggregation is unclear. In order to answer this question, we used two experimental models: zebrafish (Danio rerio) and the African clawed frog (X. laevis), which possess MCH/MCHL and only MCH, respectively ( Figure 2a ). Of note, studies have defined the hormonal status of an MCH in fish based on the immunohistochemical detection of MCH+ axon fibres that project to the neurohypophysis (Baker, 1993; Baker & Bird, 2002; Powell & Baker, 1988) and/or the biochemical detection of MCH peptides in blood Kishida et al., 1989) . Those studies, however, were performed before it was known that two pmch genes exist, and used polyclonal antibodies raised against either mammalian MCH (human or rat) or salmonid MCHL (Coho salmon), which likely cross-react in that MCH and MCHL a.a. sequences are almost identical ( Figure 2a ). In order to compare the background adaptation response between the two model systems, we grew three-dayold zebrafish and Xenopus larvae for 5 days with a white or a black background, and then measured skin pigmentation (Bertolesi et al., 2015) . Both species showed melanosome aggregation and a light skin colour with a white background ( Figure 2b ). Next, we asked whether the pmch+ and pmchl+ neurons in zebrafish and the pmch+ neurons of Xenopus are present in the hypothalamus, and project axons to the neurohypophysis. Of note, in zebrafish pmch and pmchl are expressed by different neurons (Berman et al., 2009) 

Our experimental data suggest MCHL possesses hormonal status in zebrafish, while MCH may act as a brain neurotransmitter. Together with the presence of two pmch genes in fish and one in mammals, these observations led us to hypothesize that the uni-hormonal or dual-hormonal theories of background adaptation in vertebrates might be explained by the evolution of the pomc, pmch and pmchl genes. To address this possibility, we assessed in species across evolution first the presence of a physiological background adaptation response, via an analysis of the literature, and, second, the structure and sequences of the pomc, pmch and pmchl genes and their corresponding peptides.

Thus, the evolution of the physiological background adaptation response was inferred by comparison of extant organisms grouped in 16 clades (taxonomic classes or orders) as referred to by the Integrated Taxonomic Information System (https://www.itis.gov/) (Figure 3 ).

We focused our evolutionary analysis on two assemblages in the phylum Chordata that emerged over the past 500 million years: the agnathans (bilateral organisms with an internal support system but that lack a hinged jaw; Cyclostome; Group 1) and the gnathostomes (bilateral organisms with an internal cartilaginous or bony skeleton, and a hinged jaw; Groups 2-16). The Protochordata (organisms that lack an internal cartilaginous or bony skeleton) were not considered in this study, since the mechanisms to change skin colour differ from those in Chordata (Umbers, Fabricant, Gawryszewski, Seago, & Herberstein, 2014) , and the pomc gene is not detected (Sundström, Dreborg, & Larhammar, 2010) .

Cyclostomes (Group 1) change skin colour in response to environmental light (dark/light) through melatonin secreted by the pineal gland (Joss, 1973) ; however, we are unaware of reports of quick physiological skin adaptation prompted by changing the surface colour (Figure 3 , circle with a red cross). Background adaptation does occur in extant gnathostomes starting in Group 2 (cartilaginous fish; Chondrichthyes) with sharks altering their skin pigmentation to adapt to the "background" colour generated by deep and shallow water (Lowe & Goodman-Lowe, 1996) , and continuing through to mammals (Beltran, Burns, & Breed, 2018; Zimova et al., 2018) (Figure 3 , black and white circles; see additional references in Table S1 ).

The ray-finned bony fish (Groups 3-8; Actinopterygii) constitute the largest clade of living vertebrates and distribute over Cladistei, Chondrostei, Holostei and Teleostei taxa ( Figure 3 ). Here, we maintained three of the four taxonomic classes (Cladistei, Chondrostei and Holostei, with 11, 31 and 8 living species, respectively) as independent groups (Groups 3, 4 and 5, respectively), with all containing species that background-adapt ( Figure 3 and Table S1 ). The fourth Actinopterygii taxonomic class, Teleostei, contains more than 27,000 species. We divided this clade into three groups that reflect different evolutionary lineages based on recent molecular and genomic data (Groups 6-8) (Betancur-R et al., 2013; Betancur-R et al., 2017; Near et al., 2012) .

Cypriniforme zebrafish (D. rerio) and the Anguilliforme eel (Anguilla Anguilla) with a dual-hormonal mechanism (Figure 3 , Tables S1

