The structure and evolution of the melanocortin and MCH receptors
in fish and mammals
Darren W. Logan,aRobert J. Bryson-Richardson,aKayleene E. Paga ´n,a,b
Martin S. Taylor,a,1Peter D. Currie,aand Ian J. Jacksona,*
aMRC Human Genetics Unit, Western General Hospital, Edinburgh, EH4 2XU, UK
bUniversity of Puerto Rico, Department of Biochemistry, PO Box 365067, San Juan, Puerto Rico
Zebrafish are an excellent genetic model system for studying developmental and physiological processes. Pigment patterns in zebrafish
are affected by mutations in three types of chromatophores. The behavior of these cells is influenced by alpha-melanocyte-stimulating
hormone (?MSH) and melanin-concentrating hormone (MCH). Mammals have five ?MSH receptors (melanocortin receptors) and one or
two MCH receptors. We have identified the full complement of melanocortin and MCH receptors in both zebrafish and the pufferfish, Fugu.
Zebrafish have six melanocortin receptors, including two MC5R orthologues, while Fugu, lacking MC3R, has only four. We also
demonstrate that Fugu and zebrafish have two and three MCHR genes, respectively. MC2R and MC5R are physically linked in all species
examined. Unlike other species, we find the Fugu genes contain introns, one of which is in a conserved location and is probably ancestral.
We also detail the differential expression of the zebrafish genes throughout development.
© 2003 Elsevier Science (USA). All rights reserved.
Keywords: Zebrafish; Danio rerio; Fugu rubripes; Melanocortin receptor; Melanin-concentrating hormone receptor; Molecular evolution; Pigmentation
The melanocortin receptors are a family of G-protein
coupled, 7-transmembrane receptors that respond to small
peptide hormones derived from pre-opiomelanocortin
(POMC). Humans and mice have five such receptors,
MC1R to MC5R [1–4], that have diverse functions in a
range of tissues, as shown by analysis of mutant phenotypes
[5–14]. Members of the melanocortin receptor family share
between 39% and 61% amino acid identity within a species
and it appears that MC3, MC4, and MC5 receptors are more
similar to each other than they are to MC1R or MC2R
(reviewed in ).
MC1R is expressed on the surface of mammalian mela-
nocytes and, signalling through this receptor by ?-melano-
cyte-simulating hormone (?MSH), regulates the synthesis
of dark eumelanin. Lack of signalling by inactivation of the
ligand or receptor or by binding of an inverse agonist, agouti
signalling protein (ASP), results in synthesis of red or yel-
low phaeomelanin [5–7,14,15]. MC2R is the receptor for
adrenocorticotrophic hormone (ACTH), another derivative
of POMC, and is essential for normal adrenal function
[8,16]. Two receptors, MC3R and MC4R, are expressed in
a number of different locations in the brain, including the
hypothalamus where activation by ?MSH and other POMC
peptides regulates feeding behavior, energy metabolism,
and the partitioning of fuel stores into fat [9,10,17]. The
agouti-related protein, AGRP, can antagonize activation of
these receptors in the brain in an analogous fashion to the
action of ASP on MC1R . The fifth member of the
family, MC5R, is expressed in a range of tissues including
the skin, where it is required for the synthesis of sebum by
the sebaceous glands .
The role of ?MSH on pigmentation was first identified in
lower vertebrates [19,20], although the cellular response to
the hormone is different from the response by mammalian
melanocytes. Other physiological functions of melanocort-
* Corresponding author. Fax: ?44-0-131-467-8456.
E-mail address: email@example.com (I.J. Jackson).
1Present address: Wellcome Trust Centre for Human Genetics, Oxford
University, Oxford, OX3 7BN, UK.
ins in these animals are largely unexplored, and differences
or similarities with mammals will be instructive. The ze-
brafish, Danio rerio, is an excellent vertebrate model organ-
ism in which many mutations have been identified in de-
particular, there are a large number of mutations affecting
zebrafish pigmentation [23,24]. Besides being an important
model in biological research, a smaller genome and exten-
sive regions of conserved synteny with the human genome
mean it is a useful system for comparative genomics .
