genetics of sex-linked yellow in the syrian hamster...2009/02/02 · allele in both cats and...
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Genetics of Sex-linked yellow in the Syrian hamster
Azita Alizadeh1, Lewis Z. Hong1, Christopher B. Kaelin1, Terje Raudsepp2, Hermogenes Manuel1, and Gregory S. Barsh1
1Departments of Genetics and Pediatrics, Stanford University, Stanford, CA, USA 94305; 2Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX.
Genetics: Published Articles Ahead of Print, published on February 2, 2009 as 10.1534/genetics.108.095018
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Running title: Sex-linked yellow in Syrian hamsters
Keywords: Agouti gene Syrian hamster Melanocortin signaling Pigmentation patterns Tortoiseshell
Corresponding author:
Greg Barsh Beckman Center B271A Stanford University School of Medicine Stanford, CA 94305 650 723 5035 FAX 650 723 1399 [email protected]
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ABSTRACT
Alternating patches of black and yellow pigment are a ubiquitous feature of mammalian color
variation that contributes to camouflage, species recognition, and morphologic diversity. X-
linked determinants of this pattern—recognized by variegation in females but not males—have
been described in the domestic cat as Orange, and in the Syrian hamster as Sex-linked yellow
(Sly), but are curiously absent from other vertebrate species. Using a comparative genomic
approach, we develop molecular markers and a linkage map for the euchromatic region of the
Syrian hamster X chromosome that places Sly in a region homologous to the centromere-
proximal region of human Xp. Comparison to analogous work carried out for Orange in
domestic cats indicates, surprisingly, that the cat and hamster mutations lie in non-homologous
regions of the X chromosome. We also identify the molecular cause of recessively inherited
black coat color in hamsters (historically referred to as nonagouti) as a Cys115Tyr mutation in
the Agouti gene. Animals doubly mutant for Sly and nonagouti exhibit a Sly phenotype. Our
results indicate that Sly represents a melanocortin pathway component that acts similarly to, but
is genetically distinct from Mc1r, and which has implications for understanding both the
evolutionary history and the mutational mechanisms of pigment-type switching.
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INTRODUCTION
The genetics of coat color is a longstanding and rich model system for studying gene action and
cell signaling. Like classical genetic systems in invertebrate model organisms, the cell types and
tissues that give rise to mammalian hair color are well-characterized and experimentally
accessible, and alterations in gene activity can be easily detected. Coat color mutations are
especially useful for studying genes and pathways unique to vertebrate genomes, and have led to
a deeper understanding of diverse biological processes because much of the molecular machinery
used by the pigmentary system is either shared by, or homologous to, molecules used in other
physiological pathways (BENNETT and LAMOREUX 2003; JACKSON 1997; STEINGRIMSSON et al.
2006).
This approach has proven particularly useful for dissecting the molecular mechanisms of
pigment-type switching, a phenomenon in which melanocytes choose between synthesizing
eumelanin (a relatively insoluble black or brown pigment) or pheomelanin (a cysteine-rich red or
yellow pigment that is soluble in dilute alkali) (SILVERS 1979). A focal point for pigment-type
switching is the Agouti—melanocortin 1 receptor (Mc1r) pathway. Mc1r is a G-protein coupled
receptor expressed in melanocytes, whereas Agouti protein is a paracrine signaling molecule
secreted by specialized dermal cells that inhibits Mc1r signaling (reviewed in BARSH 2006;
CONE et al. 1996). In laboratory mice, gain-of-function mutations that constitutively activate the
Mc1r (e.g. sombre, Mc1rso), cause exclusive production of eumelanin, whereas loss-of-function
Mc1r mutations (e.g. recessive yellow, Mc1re), cause exclusive production of pheomelanin
(ROBBINS et al. 1993). On the other hand, because Agouti is a Mc1r antagonist, gain-of-function
Agouti mutations (e.g. lethal yellow, Ay) cause exclusive production of pheomelanin, whereas
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loss-of-function mutations (e.g. extreme nonagouti, ae) cause exclusive production of eumelanin
(HUSTAD et al. 1995; SIRACUSA 1994).