and S2). Group 7 contains the Euteleostei plus Neoteleostei ( Figure 3 and Table S2 ). Background adaptation occurs in the Esociform Esox lucius, as well as in several species of salmonids, including Oncorhynchus mykiss, and the Chum salmon (O. keta) from which an MCH peptide (we call MCHL) was originally purified (Kawauchi et al., 1983) . Studies in salmon helped reveal the dual-hormonal mechanism, in that circulating MCH/MCHL and α-MSH levels increase with a white and black background, respectively Kishida et al., 1989) (Figure 3 and Table   S1 ). Finally, Group 8, Acanthomorpha, are collectively known as the spiny-rayed fish ( Figure 3) . Percomorphas, the clade that encompasses most of the diversity of modern Acanthomorpha, is the major evolutionary lineage at the top of the teleost tree ( Figure 3 and Table S2 ). Organisms of this group backgroundadapt and include the important models Beloniforme medaka (Oryzias latipes), Tetraodontiforme fugu (Takifugu rubripes) and the Pleuronectiforme flounder (Verasper moseri) (Klovins et al., 2004; Mizusawa, Kobayashi, Yamanome, Saito, & Takahashi, 2013; Sugimoto et al., 1997) . Interestingly, several species in this group lack the dual-hormonal mechanism, in that MCH/MCHL plasma levels but not α-MSH vary with background adaptation, including flounders (Verasper moseri and Platichthys flesus) (Gilham & Baker, 1984; Kang & Kim, 2013; Mizusawa et al., 2011) and tilapias from the genus Oreochromis (Nile and Mozambique) (Gröneveld, Balm, Martens et al., 1995a; Gröneveld, Balm, & Wendelaar Bonga, 1995b; van der Salm, Metz, Wendelaar Bonga, & Flik, 2005) .

Group 9 consists of the Coelacanthi and Dipnoi lobe-finned fish (lung fish), with only eight living species (Figure 3) . Captive Dipnoi adapt their skin colour to the background (Smith & Coates, 1936) , while no information is available for coelacanths.

To best characterize evolutionary changes in pomc and pmch, we grouped the species in the taxonomic classes Amphibia and Reptilia according to their taxonomic orders (Figure 3 ): the amphibian frogs (Anura; Group 10), salamanders (Caudata; Group 11) and caecilians (Gymnophiona; not analysed, grey circle), and the Reptilia Squamata (lizards and snakes; Group 12), Testudine (turtles; Group 13), Crocodilia (crocodiles; Group 14) and Rhynchocephalia (tuataras not analysed, grey circle) (Figure 3) . Although colour skin pigmentation of several species of caecilians mimics the above-ground soil where they move (Wollenberg & Measey, 2009) , is not clear if it is a genetically mediated process (background matching) or driven by physiological colour plasticity. Therefore, caecilians were not grouped for further analysis. In contrast, background adaptation in amphibian frogs and salamanders is well documented (Table S1 ) and appears to be driven by a uni-humoral mechanism, since MCH/MCHL do not trigger melanosome aggregation (Ferroni & Castrucci, 1987; Wilkes et al., 1984) . Background adaptation is also present in species in the clade Reptilia ( Figure 3 and Table S1 ).

Finally, the homoeotherms of the taxonomic classes Aves (birds;

Group 15) and Mammals (Group 16) 

We next performed an evolutionary analysis of the MCH and MCHL peptides and genes. We focused first on the gene structure of pmch, analysing mainly organisms whose genome was fully sequenced and listed in the NCBI gene database with the exon-intron structure and gene identification number (Table S3 ). The three-exon gene appears conserved during evolution, as it is present in all groups in which we were able to find the gene, from Chondrichthyes (Group 2) to Mammals (Group 16). In contrast, the intron-less gene is present only in teleost fish (Groups 6-8; Figure 4 and Table S3 ). Teleosts possess ~30% more pigmentation-related genes than tetrapods, most of which were duplicated and retained during the TSD and are involved in specification and differentiation of pigment cells (Braasch, Brunet, Volff, & Schartl, 2010; Lorin, Brunet, Laudet, & Volff, 2018) . Thus, the idea that pmchl was retained in Teleostei by evolutionary pressure selection on a gene with a key role in pigmentation regulation (e.g., background adaptation) is tempting. (Minth, Qiu, Akil, Watson, & Dixon, 1989) and O. kisutch (MCH1) (Nahon, Presset, Schoepfer, & Vale, 1991) . We find four variants in the Arctic char (Salvelinus alpinus) and the Atlantic salmon (Salmo salar), which express the mRNAs (Leong et al., 2010) ( Figure 5 and Table S3 ). Phylogenetic analysis of the predicted proteins of the Group 7 salmonids and the Esociform Esox lucius shows that PMCHA and B, and PMCHLA and PMCHLB are related to PMCH and PMCHL from Esox lucius, respectively, but appeared more recently in evolution ( Figure 5 ). This result, together with the ab- (Courseaux & Nahon, 2001) .

We next analysed PMCH and PMCHL at the a.a. level. Both PMCH and PMCHL contain a predicted MCH and MCHL sequence able to generate a cyclic ring with 8 a.a. between two internal cysteines that mediate a disulphide bond essential for receptor binding (Audinot et al., 2001; Matsunaga, Hruby, Lebl, Castrucci, & Hadley, 1992) , as well as two consecutive basic residues (e.g., Arg 145 -Arg 146 in mammals and rodents) critical for cleavage and release (Viale et al., 1999) ( Figures 2a and 4) . MCH peptides are at least two a.a. longer (19-25 a.a. 64/65 species) than MCHL (17 a.a. 41/41 species in Groups 6-8).