In contrast to mammals, most fish, reptiles, and amphibia
have a highly complex and sophisticated pigmentary sys-
tem. There are at least six distinct pigment cell types iden-
tified in fish, although zebrafish have only three: iri-
dophores, xanthophores, and melanophores. It is now
known that ?MSH exerts an influence on each of these cell
types, but its role in melanophore function has been best
characterized. Fish melanophores respond to ?MSH by rap-
idly dispersing aggregated, peri-nuclear localized melanin
pigment throughout the cell body and into the dendritic
extensions (reviewed in ).
The reverse function, by which dispersed pigment is
induced to aggregate, is due to the action of a second
peptide hormone,a melanin-concentrating
(MCH), on melanophores [27,28]. The rapid redistribution
of pigment allows the zebrafish to adapt its color to the
general tone of its background. This dynamic response to
?MSH and MCH is quite different from that of mammalian
melanocytes. When administered in vivo to mice, ?MSH
produces a change in melanin biosynthesis while MCH
appears to have no effect, because mice lacking MCH or its
receptor are normally pigmented [29–31]. However, recent
work has shown that a MCH receptor is expressed on
human melanocytes and MCH can antagonize the action of
?MSH on these cells in culture . Therefore, in humans
at least, MCH may have a role in melanogenic regulation.
MCH has been observed in the brains of lampreys, elasmo-
branches, amphibians, mammals, and birds where it appears
to have a neuromodulatory role without melanin-aggregat-
ing activity (reviewed in ). The hormone has a single
receptor in mice (which we will term MCHR1, but has been
identified as GPR24 previously) expressed in the hypothal-
amus and the mutation of which results in mice that are lean
yet hyperphagic. The lean phenotype is due to a combina-
tion of hyperactivity and increased metabolic rate .
Humans have two MCH receptors, which we will term
MCHR1 and MCHR2 [34–37]. Other mammals also have
two MCH receptors, although rats have only one. Whether
rodents have lost one of the receptors or other mammals
have duplicated their is not known, although the presence of
a pseudogene in rabbits and guinea pigs may suggest that
the rodent lineage has lost one functional copy . It is not
known if MCH plays a role in feeding and metabolism of
fish, but a full comprehension of fish melanocortin function
will require an understanding of MCH function both on
melanophores and in the brain.
Recently three melanocortin receptors have been identi-
fied in zebrafish, one of which appears to be orthologous to
MC4R, and two orthologues of MC5R . We wanted to
analyze zebrafish melanocortin receptors, not only to gain
an understanding of the evolution of this receptor family,
but also to provide the foundation for work on the genetics
of a variety of physiological systems.
A second fish model organism, the pufferfish, Fugu ru-
bripes, has been well studied as a genomic model. It has a
genome approximately eight-fold smaller than mammals,
but has been shown to encode a similar gene repertoire to
other vertebrates . The compact nature of the Fugu
genome is reflected by a dearth of interspersed repetitive
elements, and small intronic and intergenic distances rela-
tive to other vertebrates. The teleost lineage that includes
both Fugu and zebrafish diverged from the mammalian
lineage approximately 450 million years ago. Analysis of
the melanocortin receptors in Fugu provides an additional,
well diverged set of sequences for evolutionary comparison.
The compact genome may also permit identification of
genomic relationships more readily than other larger ge-
We have identified the full repetoire of melanocortin and
MCH receptor genes from both zebrafish and Fugu, which
will provide a basis for further studies. Additionally, the
sequences provide useful information about the evolution of
these gene families.
Results and discussion
Sequence analysis of the melanocortin receptor gene
Extensive DNA sequence data are available for both
zebrafish and Fugu. We screened shotgun sequence data-
bases of both genomes using iterated BLAST analysis and
assembly. The assembled DNA sequences were confirmed
by PCR and resequencing.
We identified six melanocortin receptors in zebrafish and
four in Fugu. Table 1 shows the degree of amino acid
identity between the fish and human receptors. To examine
the evolutionary relationship of the receptors, the phylogeny
of the proteins was examined. A tree summarizing the
phylogenetic relationship of zebrafish and Fugu receptors,
along with the five receptors from human, mouse, and
chicken, is shown in Fig. 1. The tree shows that the two
zebrafish sequences identified previously as two MC5R
sequences indeed appear to be a recently duplicated pair of
receptors in the fish lineage. It is interesting that the dupli-
cation seems to have occurred before zebrafish and Fugu
diverged, yet we identify only one MC5R sequence in Fugu.