In the early 1900s, Sewall Wright (1917) concluded that genetic mechanisms that control
pigmentary variation have been largely conserved during mammalian evolution. In particular, the
Agouti phenotype—in which individual hairs display a subapical band of pheomelanin on an
otherwise eumelanic background—is observed across a wide range of mammalian phyla. In the
last decade, gain- and loss-of-function Mc1r mutations have been identified in many domestic
animals (ANDERSSON 2003; KLUNGLAND and VAGE 2003), and Agouti and/or Mc1r variation
have been shown to contribute to pigmentary variation in several natural populations (EIZIRIK et
al. 2003; HOEKSTRA et al. 2006; MUNDY et al. 2004; NACHMAN et al. 2003; STEINER et al.
2007). Most experimental work on the Agouti –Mc1r pathway has been carried out in laboratory
mice. Nonetheless, comparative zoologic studies by Wright (1918) and others (LITTLE 1957;
SEARLE 1968) suggested that some components of mammalian pigment type-switching are not
represented as coat color mutations in laboratory mice. In addition to a regular black and yellow
stripe pattern found in many carnivores that is usually attributed to the Tabby gene (SEARLE
1968), irregular black and orange patches due to the activity of an X-linked gene is also present
in the domestic cat and the Syrian hamster; neither of these phenomena have been described in
laboratory mice.
In the domestic cat, female-specific variegation of black and orange coat color patches has been
appreciated for more than a century, and helped support the initial hypothesis that random and
epigenetically heritable X-inactivation is a universal feature of placental mammals (LYON 1962).
A similar phenotype thought to be caused by mutation of a homologous X-linked gene was
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described in Syrian hamsters more than 40 years ago (ROBINSON 1966). As in cats, hemizygous
male hamsters and homozygous female hamsters are completely yellow, while heterozygous
females exhibit a characteristic tortoiseshell pattern of yellow patches on a black background.
(The terms tortoiseshell and calico are often used to refer to the phenotype, the gene, and/or the
allele in both cats and hamsters; here we use Orange, O, to describe the gene in cats, and
Robinson’s original designation, Sex-linked yellow, Sly, to describe the gene in hamsters).
More than 100 mouse coat color mutations have been characterized over the last century, and
there exist multiple alleles for those directly implicated in pigment-type switching (Agouti, Mc1r,
Atrn, Mgrn, Sox18), none of which lie on the X chromosome (BENNETT and LAMOREUX 2003).
The apparent absence from laboratory mice of an X-linked pigment-type switching mutation is
perplexing in the face of Ohno’s law, which predicts conservation of synteny on the X
chromosome across different mammalian species due to selection against altered gene dosage
(OHNO 1969). Genetic characterization of Sex-linked yellow in Syrian hamsters could shed light
on this paradox, and identify additional components of the pigment-type switching pathway.
However, by contrast to laboratory mice, very little of the classical work on coat color variation
in Syrian hamsters (NIXON et al. 1970) has been developed at the molecular genetic level.
We have established a laboratory-based colony of Syrian hamsters to study the biology and
genetics of Sex-linked yellow together with other coat color variants, and confirmed that the
mutation behaves similarly to what was originally reported (ROBINSON 1966; ROBINSON 1972).
Here we use a comparative genomic approach to generate a molecular genetic map of the Syrian
hamster X chromosome that includes Sex-linked yellow, which provide a basis for molecular
genetic and epistasis studies revealing that Sly represents a component of the melanocortin
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pathway similar to, but independent of, the Mc1r. Comparison to analogous work carried out for
Orange in domestic cats (Schmidt-Küntzel et al.) indicates that the cat and hamster mutations
likely lie in non-homologous regions of the X chromosome, which has implications for
understanding both evolutionary history and the mutational mechanisms of pigment-type
switching.