The latter is highly conserved (>94% identity, 16/17 a.a.) in all species of Groups 6-8 (Figure 4 and Table S3 ). Also, highly conserved in F I G U R E 4 Carboxy-end amino acid sequence of the evolutionary conserved PMCH and the teleost-specific PMCHL. Structure (top) and alignment of the carboxy-end region of PMCH and PMCHL of several species grouped as indicated in Figure 3 . The putative peptides processed from PMCH include MCH and neuropeptide glutamic acid-isoleucine (NEI), and from PMCHL include MCHL, neuropeptide glutamic acid-valine (NEV) and MCH gene-related peptide (Mgrp). The length of the a.a. sequences and the number of exons (Ex; red) that contribute to the open reading frame are indicated on the right. Note three (or more) exons and one exon contribute to PMCH and PMCHL, respectively. Multiple protein sequence alignments were performed by using MUSCLE and correspond to PMCH from Group MCHL is a valine 7 inside the ring (41/41 species from Groups 6-8).

In MCH, the a.a. at this site is isoleucine or leucine, except for in two Group 6 species, D. rerio and Paramormyrops kingsleyae, in which valine is present (Figure 4) .

We next analysed the presence of other putative peptides in the PMCH and PMCHL prohormones. In mammals, PMCH is processed to MCH and neuropeptide glutamic acid-isoleucine (NEI) (Viale et al., 1999) , a 13-a.a. peptide that affects the hypothalamic-gonadal axis (Bittencourt & Celis, 2008; Fujimoto, Fukuda, Sakamoto, Takata, & Sawamura, 2017) . Conserved sequence for the mammalian NEI is present in amphibians and reptiles (Groups 10-14), while several a.a. are absent from Group 6 species and missing completely from Group 7 and 8 species (Figure 4) . These data suggest that NEI has no biological function in teleosts. Similarly, in birds (Group 15), the predicted NEI peptide is highly modified, and the loss of the K/R R site for cleavage in chicken and Japanese quail (Figure 4) suggests that the peptide is not processed and released. Thus, NEI is conserved in land organisms, but not aquatic organisms or birds. Interestingly, a arginine-valine (NEV) neuropeptide from the rainbow trout sequence was suggested as a homologue of the mammalian NEI, based mainly on the topological position and the presence of a dibasic RR motif Nahon et al., 1989) . Our analysis shows that NEV is predicted to be processed from PMCHL but not PMCH, and in species from Groups 6 and 7, but not Group 8 (Figure 4 ). An additional putative peptide, named MCH gene-related peptide (Mgrp) (Figure 4) , is present in Nile tilapia, but does not participate in background adaptation (Gröneveld, Balm, & Wendelaar Bonga, 1995c) . (Hu et al., 2016; Takahashi et al., 2004) .

Thus, the three-exon gene is translated to PMCH, which then releases MCH after processing. In mammals, and probably in fish (Berman et al., 2009; Mizusawa et al., 2012) , MCH acts as a neurotransmitter to regulate feeding. The paralogue pmchl intron-less gene (originally named pmch), and the MCHL peptide to which it

gives rise, appears only in Teleostei. Its existence, coupled with the potential of MCHL to reach the circulation, associates with the dualhormonal mechanism for background adaptation in teleosts.

With α-MSH as a key regulator of background adaptation, and data which suggest that pomc and mcr1 in chordates are linked evolutionarily (Dores & Baron, 2011; Dores et al., 2016) , we next analysed the a.a. sequences of POMC, α-MSH and ACTH. ACTH and α-MSH arise from processing of POMC in the anterior pituitary and pars intermedia pituitary, respectively, and are utilized in distinct neuroendocrine circuits (Zhou, Bloomquist, & Mains, 1993) (Figure 6a ). β-, γ-and δ-MSH are also processed from POMC, but have no known role in F I G U R E 6 Topological organization of POMC and amino acid sequence alignment of ACTH/α-MSH. (a) Schematic representation of POMC with the positioning of the different processed peptides. γ-MSH, β-MSH, ACTH (green) and α-MSH (red), which is at the amino-end of ACTH, are indicated and positioned based on the presence of the conserved HFRW sequence. β-Endorphin is located at the carboxy-end. The a.a. length and the position of the first a.a. of HFRW are indicated on the right and inside the ACTH/α-MSH domain, respectively. Note that δ-MSH is exclusive to Group 2 (sharks) and γ-MSH is not present in teleosts (Groups 6, 7 and 8), cyclostomes (Group 1) and the lineage from which arose the snakes (Group 12), but is present in lizards (half blue rectangle). Also indicated are mutations in HFRW in Groups 4 and 5 species. In lamprey, ACTH and α-MSH are encoded by different genes, named pom and poc, respectively (Heinig et al., 1995; Takahashi et al., 1995) . (b) Aligned sequences of ACTH and α-MSH (green and red box, respectively). Amino acids in α-MSH are coloured. Note similarity between Groups 2 to 16. Topological organization and sequence alignment were performed by using the following POMC prohormones (species, GenBank Acc. background adaptation (Gilham & Baker, 1984; Sumpter & Lowry, 1984) . For Group 1 cyclostomes, we analysed the peptides that would arise from two genes with homology to pomc, pom (pro-opiomelanotropin) and poc (pro-opiocortin), which contain the ACTH and the α-MSH sequences, respectively (Heinig, Keeley, Robson, Sower, & Youson, 1995; Takahashi et al., 1995) . Common to ACTH and all three MSH peptides is the presence of a His-Phe-Arg-Trp (HFRW) sequence, critical for binding and activation of MCRs (Dores et al., 2016; Schwyzer, 1977) . We used this motif to align the various POMC peptides of the different taxonomic groups (Figure 6a ).