The Fugu genome sequence is not yet complete. However,
the coverage afforded by the available sequence data is
estimated to be 5.6? , giving a ?95% chance of a read
containing the sequence. Furthermore, an extensive dataset
D.W. Logan et al. / Genomics 81 (2003) 184–191
consisting of a 6? coverage of DNA sequence from a
closely related pufferfish, Tetraodon nigroviridis, also con-
tains a sequence from only a single MC5R gene. The prob-
ability that these combined databases lack the second MC5R
gene by chance is less than 0.002, and it is highly probable
that MC5RB is absent from the Fugu lineage. The phylo-
genetic relationship of the zebrafish and pufferfish MC5R
proteins suggests that the genes duplicated early in fish
evolution, and that one of them (orthologous to zebrafish
mc5ra) was lost in the pufferfish lineage. However, it is
possible that the duplication occurred after the zebrafish/
pufferfish lineages separated, and selective constraints on
one of the pair may have relaxed, allowing it to diverge
Both zebrafish and Fugu contain single orthologues of
MC1R, MC2R, and MC4R, MC3R, however, appears to be
absent from Fugu. Again, consideration must be given to
the depth of sequence coverage of the Fugu data, but we
also do not identify MC3R gene sequences in the Tetraodon
sequence trace dataset. It is probable that the pufferfish has
lost the MC3R gene, which must be considered when the
role of MC3R in fish and other vertebrates is studied.
We believe that we have identified the complete mela-
nocortin receptor family from both euteleost (pufferfish)
and otocephela (zebrafish) lineages of teleost fish. These
will provide the underpinning for further work on the phys-
iological function of these genes in zebrafish.
Genome structure and chromosomal location of
The chromosomal location of the six zebrafish genes was
determined by analysis of radiation hybrids. Each gene was
successfully mapped in duplicate with a LOD score ?11
and a net LOD difference ?6, indicating significant linkage.
The six genes localize to five linkage groups (Table 2). In
most cases the zebrafish orthologues were located in regions
that showed conserved synteny with both their mouse and
human counterparts. For example, zebrafish mc1r maps to
linkage group (LG) 18, very close to cytochrome oxidase
subunit 4 (cox4), and distal of tyrosine aminotransferase
(tat). Orthologous genes are found in the same order within
a 15 Mb region on human chromosome 16 and mouse
The duplicated mc5r genes, as has been previously re-
ported, are both linked to genes that are also duplicated in
the zebrafish genome, which have themselves a single or-
thologue in both the mouse and humans, but none of these
genes are synthenic with MC5R in the mouse, and human
genomes . However, radiation hybrid mapping of ze-
brafish mc2r places it within 5 cR (approx. 740 kb) of
mc5ra. This gene pair has also been closely linked by
genetic mapping in the mouse to chromosome 18 and by
cytogenetic analysis on human chromosomes to 18p11.2. It
appears that the duplicated chromosomal segment that con-
tains the zebrafish mc5r paralogue has subsequently lost
We examined the genome sequence assemblies for both
the mouse and human, and found that the MC2R and MC5R
genes are very closely linked, and are convergently tran-
scribed from opposite DNA strands (Fig. 2). They are sep-
arated by approximately 40 kb (human) and 67 kb (mouse).
We made an assembly of Fugu genomic sequence around
MC5R and MC2R and found that in this organism they were
also convergently transcribed, and the termination codons
of the two coding regions were separated by only a few
Fig. 1. Phylogenetic analysis of the melanocortin receptor protein family
by neighbor-joining. The receptor sequences from human (Hs), mouse
(Mm), chicken (Gg), zebrafish (Dr), and Fugu (Fr) are shown. The num-
bers at the nodes indicate percentage of 1000 bootstrap replicates. The
human melanin-concentrating hormone receptor was used to root the tree.
Protein sequence identities of melanocortin receptors
Hs MC1R Hs MC2R Hs MC3R Hs MC4R Hs MC5R
Dr MC5RA 45.2
Note. Human (Hs) full-length sequences are compared with zebrafish
(Dr) and Fugu (Fr) sequences. The best human match to each fish gene is
indicated by a shaded box. The values are percent identity.
D.W. Logan et al. / Genomics 81 (2003) 184–191
kilobases. The intergenic distance between this gene pair
has thus been compressed by 20–30 fold relative to mam-
mals (Fig. 2).