MATERIALS AND METHODS
Animal husbandry: Sly and other coat color variants in the Syrian hamster are well-recognized
in the hobbyist community, but are not maintained in an academic- or research-oriented setting;
none of the laboratory animal vendors we contacted were aware of a sex-linked and/or variegated
phenotype. We obtained animals with a diverse set of coat color phenotypes from San Joaquin
Valley Fisheries (Fresno, CA), including belted, black, cream, golden, red-eyed dilute,
tortoiseshell, and tricolor (analogous to calico in the domestic cat). For phenotypic
characterization and linkage mapping of Sly, we used tortoiseshell females and black males, and
confirmed that tortoiseshell segregated as an X-linked trait in accord with Mendelian
expectations as indicated in Figures 1, 2, and Table 2. All animal work was carried out under an
APLAC-approved protocol.
Genetic markers: We first developed simple sequence length polymorphism (SSLP) markers
using a comparative genomic strategy in which aligned regions of the mouse and human X
chromosomes were used to identify potential SSLP targets for PCR amplification using Primer 3
(ROZEN and SKALETSKY 2000). Precomputed alignments of the human and mouse genomes were
obtained from ftp://genome.ucsc.edu/goldenPath/hg16/vsMm4/axtTight/. From 39889 regions of
human-mouse X chromosome alignments, we used a custom perl script to identify 7204 pairs of
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alignments in which each pair was separated by a 100 – 300 bp gap (in which no alignment was
recorded, and therefore may have contained an SSLP. We filtered the 7204 pairs of alignments
by the extent of similarity, allowing only those alignments in which there was a perfect human-
mouse match of at least 20 bp, yielding 943 alignment pairs. Among these, suitable primers (that
met default criteria for Primer3) could be designed for 452. Among the 452 alignment pairs, the
intervening (to be amplified) sequence contained one or more SSLP targets in both the mouse
and the rat genomic sequence in ~30 alignments, all of which were used for PCR of hamster
genomic DNA. This approach yielded 7 amplicons that contained polymorphic SSLPs; we then
designed new internal primers directly from the hamster sequence for subsequent amplification
of hamster genomic DNA.
We supplemented the SSLP framework map with SNP-based markers ascertained by
resequencing introns, or between pairs of conserved non-coding sequences. For resequencing
introns, exon-based primer pairs were chosen using a perl script designed to target
oligonucleotides corresponding to little or no codon degeneracy (and therefore likely to be
conserved between mouse and hamster). For resequencing between pairs of conserved non-
coding sequences, we modified the strategy described above to start with precomputed mouse-
human alignments (BRUDNO et al. 2004; KENT et al. 2003). From ~300 amplicons from hamster
DNA, we identified 9 SNPs used to supplement the SSLP-based linkage map. For 7 of the 9 SNP
markers, primers based on mouse sequence yielded robust and specific amplification products
from hamster genomic DNA; in 2 cases (DBarX111 and DBarX118) we developed new internal
primers directly from the hamster sequence.
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Identity of the amplicons for all 16 primer pairs was investigated by aligning the amplified
hamster sequence to the predicted mouse amplicon. In four cases (DXBar53, DXBar 54, DXBar
DXBar 57, and DXBar 119), no significant similarity was observed between mouse and hamster
amplicons, probably due to internal repetitive sequence and/or lack of evolutionary constraint;
therefore the identity of these amplicons relies on comparative mapping results (Figure 3).
Molecular genetic and linkage analysis: SSLP-based markers were amplified with dye-labeled
primers; SNP-based markers were sequenced with dye terminators after amplification; all
markers were separated on a capillary instrument equipped for fluorescence detection. Genotypes
for SSLP markers were called manually; genotypes for SNPs were called with an automated
commercial detection platform (CodonCode). Prior to linkage analysis, Mendelian error-
checking was performed; apparent instances of non-Mendelian transmission were inspected for
potential genotype errors, and either corrected, or dropped from the analysis. An initial map
order was established by minimizing the number of double crossovers, and subsequently refined
with a regression-based analysis for intermarker distance implemented in JoinMap (STAM 1993).