The comparison of MSH peptides between groups supported our taxonomic grouping of phylogenetic clusters. First, in agreement with previously described differences (Dores, Cameron, Lecaude, & Danielson, 2003; Takahashi, Amemiya, Nozaki, Sower, & Kawauchi, 2001) , the δ-MSH sequence is detected only in species of Group 2 (7/7 species) (Figure 6a and Table S4 ). Second, γ-MSH exists in cartilaginous fish and tetrapods, but is absent in the agnathan POC and POM (Amemiya, Takahashi, Suzuki, Sasayama, & Kawauchi, 1999) ( Figure 6a and Table S4 ). In agreement with previous studies (Dores & Baron, 2011; Kitahara et al., 1988) , we found γ-MSH is also absent from Groups 6, 7 and 8. Novel is that γ-MSH in Squamata is missing in the lineage giving rise to snakes (4/4 species), but is present in lineages from which lizards and geckos arose (4/4 species) ( Figure 6a and Table S4 ). Further, we find mutations in the functionally relevant HFRW motif of γ-MSH in species from Groups 4 and 5 [e.g., HFHW

(1/2 species) and HFRS (2/2 species), respectively) ( Figure 6a and Table S4 ), which may result in non-functional peptides. Together, our analysis suggests that while γ-MSH was lost in teleosts and snakes, it may already have become non-functional earlier in evolution (Chondrostei and Holostei lineages).

The analysis of ACTH/α-MSH sequences between the agnathan POC/POM and gnathostome POMC indicates an association with the ability to background-adapt. The α-MSH sequence is similar in length (13 a.a.) and homology (identity between 92% and 100%; 12/13 and 13/13 a.a., respectively) only between groups that background-adapt (Groups 2 to 16), but is remarkably different in cyclostomes (Figure 6b ). These differences in cyclostome α-MSH may explain the lack of background adaptation in lamprey.

First, while the α-MSH peptide predicted from the POM gene is identical to the MSH-B peptide isolated from the pituitary glands of the Sea lamprey Petromyzon marinus , it is longer than gnathostome α-MSH, with additional a.a. located near two amino terminal basic a.a. essential for endopeptidase cleavage ( Figure 6b) . Second, the homology in the region surrounding HFRW is low in agnathans (62%; 8/13 a.a. as compared to more than 92% in gnathostomes) (Figure 6b) . Finally, the affinity of α-MSH to the lamprey Mcr1 is 1,300 times lower than to the human receptor (Haitina et al., 2007; Schiöth, Petersson, Muceniece, Szardenings, & Wikberg, 1997) . The fact that α-MSH and ACTH bind to the lamprey receptor with similar affinities, while the affinity of α-MSH for the human MCR1 is 20 times higher than that of ACTH (Haitina et al., 2007; Schiöth et al., 1997) , suggests that ACTH may be the biological and the "ancestral" ligand for the Mcr1 in agnathans. Interestingly, the localization of α-MSH and ACTH to the intermedia and anterior pituitary, respectively, is nonetheless conserved between agnathans and gnathostomes , as is the tetrapeptide HFRW required to bind Mcr1 (Figure 6b ) (Costa et al., 2004; Schwyzer, 1977) .

As mentioned, species of Groups 2 to 16 all show background adaptation. The literature supports the idea that this pigmentation response associates with the conservation of α-MSH sequence between these groups. Particularly relevant in this regard, given their evolutionary separation from mammals, are Group 2 species, which also lack MCHL. The dogfish Squalus acanthias α-MSH (Figure 6b ) (Lowry, Bennett, & McMartin, 1974) is almost identical to that of human (identity 12/13 a.a; Figure 6b ). Interestingly, in sharks, background adaptation is reversed by intravenous administration of α-MSH antibodies (Rodrigues & Sumpter, 1984) , and the marine elasmobranch and mammalian α-MSH both induce melanosome dispersion after intravenous injection (Sumpter & Lowry, 1984) .

Indeed, α-MSH regulates pigmentation in species from Groups 2 (Potamotrygon reticulatus) (Visconti & de L. Castrucci, 1993) to Group 16 (Abdel-Malek et al., 1995) , suggesting a conserved role (supporting references in Table S1 ).

Of note, while α-MSH functions in background adaptation in homoeotherms (Groups 15-16), the peptide source may differ in key ways from that in Groups 10-14. In birds, α-MSH is likely produced in a paracrine manner in the integument, and not from the pars intermedia pituitary, which birds lack (Boswell & Takeuchi, 2005) . In mammals, α-MSH levels change seasonally, rising during spring/summer and decreasing in autumn/winter (Altmeyer, Stöhr, & Holzmann, 1986; Lincoln & Baker, 1995; McFarlane et al., 2011) , but the mechanisms and the source of α-MSH are unknown. A possible explanation for the differences with Groups 10-14 is that species therein require rapid colour plasticity for thermoregulation (Fan, Stuart-Fox, & Cadena, 2014; Tattersall, Eterovick, & Andrade, 2006) , a need minimized in birds and mammals with an "insulated" integument.