None of the full-length melanocortin receptor genes thus
far reported, of which there are more than 40, have introns
in the coding region, with the possible exception of a spliced
variant of human MC1R that is of unknown significance.
However, when we examined the assembled sequences
from Fugu, we find that two of the melanocortin receptor
genes, MC2R and MC5R, do contain introns. The Fugu
MC5R gene contains three introns in the coding region
(situated 156, 263, and 452 nucleotides downstream from
the ATG initiator). Fugu MC2R contains only one intron
(after nucleotide 353), but is in an identical position and
phase relative to coding sequence as MC5R intron 3 (Fig. 2).
There have been previous reports of intronless genes the
Fugu orthologues of which contain introns, and of addi-
tional introns present in Fugu [40,41]. This example, how-
ever, gives a useful insight into the evolution of the mela-
nocortin receptor genes. MC2R and MC5R are among the
most diverged in the gene family, and yet share a common
intron in Fugu that is absent from the other family members
in this species and from all family members currently iden-
tified in other species. It seems probable that this is an
ancient intron that has been lost from virtually all members
of the gene family, but has been retained in two in Fugu.
Extensive examination of other GPCR genes in several
species, including invertebrates, reveals an intron in the
same phase in the equivalent codon in a small but phyoge-
netically diverse number of these genes, which suggests that
the intron is very ancient, but has been lost in the majority
of GPCR genes (manuscript in preparation).
Expression of melanocortin receptor genes in zebrafish
In order to assess whether all the zebrafish genes we have
identified are transcribed, we performed a reverse-transcrip-
tion-PCR on mRNA from different stages of zebrafish de-
velopment and from adults. Fig. 3 shows that all of the
genes are transcribed. Transcripts from all genes except
mc2r can be detected at the earliest time point of two days,
although mc4r has a particularly weak signal.
Throughout larval development mc1r is expressed. It is
during this period that fish melanophores differentiate and
develop. In adult fish this gene has a lower apparent signal
from head RNA than body. This observation is consistent
with the smaller number of melanophores found in the head,
although quantitative RT-PCR would be required to confirm
this difference. The pair of mc5r genes do not have the same
pattern of expression as each other. It appears that mc5ra is
largely embryo-specific; no transcript can be detected from
the adult head and the adult body gives a relatively weak
signal. By contrast, mc5rb has a robust signal in both em-
bryonic and adult RNA.
MC3R and MC4R in mammals have exclusively postna-
tal functions. However, the level of transcription of their
orthologues in zebrafish appears much higher in embryonic
RNA than in adults. Nevertheless, transcription of both
genes in adult fish appears to be higher in the head than the
body tissues, which is consistent with these receptors having
a role similar to their mammalian function in the hypothal-
A more restricted pattern of transcription is shown in
mc2r. In mammals the gene expression is found only in the
adrenals, and we see in zebrafish that the gene is expressed
only from the fifth day of development, and is found only in
the torso and not the head of adults.
All six zebrafish melanocortin receptor genes are tran-
scribed during embryonic development. Zebrafish offer a
Chromosomal position of melanocortin and MCH receptors
Gene Mouse map Human mapZebrafish map
8, 123.5 Mb
18, 68.7 Mb
2, 173.5 Mb
18, 67.2 Mb
18, 68.7 Mb
15, 82 Mb
16q24.3, 91.8 Mb
18p11.2, 14.0 Mb
20q13.2, 54.6 Mb
18q22, 57.9 Mb
18p11.2, 13.9 Mb
22q13.3, 37.7 Mb
6q16.3, 100.2 Mb
18, 318.1 cR
16, 313.0 cR
8, 320.4 cR
2, 365.8 cR
16, 308.0 cR
19, 174.2 cR
6, 291.5 cR
3, 184.4 cR
20, 340.0 cR
Note. Human cytogenetic locations are shown for each gene followed by
position on the Ensembl genomic assembly. Mouse gene locations are
shown as chromosome number followed by position on genomic assembly.