Molecular cytogenetics: Fluorescence in situ hybridization (FISH) of bacterial artificial
chromosome (BAC) probes to metaphase chromosomes from cultured mouse and hamster
fibroblast cells was carried out as described previously (RAUDSEPP and CHOWDHARY 2008). We
selected three mouse BACs (RP24-77C5, RP24-186P1, and RP24-96H13) based on their gene
content as inferred from the July 2007 (Build 37) assembly on the UCSC genome browser,
http://genome.cse.ucsc.edu/ (KAROLCHIK et al. 2003). Each BAC contains one or more genes
that are found in segments whose order is physically conserved among mice, humans, dogs, and
cats; the genes (and BACs) were chosen to lie close to, and possibly flanking, the cat Orange
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gene as depicted in Figure 5. BACs were labeled by nick-translation with digoxigenin (77C5 and
196H13)- or biotin (186P1)-coupled UTP, hybridized to nuclear chromatin in pairwise
combinations (77C5 + 186P1 or 196H13 + 186P1), and detected with indirect
immunofluorescence (RAUDSEPP and CHOWDHARY 2008). For cross-species detection (mouse
probes hybridized to hamster chromosomes), probe concentration was increased by ~ 5 – 10 fold,
and hybridization was carried out for 72 rather than 24 hours.
RESULTS
Sex-linked yellow in hamsters: phenotype and genetics: Commercial suppliers of Syrian
hamsters to research laboratories carry few, if any, coat color variants. We identified a major pet
store supplier in the California central valley, imported several different coat color variants, and
established standardized pedigrees from 7 tortoiseshell females and 6 black males to confirm and
characterize the phenotype and inheritance pattern. In what follows, we refer to mutant and non-
mutant alleles for Sly as SlyTo and Sly+, respectively.
As described originally (ROBINSON 1966), mutant animals (SlyTo/Y or SlyTo/SlyTo) are orange-
yellow in color with a darker dorsum than ventrum, and a sooty appearance, whereas non-mutant
animals (Sly+/Sly+ or Sly+/Y) are “golden”, presumably due to the presence of an Agouti allele
that promotes an extended band of pheomelanin. However, in the hobbyist community today,
SlyTo is almost always described on a presumptive nonagouti (a/a) background such that non-
mutant animals (Sly+/Sly+ or Sly+/Y) are black.
We examined dorsal hair pigment distribution from yellow (SlyTo/Y; a/a), black (Sly+/Y; a/a),
and golden (Sly+/Y; AW/AW) animals. As in the mouse (SUNDBERG 1994), the hamster coat
contains three major types of hair: long guard hairs, and shorter hairs, awls and zigzags, that
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constitute the underfur, and whose relative proportions are similar among yellow, black, and
golden hamsters (Table 1). Pigment-type switching phenotypes are most evident in the zigzag
hairs, which comprise the majority of the underfur. Hairs from golden animals exhibit a dark tip
and base separated by a wide pheomelanic band that extends over ~80% of the hair length
(thereby accounting for a golden rather than a typical brushed Agouti appearance). Hairs from
yellow animals exhibit a dark tip (but less dark than in golden animals), a pale yellow shaft (less
intense than the pheomelanic band of golden hairs), and a white base (Figure 1).
To characterize the inheritance of Sly, we used a three-generation breeding scheme in which
phase could be inferred unambiguously for X-linked markers; tortoiseshell females were always
mated to black males, such that only a single Sly allele was segregating among F3 progeny
(Figure 2A). A total of 155 F3 progeny were obtained from 17 pedigrees. X-linked inheritance of
Sly was confirmed by the absence of yellow females in each pedigree, and a distribution of F3
progeny that did not deviate significantly from that expected for X-linkage (Table 2). These
observations confirmed that each kindred is segregating a single X-linked allele that results in
constitutive pheomelanin production, and provided a sufficient number of meiotic events to carry
out a linkage scan of the hamster X chromosome.