In summary, our analysis reveals that α-MSH (and β-MSH) was conserved during evolution, while γ-MSH and δ-MSH were not.

Interestingly, the differences in α-MSH a.a. sequence between agnathans and gnathostomes associate with the capacity to background-adapt. In ectotherms, background adaptation (and thermoregulation) is regulated by α-MSH secreted from the pars intermedia pituitary. In homoeotherms, however, a role of a pituitary-derived α-MSH in background adaptation is unclear.

We used both the structure and presence of pomc, pmch and pmchl as a "genetic basis," alongside MCH/MCHL "hormonal status," to explain differences in the physiological mechanisms for background adaptation over evolution. The genetic basis includes a conserved α-MSH sequence and function between Groups 2 and 16, but not Group 1 (Figure 7) . A conserved three-exon pmch gene, whose function appears conserved and relates to feeding behaviours and energy homoeostasis, is present in species of all 16 Groups. A pmchl gene appears in teleosts (Groups 6-8) and associates with hormonal status for MCHL ( Figure 7 ) and axon projections from pmchl+, but not pmch+, neurons to the hypophysis. Therefore, we suggest MCHL arose evolutionarily in the Teleostei lineage, acquiring hormonal status and a new role in skin pigmentation. Finally, the "genetic basis" and the "hormonal status" together explain the following differences in the mechanisms that regulate background adaptation in vertebrates (Figure 7 ; conclusions):

1. Group 1: No significant role for α-MSH and MCH. The absence of a background adaptation response in cyclostomes likely reflects the inability of MCH and α-MSH to regulate skin pigment dispersion or aggregation, with a distinct α-MSH sequence and affinity to Mcr1 from that in gnathostomes. Lamprey skin does not respond to synthetic salmon MCHL (Baker & Bird, 2002) , and a "melanophore-aggregating factor" is detected in hypothalamic but not pituitary extracts (Baker & Rance, 1983) , suggesting that MCH does not possess hormonal status.

Additionally, MCHL is not present in cyclostomes (Figure 7 ).

Groups 2-4: α-MSH but not MCH regulates background adaptation. MCHL is not detected in these groups, and MCH does not appear to have hormonal status. For instance, a shark MCH antibody detects cells in the hypothalamus, but not the pituitary gland, of Sphyrna lewini (Group 2) (Mizusawa et al., 2012) .

Additionally, neither human MCH (95% identity to shark MCH; 18/19 a.a.), nor synthetic MCHL, produce aggregation in Triakis scyllium and Potamotrygon reticulatus (Group 2) (Mizusawa et al., 2012; Visconti & de L. Castrucci, 1993 ). Yet, pituitary-derived α-MSH regulates background adaptation in several shark species (Visconti & de L. Castrucci, 1993) (Figure 7 ; supporting references in Table S1 ). Interestingly, skin melanophores from Erpetoichthys calabaricus (Group 3), and Acipenser stellate and Polyodon spathula (Group 4), do not respond to synthetic MCH and MCHL, and MCH antibodies fail to label fibres in the posterior pituitary (Baker & Bird, 2002; Sherbrooke & Hadley, 1988) , arguing against hormonal status for MCH in Groups 3 and 4. Background adaptation in species from these groups, however, requires a functional pituitary and involves α-MSH-induced dispersion of melanosomes, pointing to α-MSH, but not MCH, as the key regulator of skin pigmentation (Figure 7 and additional references in Table S1 ).

Group 5: Hormonal status and skin response to MCH appears.

In holosteans, MCH+ fibres extend to the pituitary in Lepisosteus osseus and Amia calva, and MCHL causes melanosome aggregation (Baker & Bird, 2002; Sherbrooke & Hadley, 1988) . Therefore, a dual hormonal role of α-MSH and MCH in regulating skin pigmentation is possible. Whether MCHL is responsible is unclear, in that we were unable to find pcmhl in any holostean genome, including that of the fully sequenced Lepisosteus oculatus (Figure 7 ). Thus, it is possible that the dualhormonal system was present in the common ancestor of holosteans and teleosts. A key question that needs to be addressed is what selective advantage, if any, was provided by the duplicated genes of teleosts? Evolutionary selection of pmchl may be explained as a case of subfunctionalization, with essentially no functional change before and after the duplication in holosteans.