Zebrafish locations are represented by linkage group followed by position
in centiRays (cR). Mammals have only a single orthologue of MC5R and
MCHR1; the table does not imply a closer relationship to either zebrafish
Fig. 2. The conserved linkage relationship between MC5R and MC2R
throughout vertebrate evolution. Species are indicated along with chromo-
some number where they are known. The direction of MC5R (black boxes)
and MC2R (grey boxes) transcription is indicated by arrows. Each box
represents a single exon and the lines between boxes of the same color
indicate introns. The single intron in Fugu MC2R is in an identical position
and phase to the third intron in Fugu MC5R. Intergenic distances (dashed
lines) are calculated from assembly of genomic sequence, except in ze-
brafish where it is estimated from centiRay distance by radiation hybrid
D.W. Logan et al. / Genomics 81 (2003) 184–191
valuable and relatively simple method to establish gene
function. Injection of chemically modified oligonucleotides
(termed “morpholinos” after the morpholine moeity that
replaces ribose), complementary to the translation initiation
codon and upstream sequence of an mRNA into fertilized
zebrafish eggs, prevents translation of the targeted mRNA
. Morpholino inhibition may be a useful method of
determining melanocortin receptor function during embry-
Identification of melanin-concentrating hormone receptor
genes in fish
The pigmentary function of ?MSH in teleost fish, pre-
sumably mediated by MC1R, is functionally antagonized by
the action of MCH (reviewed in ). It is also possible, by
analogy with the mammalian energy homeostasis pathways,
that the function of MC3R and MC4R may be balanced by
action of MCH in the central nervous system. We therefore
identified MCHR sequences from zebrafish and Fugu in the
whole genome shotgun datasets. Fugu contains two MCHR
genes and zebrafish has three.
A neighbor-joining tree showing the relationship be-
tween the fish sequences and representative mammalian
MCHR protein sequences is in Fig. 4. It is evident from this
analysis that an initial duplication of the MCHR gene oc-
curred before the divergence of the vertebrate lineage, and
both zebrafish and Fugu have clear MCHR1 and MCHR2
orthologues. The presence of a single MCH-receptor gene in
rodents must be due to the loss of one of these genes
(MCHR2) after the separation of rodents from the other
mammals. The third MCHR gene in zebrafish is a late
duplication in fish evolution. It appears that a duplication of
MCHR1 has taken place after the divergence of euteleosts
Fig. 3. RT-PCR-assayed expression of MC and MCH receptors in zebrafish embryos and adult tissue. The embryo ages, tissue origin, and controls (neg. ?
negative control, DNA ? positive control) are indicated at the top of the figure with the genes denoted at either side. Zebrafish b-actin was used as a control
for RNA quality. Zebrafish mc2r amplification shows a second, smaller, PCR fragment in all RNA samples. Sequencing indicates that this is a PCR artifact
unrelated to mc2r sequence.
Fig. 4. Phylogenetic analysis of the melanin-concentrating hormone recep-
tor protein family by neighbor-joining. The receptor family from human
(Hs), mouse (Mm), domestic dog (Cf), rat (Rn), zebrafish (Dr), and Fugu
(Fr) are shown. The numbers at the nodes indicate the percentage of 1000
bootstrap replicates. The human melanocortin 1 receptor was used to root
D.W. Logan et al. / Genomics 81 (2003) 184–191
from the zebrafish lineage, although a loss from the former
lineage is possible.
We determined the map location of the three zebrafish
genes by an analysis of a radiation hybrid panel (Table 2).
Zebrafish mchr1b maps to LG3, which is near to the ze-
brafish orthologues of human ribosomal protein L3 (RPL3),
mini-chromosome maintenance deficient 5 (MCM5), and
eukaryotic translation initiation factor 3 subunit 7 (EIF3S7).
These genes, and human MCHR1, are all located within a
5.2 Mb region of human chromosome 22. We identified no
synteny between human MCHR and zebrafish mchr1a.
However, we did find other similarly duplicated genes:
mchr1a maps slightly distal of netrin 1a (ntn1a) on LG6,
while mchr1b maps slightly distal of netrin 1 (ntn1). We
searched the Fugu genome assembly for netrin 1 ortho-
logues and could identify only one, providing further evi-
dence that this particular genomic segment may have du-
plicated after the divergence of the Fugu and zebrafish.
Fig. 3 shows the expression of the zebrafish mchr genes. It
appears that mchr1a is embryo specific. No transcripts are
detected in adult tissues, and only weak expression can be
found in embryos younger than five days. Expression of
mchr1b appears stronger than mchr1a, can be detected in
adults and young embryos, and is strongest between days
four and seven of development. The expression of mchr2
seems to parallel that of mc1r; it can be detected from the
earliest time point, throughout the period of larval melano-
phore function, and appears to be expressed at higher levels
in the adult torso compared to the head. Therefore, based
solely on RT-PCR-determined expression patterns, we
would suggest that either MCHR1B or MCHR2 is likely to
be the receptor that acts to regulate pigmentation.