Linkage scan of the X chromosome: We used a combination of comparative genomic and
PCR-based approaches to develop molecular genetic markers for the hamster X chromosome.
Among all possible combinations of markers (7 SSLP markers and 9 SNP markers) with 155 F3
individuals, 1077 assays yielded informative results, and were used to establish a genetic map
(Figures 2B, 3). The hamster X chromosome map covers 46 cM with a median intermarker
distance of 4.1 cM; by comparison, the mouse X-specific region is 72 cM in length (BLAKE et al.
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2003). The hamster map includes markers that map to both ends of the human X chromosome,
but lacks markers corresponding to two key regions in mouse (0 – 20 Mb and 85 – 129 Mb) and
one key region in humans (54 – 98 Mb). Thus, we cannot determine whether the shorter genetic
length of the hamster X chromosome relative to the mouse represents a reduced ratio of
genetic/physical distance in the hamster relative to the mouse, or a terminal region on the
hamster X that has not been captured by our genetic markers.
Comparing the relative position of homologous markers between the mouse, hamster, human,
and dog X chromosomes reveals a considerable degree of intrachromosomal rearrangement
between hamsters and mice, and between hamsters and humans or dogs (Figure 3A). However,
the relative position of markers in the hamster is almost identical to that in the rat (Figure 3B),
consistent with previous molecular cytogenetic work (KUROIWA et al. 2001a; KUROIWA et al.
2001b; KUROIWA et al. 2001c) suggesting that gene order on the X chromosome is largely
conserved between several rodent species (including the rat and the Syrian hamster), though not
in the laboratory mouse.
The Syrian hamster X chromosome is metacentric, but with the long arm composed entirely of
constitutive heterochromatin (DIPAOLO and POPESCU 1973); thus, our molecular map applies to
hamster Xp. Comparison of our results to molecular cytogenetic studies suggest an orientation in
which DXBar111 and DXBar51 lie most centromere-proximal and centromere-distal,
respectively. Sly lies in a 9.6 cM region at the centromere-proximal end, flanked by DXBar111
and DXBar56 (Figure 3). Transposed against the human and dog X chromosomes, this region
defines 8 Mb and 5 Mb intervals, respectively, that also lie close to the human and dog
centromeres.
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Candidate genes for Sly: Agouti and Mc1r: As indicated above, although SlyTo was originally
described on an Agouti (“golden”) background, most animals carrying SlyTo are presumably
nonagouti, such that Sly+/Y and Sly+/ Sly+ animals display a black coat, whereas Sly+/SlyTo
animals display patches of yellow hair on a black background, presumably in regions where
melanocyte clones have undergone epigenetic inactivation of the wild-type X chromosome
(Figure 1A). However, coat color genetics in hamsters is based almost entirely on the similarity
of phenotypes and genetic interactions to those observed in laboratory mice, and there are at least
two hamster genes that can yield recessive inheritance of a black coat (KURAMOTO et al. 2002).
To investigate the underlying molecular basis for black coat color (and, indirectly, the epistasis
relationship between black coat color and Sly), we used a PCR-based strategy to determine the
coding sequence for the hamster Agouti gene. Within exon 4, which encodes the majority of the
Agouti protein-coding region, we detected a G to A mutation that predicts a Cys115Tyr
substitution (Figure 4A) found in both black hamsters and yellow hamsters in our colony, but not
in golden hamsters. This residue is conserved among all known Agouti homologs, and helps
stabilize the key active loop responsible for melanocortin receptor antagonism (MCNULTY et al.
2005). Furthermore, a Cys115Ser mutation in a mouse Agouti transgene has the same effect as a
null allele (PERRY et al. 1996). Taken together, these results indicate that loss-of-function in
Agouti is responsible for the black coat color in our colony.