Groups 6-8: A dual control mechanism at the bottom of the Teleostei lineage (Groups 6 and 7), and a single main regulator, likely MCHL, at the top (Group 8). We suggest pcmhl appears early in the teleost lineage by retroposition or during the TSD, with MCHL-expressing neurons then selected over evolution to extend F I G U R E 7 Genetics and the hormonal status of MCHL explain the different mechanisms for background adaptation. Groups able to background-adapt are indicated with a white/black circle. The genetic basis corresponds to the presence of a highly conserved 13-a.a. α-MSH (red square), a three-exon pmch gene (three yellow bars) or a one-exon pmchl gene (yellow square), while hormonal status indicates the capacity of an MCH to be released into the bloodstream. A teleost-specific pmchl gene encodes MCHL, which acts as a hormone in background adaptation, while the conserved pmch gene encodes MCH, which we suggest acts as a neurotransmitter to regulate energy homoeostasis and food intake. The six different mechanisms for background adaptation include the following: (a) Group 1: no significant role for α-MSH and MCH; (b) Groups 2 to 4: α-MSH but not MCH participates; (c) Group 5: hormonal status appears with a dual role of α-MSH and MCH in regulating skin pigmentation; (d) Groups 6-8: a "dual control theory" mechanism for background adaptation at the bottom of Teleostei lineage (Groups 6 and 7) and a single main regulator, likely MCHL, at the top (Group 8); (e) Group 9: α-MSH but not MCH regulates background adaptation; (f) Groups 10-14: a uni-humoral α-MSH system that also is involved in thermoregulation; and (g) Groups 15 and 16: α-MSH and MCH roles changed in homoeotherms as insulation systems (fur and feathers) appear during the evolution of endothermy their axons to the pituitary. In this manner, function was split between neurotransmitter (MCH) and hormonal (MCHL) roles. In support of the dual control theory in Groups 6 and 7, physiological changes in both α-MSH (black background) and MCHL (white background) are produced when background is altered. For example, in the eel (Group 7), MCH induces melanosome aggregation, and a salmon MCHL antibody labels secretory granules in neurons with fibres that terminate on neurohypophysial blood vessels, whose density varies between white and black adapted organisms (Gilham & Baker, 1984; Powell & Baker, 1988) . Plasma α-MSH levels increase in eels with a black background . In zebrafish, in addition to the hormonal status for MCHL suggested by our expression analysis (Figure 2) , a white background increases pmchl mRNA levels, while fasting increases the expression of pmch (Berman et al., 2009) . A black background in zebrafish triggers melanosome dispersion via α-MSH interaction with Mcr1 expressed by melanophores (Richardson et al., 2008) . In two Group 8 species, Starry flounder (Platichthys stellatus (Kang & Kim, 2013) ) and Barfin flounder (Verasper moseri (Mizusawa et al., 2015) ), pmch and pmchl mRNAs increase in white backgroundadapted organisms, although most robustly for pmchl. Because MCHL, but not pomc mRNA/α-MSH, levels change with background in several Group 8 species at the top of the Teleostei lineage, it is possible that during evolution the balance between α-MSH and MCH changed from α-MSH»MCH in Groups 2-4, to a dual mechanism in Groups 6 and 7 (α-MSH = MCH2), to α-MSH«MCHL in Group 8 species (Figure 7) .

Group 9: α-MSH but not MCH regulates background adaptation. MCH+ nerve fibres are absent from the neural lobe of the pituitary of Protopterus annectens, and synthetic salmonid MCHL does not influence skin cells of Lepidosiren paradoxa (Dipnoi) (Baker, 1991; Visconti & de L. Castrucci, 1993) , suggesting that MCH does not have hormonal status and plays no role in skin pigmentation ( Figure 7 ). 6. Groups 10-14: A uni-humoral α-MSH control system for background adaptation and thermoregulation. MCHL is not present in amphibians and reptiles, and MCH appears to lack hormonal status ( Figure 7) . Indeed, while the hypothalamus contains MCH-expressing cells in frogs (Rana esculenta; Group 10), Squamata (Podarcis muralis and Natrix natrix; Group 12) and turtles (Trachemys scripta; Group 13), their axons do not project to the posterior pituitary (Cardot et al., 1994; Lázár et al., 2002) . Thus, α-MSH is the main regulator of background adaptation and may also be important for temperature regulation of ectotherms though colour plasticity (Fernandez & Bagnara, 1991) .

Groups 15 and 16: New roles for α-MSH and MCH. In birds and mammals, MCHL does not exist, MCH+ cells do not extend axons to the pituitary (Cardot et al., 1999; Cortés et al., 2014) , and the pars intermedia pituitary disappears in birds, arguing against a hormonal status for MCH (Figure 7) . Birds rely on carotenoid ingestion for background adaptation, while in mammals α-MSH possibly associates with seasonal colour moulting. Thus, while colour plasticity is important for thermoregulation in ectotherms, in feather/hair covered homoeotherms, skin pigment cells changed with regard to cell type (only melanocytes in birds and mammals), pigment type (eumelanin and pheomelanin) and properties (pigment secretion vs. dispersion/aggregation).

Together, our experimental data and comparison of evolutionary studies suggest that MCHL but not MCH has hormonal status in teleosts. A conserved α-MSH peptide and the presence of MCH or MCHL constitute the "genetic basis" for background adaptation that together with the "hormonal status" for MCHL explains the different background adaptation mechanisms observed in vertebrates. 

The authors declare no conflict of interest.