The presence and possible functional overlap of three
MCH receptor genes in zebrafish may make the analysis of
their function by mutagenesis or by morpholino-inhibition
problematic. The role of a single mouse melanin-concen-
trating hormone receptor in feeding behavior is well estab-
lished . In humans, however, the action of MCHR2
remains to be elucidated and there is some evidence that
MCHR1 can regulate pigmentation in addition to its neuro-
modulatory role . We believe further characterization of
the spatial expression of the zebrafish receptors may provide
an insight into the full extent of MCH function in both
humans and fish.
Our data suggest that the entire MC and MCH receptor
families were present before the divergence of the ray-
finned fish from the tetrapod lineage approximately 450
million years ago. Using the same search methods in the
genomes of the invertebrates Anopheles gambiae, Drosoph-
ila melanogaster, Caenorhabditis elegans, and Ciona intes-
tinalis, we have failed to find MC/MCH receptors, suggest-
ing that this subfamily of seven transmembrane GPCRs may
be vertebrate specific. However, the remarkable conserva-
tion of amino acid sequence in many of these receptors
throughout more than 400 million years of vertebrate evo-
lution implicate an important role in normal physiological
The duplication, and potential functional complementa-
tion, of mc5r in the zebrafish is consistent with a hypothesis
of tetraploidization in the teleost lineage . However, an
independent duplication in the zebrafish lineage could also
explain the existence of two mchr1 orthologues in zebrafish
but only one in Fugu. The identification of a common intron
in mc2r and mc5r in Fugu, combined with the linkage of
both genes in both mammals and teleosts, suggests that an
ancestral melanocortin receptor may not have been intron-
less. Indeed, this intron may have been present even earlier,
because the conserved intron is seen in other G-protein-
coupled receptors, including human, zebrafish, and Fugu
MCHR2 (manuscript in preparation).
In conclusion, we have identified, mapped, and charac-
terized the expression of the melanocortin receptor and
melanin-concentrating hormone receptor families in teleost
fish. These receptors are good candidates for involvement in
a range of processes, including background adaptation and
feeding regulation. Their identification in zebrafish provides
an excellent system in which to investigate their function
Materials and methods
Identification of gene sequences
TBLASTN analysis of the Danio rerio, Fugu rubripes,
and Tetraodon nigroviridis whole genome shotgun data-
bases was carried out using the chicken MC1R amino acid
sequence (accession number D78272) and the mouse
MCHR1 sequence (NM_145132). Matching reads were as-
sembled using Phred/Phrap  and extended by repeated
BLAST searching and assembly.
The predicted coding sequence of each gene was ampli-
fied by PCR using oligonucleotide primers designed using
Primer3 . These primer sequences are available on re-
quest. PCR products were purified using a QIAquick PCR
purification kit (Qiagen, Crawley, UK) and sequenced.
Sequencing reactions were performed using the ABI
PRISM BigDye Terminator cycle sequencing kit according
to the manufacturer’s instructions and analyzed on a ABI-
377 automated sequencer (Applied Biosystems, War-
rington, UK). The sequences were aligned and compared
with the database assemblies using Sequencher v.4.0.5
(Gene Codes, Ann Arbor, MI, USA).
D.W. Logan et al. / Genomics 81 (2003) 184–191
Mapping was performed by PCR on the LN54 radiation
hybrid panel . Using the following primers, orientated
to3?: MC1Rf, TCAAAAGGACTGTGGAAGGG;
MC1Rr, AAAGTCACGAGACAGGCGAT; MC2Rf, CAC-
CAGCTGGAACTCTCTGA; MC2Rr, GCCACAATCAC-
CAAGAGGTT; MC3Rf, GCTGCAACATCTGACTCTGC;
MC3Rr, CAAACGCACAAATTGGTCAC; MC4Rf, TCT-
MC5Rar, CAGGCTGTGTGTCCGAGTAG; MC5Rbf, GCT-
TGTGGTGGAAGACCATT; MC5Rbr, GGGACAGGAAA-
MCHR1bf, TGACTTTGGACCGATACTTGG; MCHR1br,
CGTGCTCATTACGGACACAA; MCHR2f, TTGCAAT-
CGTCCATCCTACA; MCHR2r CTGGTGGGATGCTG-
GATACT. Each assay was performed twice and discor-
dance between duplicate assays was less than 5%. Map
placement was calculated using RHMAPPER (World Wide
Web URL: http://mgchd1.nichd.nih.gov:8000/zfrh/beta.cgi).