Similar to what has been described for Orange in tortoiseshell and calico cats (SEARLE 1968), the
patches of black and yellow pigment in Sly+/SlyTo hamsters become larger in the presence of
white spotting mutations (ROBINSON 1972), suggesting that SlyTo acts in a melanocyte-
autonomous manner, similar to Mc1re in laboratory mice. To investigate the possibility that SlyTo
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might represent an Mc1r loss-of-function allele that had been transposed to the hamster X
chromosome, we used oligonucleotide primers based on the mouse and rat genome to PCR-
amplify the hamster Mc1r gene and flanking sequences from yellow and black hamsters. We
identified several synonymous and/or flanking sequence SNPs that were heterozygous in at least
one male animal (data not shown), but did not observe any alterations expected to impair Mc1r
function; thus, as in all other mammals, including cats (EIZIRIK et al. 2003), the hamster Mc1r
appears to be autosomal. We conclude that, similar to Orange in cats, Sly is epistatic to Agouti
but genetically distinct from Mc1r (Figure 4B).
Relationship to Orange in domestic cats: The puzzling similarities between cat Orange and
hamster Sly—puzzling given the evolutionary distance between phyla and the absence of a
similar mutation in any other mammal—prompted us to investigate whether the two mutations
lie in homologous locations. In an accompanying manuscript, Schmidt-Küntzel et al.
demonstrate that Orange lies in a 6 cM, ~11 Mb interval, that corresponds to 96 Mb – 106 Mb,
and 120 Mb – 131 Mb, in the dog and human genomes, respectively. By contrast, flanking
markers for hamster Sly delineate a different interval, on the short arm of the dog (40 Mb – 45
Mb) and human (46 Mb – 54 Mb) X chromosomes (Figure 5C).
A potential caveat to the conclusion that the hamster Sly and cat Orange mutations lie in non-
homologous locations is that the large number of evolutionary breakpoints apparent from
comparing the mouse and hamster X chromosomes to those of other mammals might mask one
or more chromosomal rearrangements in which segments harboring the Orange gene were
transferred to what we currently recognize as the Sly region. To investigate this possibility, we
carried out cytogenetic FISH experiments to evaluate the location of the cat Orange interval in
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mouse and hamster chromosomes. We selected 3 bacterial artificial chromosome (BAC) clones
from a mouse genomic DNA library that each carried genes found in homologous locations in
humans, dogs, and cats, and whose positions were likely to flank the Orange mutation (Figure
5C). Each of the three BACs was hybridized to metaphase spreads from an X/X mouse, an
Sly+/Y hamster, and an SlyTo/Y hamster. As expected, the cytogenetic locations for the three
BACs on the mouse X chromosome correspond to their positions on the physical map, at ~38
Mb (BAC1: 77C5), 39 Mb (BAC2: 186P1), and 47 Mb (BAC3: 196H13) (Figure 5A).
Hybridization of the same BAC probes to hamster chromosomes yielded weaker signals, and it
was not possible to order the probes in double labeling experiments. However, all three probes
hybridized to the same region at the telomeric end of hamster Xp for both the Sly+- and the SlyTo-
bearing chromosomes (Figures 5A, 5B). These results suggest that the X chromosomal region
that contains Orange (as defined by genetic mapping experiments carried out by Schmidt-
Küntzel et al.) remained intact during rodent evolution, and is distinct from the region where Sly
maps in hamsters.
DISCUSSION
In most respects, the effects of Sly in hamsters and Orange in domestic cats are similar to those
caused by Mc1r loss-of-function mutations in other mammals. Both Sly and Orange yield a
uniformly pheomelanic pelage whose effects are epistatic to those of nonagouti, as is the case for
Mc1r loss-of-function in mice (SILVERS 1979), horses (MARKLUND et al. 1996; RIEDER et al.