GEB designed the experiments. GEB and JZZ performed the experiments and analysed the data. GEB and SM wrote the paper.

https://orcid.org/0000-0001-6154-9513

Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides

Seasonal rhythm of the plasma level of alpha-melanocyte stimulating hormone

Melanin-concentrating hormone: A neuropeptide hormone affecting the relationship between photic environment and fish with special reference to background color and food intake regulation

A Newly characterized melanotropin in proopiomelanocortin in pituitaries of an elasmobranch, Squalus acanthias

Structure-activity relationship studies of melanin-concentrating hormone (MCH)-related peptide ligands at SLC-1, the human MCH receptor

Zur chromatischen Hautfunktion der Amphibien. Pflüger, Archiv Für Die Gesammte Physiologie Des Menschen Und Der Thiere

Melanin-concentrating hormone: A general vertebrate neuropeptide

The role of melanin-concentrating hormone in color change

Neuronal organization of the melanin-concentrating hormone system in primitive actinopterygians: Evolutionary changes leading to teleosts

Cloning and expression of melanin-concentrating hormone genes in the rainbow trout brain

Further observations on the distribution and properties of teleost melanin concentrating hormone

Changes in pituitary and plasma levels of MSH in teleosts during physiological colour change

Convergence of biannual moulting strategies across birds and mammals

Characterization of two melanin-concentrating hormone genes in zebrafish reveals evolutionary and physiological links with the mammalian MCH system

Melanopsin photoreception in the eye regulates light-induced skin colour changes through the production of α-MSH in the pituitary gland

Seeing the light to change colour: An evolutionary perspective on the role of melanopsin in neuroendocrine circuits regulating light-mediated skin pigmentation

The tree of life and a new classification of bony fishes

Phylogenetic classification of bony fishes

Anatomy, function and regulation of neuropeptide EI (NEI)

Recent developments in our understanding of the avian melanocortin system: Its involvement in the regulation of pigmentation and energy homeostasis

Pigmentation pathway evolution after whole-genome duplication in fish

Structure and regulation of the mouse melanin-concentrating hormone mRNA and gene

Melanin-concentrating hormone-producing neurons in reptiles

Melaninconcentrating hormone-producing neurons in birds

Evolution of the melanocortin system

Mutational analysis of evolutionarily conserved ACTH residues

Birth of two chimeric genes in the hominidae lineage

Characterization of melanin-concentrating hormone (MCH) and its receptor in s: Tissue expression, functional analysis, and fasting-induced up-regulation of hypothalamic MCH expression

Morphology of the pars intermedia and the melanophore-stimulating cells in Xenopus laevis in relation to background adaptation

Evolution of POMC: Origin, phylogeny, posttranslational processing, and the melanocortins

Presence of the delta-MSH sequence in a proopiomelanocortin cDNA cloned from the pituitary of the galeoid shark, Heterodontus portusjacksoni

60 YEARS OF POMC: Melanocortin receptors: Evolution of ligand selectivity for melanocortin peptides

Melanophore-contracting hormone (MCH) of possible hypothalamic origin in the catfish

Tadpoles respond to background colour under threat

Cyclic colour change in the bearded dragon Pogona vitticeps under different photoperiods

Effect of background color and low temperature on skin color and circulating alpha-MSH in two species of leopard frog

Alpha-MSH (melanocyte stimulating hormone) and MCH (melanin concentrating hormone) actions in Bufo ictericus ictericus melanophores

Neuropeptide glutamic acid-isoleucine (NEI)-induced paradoxical sleep in rats

Evidence for the participation of a melanin-concentrating hormone in physiological colour change in the eel

Differential melanin-concentrating hormone gene expression in two hypothalamic nuclei of the teleost tilapia in response to environmental changes

Biphasic effect of MCH on alpha-MSH release from the tilapia (Oreochromis mossambicus) pituitary

Identification, cellular localization and in vitro release of a novel teleost melanin-concentrating hormone gene-related peptide

Functional characterization of two melanocortin (MC) receptors in lamprey showing orthology to the MC1 and MC4 receptor subtypes

The appearance of proopiomelanocortin early in vertebrate evolution: cloning and sequencing of POMC from a lamprey pituitary cDNA library

Phylogenetic timing of the fish-specific genome duplication correlates with the diversification of teleost fish

Genetics, development and evolution of adaptive pigmentation in vertebrates

The pigmentary effector system. VIII. The dual receptive mechanism of the amphibian background response

Identification of prohormones and pituitary neuropeptides in the African cichlid

Synthesis, storage, and release of MSH in the pars intermedia of the pituitary gland of Xenopus laevis during background adaptation

A swimming mammaliaform from the Middle Jurassic and ecomorphological diversification of early mammals

The pineal complex, melatonin, and color change in the lamprey Lampetra

Functional characterization of two melanin-concentrating hormone genes in the color camouflage, hypermelanosis, and appetite of starry flounder

Functions of melanin-concentrating hormone in fish

Characterization of melanin-concentrating hormone in chum salmon pituitaries

Transgenic medaka overexpressing a melanin-concentrating hormone exhibit lightened body color but no remarkable abnormality

The measurement of melanin-concentrating hormone in trout blood solid-phase

Absence of a gamma-melanocyte-stimulating hormone sequence in proopiomelanocortin mRNA of chum salmon Oncorhynchus keta

The melanocortin system in fugu: determination of POMC/AGRP/MCR gene repertoire and synteny, as well as pharmacology and anatomical distribution of the MCRs

MEGA X: Molecular evolutionary genetics analysis across computing platforms

Distribution of melanin-concentrating hormone-like immunoreactivity in the central nervous system of Rana esculenta

Salmo salar and Esox lucius full-length cDNA sequences reveal changes in evolutionary pressures on a post-tetraploidization genome

Evolution of melanocortin receptors in cartilaginous fish: Melanocortin receptors and the stress axis in elasmobranches