Genes neighboring zebrafish melanocortin and melanin-
concentrating hormone receptors were identified from the
Zebrafish Information Network (ZFIN), (World Wide Web
URL: http://zfin.org/). They were used in a TBLASTX
search against the human and mouse whole genome assem-
bly using Ensembl  (World Wide Web URL: http://
www.ensembl.org/). The best hit was then used in a recip-
rocal TBLASTN search against the zebrafish assembly. If
this identified the original zebrafish sequence as the best hit,
the genes were designated as orthologues. Syntenic relation-
ships were established by cross referencing neighboring
genes on ZFIN, with the position of their mammalian or-
thologues using Ensembl. All data from Ensembl were from
the most current data assembly on 2 October 2002.
Sequence alignment and phylogenetic analysis
The Fugu and zebrafish melanocortin receptor full-
length amino acid sequences were aligned with their mouse,
human, and chicken orthologues using ClustalW . Mel-
aligned with their human, mouse, rat, and dog orthologues.
These sequences were obtained from Genbank. Melanocor-
tin receptor accession numbers: NM_002386, NM_000529,
XM_009545, NM_005912, XM_008685, NM_008559,
D78272, AB009605, AB017137, AB012211, AB012868.
Melanin-concentrating hormone receptor accession num-
bers: NM_005297, NM_032503, AY112658, AY112659,
Phylogenetic trees were built by MEGA v.2.2  using
a neighbor-joining method. Phylogeny was tested using a
bootstrap resampling strategy with 1000 replicates. The
human MCHR1 sequence was used to root the melanocortin
receptor tree and the human MC1R sequence was used to
root the melanin-concentrating hormone receptor tree.
One male and one female AB strain zebrafish was de-
capitated and total RNA isolated from the pooled head
tissues and body tissues using the RNAgents system (Pro-
mega, Southampton, UK). Zebrafish embryos from AB
strain matings were raised at 28.5°C and total RNA isolated
at defined days after fertilization. The RNA was then
RNase-Free DNase treated at 37°C for 15 minutes and
purified according to the manufacturer’s instructions. The
quantity of RNA was controlled using a GeneQuant spec-
trophotometer (Amersham, Little Chalfont, UK). RNA was
reverse transcribed using a First Strand cDNA Synthesis kit
(Roche, Lewes, UK) and the product used as a template for
PCR. The specific primers for each gene were identical to
those used for genetic mapping, with the following excep-
tions: MC2Rf, CTCTGCTCCTGATCCTCCTG; MC2Rr,
CCTGGTTCTCATTCAAGCAC; MC3Rf, TGCATCTCT-
CTTGTGGCATC; MC3Rr, GGTGAGGACAGGACAC-
CAGT; MCHR1af, AAATGCCAGGCTAAACAAACA;
MCHR1ar, AAGACGAAGGGACACAGTGG; MCHR1bf,
TGGTGTGGATCCTCTCACTG; MCHR1br, CCGGATG-
GCAACAATAAACT. Primers specific to zebrafish beta-
actin (NM_131031) were used as a positive RNA control:
GAAGGTGGTCTCGTGGAT. A PCR with total RNA
from each sample as a template was used as a negative
control. No DNA contamination was detected. The condi-
tions for PCR were 5 minute denaturation, then 60 s. at
94°C, 60 s. at 60°C, and 90 s. at 72°C for 35 cycles, then a
final 10 minutes at 72°C. The PCR products were analyzed
on an ethidium bromide-stained, 1.2% agarose gel, and
reverse images recorded. Each PCR was carried out at least
twice, with the same result achieved each time.
We thank Marc Ekker (Ottawa Health Research Insti-
tute) for providing radiation hybrid DNA and are grateful to
Sally Cross, Ian Smyth and Tom Van Agtmael for energetic
discussions. K.E.P. is the recipient of a Minority Interna-
tional Research Training Grant from the John E. Fogarty
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