The Atlantic salmon genome provides insights into rediploidization

Topology of feather melanocyte progenitor niche allows complex pigment patterns to emerge

Seasonal and photoperiod-induced changes in the secretion of a-melanocyte-stimulating hormone in Soay sheep: Temporal relationship with changes in b-endorphin, prolactin, follicle-stimulating hormone, activity of the gonads and growth of wool and horns

Adaptive evolution of multiple traits through multiple mutations at a single gene

Teleost fish-specific preferential retention of pigmentation gene-containing families after whole genome duplications in vertebrates

A phenology of the evolution of endothermy in birds and mammals

Suntanning in hammerhead sharks

The isolation and amino acid sequence of an adrenocorticotrophin from the pars distalis and a corticotrophin-like intermediate-lobe peptide from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias

Melanin-concentrating hormone overexpression in transgenic mice leads to obesity and insulin resistance

Feeding-induced changes of melanin-concentrating hormone (MCH)-like immunoreactivity in goldfish brain

Synthesis and bioactivity studies of two isosteric acyclic analogues of melanin concentrating hormone

The effect of geographic location, breed, and pituitary dysfunction on seasonal adrenocorticotropin and α-melanocyte-stimulating hormone plasma concentrations in horses

Two precursors of melanin-concentrating hormone: DNA sequence analysis and in situ immunochemical localization

Identification of mRNAs coding for mammalian-type melanin-concentrating hormone and its receptors in the scalloped hammerhead shark Sphyrna lewini

Involvement of melanin-concentrating hormone 2 in background color adaptation of barfin flounder Verasper moseri

Inhibiting roles of melanin-concentrating hormone for skin pigment dispersion in barfin flounder, Verasper moseri

Interrelation between melanocyte-stimulating hormone and melanin-concentrating hormone in physiological body color change: Roles emerging from barfin flounder Verasper moseri

The rat melanin-concentrating hormone messenger ribonucleic acid encodes multiple putative neuropeptides coexpressed in the dorsolateral hypothalamus

Identification of a single melanin-concentrating hormone messenger ribonucleic acid in coho salmon: Structural relatedness with 7SL ribonucleic acid

Resolution of ray-finned fish phylogeny and timing of diversification

Distribution of lamprey adrenocorticotropin and melanotropins in the pituitary of the adult sea lamprey, Petromyzon marinus

Neuroendocrine control of physiological color change in Chameleo gracilis

Structures of two kinds of mRNA encoding the chum salmon melanin-concentrating hormone

Origin of adult pigmentation: Diversity of pigment stem cell lineages and implications for pattern evolution

Structural studies of nerve terminals containing melanin-concentrating hormone in the eel, Anguilla anguilla

Evolutionary genetics: The evolution of plumage patterns

The evolutionary origin and diversification of feathers

mc1r pathway regulation of zebrafish melanosome dispersion

Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function

Effects of background adaptation on the pituitary and plasma concentrations of some pro-opiomelanocortin-related peptides in the rainbow trout (Salmo gairdneri)

Adaptive reptile color variation and the evolution of the MC1R gene

Genetics of colouration in birds

Deletions of the N-terminal regions of the human melanocortin receptors

ACTH: A short introductory review

Exploring the evolutionary history of melanin-concentrating and melanin-stimulating hormone receptors on melanophores: Neopterygian (holostean) and chondrostean fishes

Cognition and the evolution of camouflage

Cutaneous melanosis in lungfishes (LEPIDOSIRENIDAE)

Mass spectrometric map of neuropeptide expression and analysis of the gamma-prepro-tachykinin gene expression in the medaka (Oryzias latipes) brain

Regulation of melanophore responsiveness in the background-adapted medaka, Oryzias latipes: Change in the intracellular signaling system

The involvement of melanotrophins in physiological in the dogfish Scyliorhinus canicula colour change

Concomitant duplications of opioid peptide and receptor genes before the origin of jawed vertebrates DeSalle R

Isolation and characterization of melanotropins from lamprey pituitary glands

Evolutionary significance of proopiomelanocortin in agnatha and chondrichthyes

Possible involvement of melanin-concentrating hormone in food intake in a teleost fish, barfin flounder

Structures of two genes coding for melanin-concentrating hormone of chum salmon

Tribute to R. G. Boutilier: Skin colour and body temperature changes in basking Bokermannohyla alvarengai (Bokermann 1956)

Reversible colour change in Arthropoda

Alpha-MSH, the melanocortin-1 receptor and background adaptation in the Mozambique tilapia

Cellular localization and role of prohormone convertases in the processing of pro-melanin concentrating hormone in mammals

Melanotropin receptors in the cartilaginous fish, Potamotrygon reticulatus and in the lungfish, Lepidosiren paradoxa

Synthesis of a cyclic melanotropic peptide exhibiting both melaninconcentrating and -dispersing activities

Why colour in subterranean vertebrates? Exploring the evolution of colour patterns in caecilian amphibians

Pineal-specific agouti protein regulates teleost background adaptation

The prohormone convertases PC1 and PC2 mediate distinct endoproteolytic cleavages in a strict temporal order during proopiomelanocortin biosynthetic processing

Function and underlying mechanisms of seasonal colour moulting in mammals and birds: What keeps them changing in a warming world