2001), and dogs (KERNS et al. 2004; NEWTON et al. 2000). Furthermore, both Sly and Orange
interact with white-spotting mutations in a manner that suggests they are melanocyte
autonomous, as is the case for mosaic Mc1r mutations in pigs (KIJAS et al. 2001), in mice
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(LAMOREUX and MAYER 1975), and presumptive Mc1r mutations in guinea pigs and rabbits
(SEARLE 1968). However, both Sly and Orange are genetically distinct from the Mc1r, and
possibly from each other. In what follows, we consider two explanations that could account for
these observations.
Sly and Orange may be orthologs, representing loss-of-function mutations in the same
(previously unrecognized) component of melanocortin signaling that acts in a manner and cell
type similar to that of the Mc1r, but lies genetically upstream, such as a transcription factor
required for Mc1r expression, or a receptor-associated protein required for proper cell surface
expression and/or targeting of the Mc1r. Absence of a similar X-linked phenotype in mammals
other than hamsters and cats could be explained by redundancy, e.g. if the Sly/Orange gene was
duplicated early in mammalian evolution, and that the duplicated genes were redundant with
regard to pigmentary function in most mammals. According to this scenario, most mammals
would then have two Sly/Orange genes, but one of the duplicated genes would have been lost, by
chance, from the hamster and, independently, from the cat lineage.
If Sly and Orange are orthologs, the apparent lack of “homology” in their respective
chromosomal locations (homology as assessed with the comparative mapping approach in Figure
5) likely reflects additional chromosomal rearrangements during rodent evolution, in which the
the Orange gene in an ancestral mammal translocated to the centromere-proximal region of
hamster Xp. Although the FISH-based cytogenetics do not support such a rearrangement, the
sequences tested (3 BACs in an ~10 Mb region) represent only a fraction of the interval, and
cannot exclude the possibility of small chromosomal rearrangements that will only become
apparent from additional genome sequences.
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Alternatively, Sly and/or Orange could represent unusual gain-of-function mutations in which a
(perhaps previously known) component of melanocortin signaling has been activated in
melanocytes. For example, retrotransposition of Agouti into a melanocyte-specific locus on the X
chromosome, or a point mutation of a melanocyte-specific X-linked Gi-coupled receptor that
causes constitutive activation would be expected to yield a phenotype and set of genetic
interactions similar to those observed for Sly and Orange. Both sorts of events would be very
rare—much less frequent than loss-of-function mutations—and therefore might explain why X-
linked pheomelanism has not been observed in species where very large numbers of animals
have been subjected to a natural screen, i.e. laboratory mice and humans. This idea is also
consistent with preliminary analyses of the cat and dog X chromosome sequences, in which the
dog region homologous to that which carries Orange also harbors several pseudogenes whose
origin is autosomal.
A consideration of SlyTo candidate genes must also account for the epistatic relationship between
Sly and Agouti; for example, Sly is unlikely to represent a gene such as Sox18 which acts in the
dermal papilla to promote Agouti expression (FITCH et al. 2003; PENNISI et al. 2000). From this
perspective, genetic interactions between Mc1r and Sly would also be helpful in evaluating
potential candidate genes. Variation at Mc1r is curiously absent from the cat, but prospects for
determining whether Sly lies upstream, downstream, or parallel to Mc1r in the hamster are
encouraging, since recessively inherited cream coat color in the hamster is thought to represent
the action of Mc1r (MAGALHAES 1954; ROBINSON 1964).
These questions can be resolved, of course, by molecular identification of the Sly and Orange
genes, and should be facilitated by availability of additional mammalian genome sequences.
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Defining the molecular genetics of Sly and Orange may also be useful as a tool for understanding
the biology of other color variation patterns thought to affect the distribution of pheomelanin and
eumelanin, such as tabby striping in domestic and wild cats, zebra striping in horse x zebra
hybrids, and rostro-caudal striping seen in squirrels and/or chipmunks (SEARLE 1968).
Acknowledgments
We thank Tyler Vogt for collecting data on hamster hair, Anne Schmidt-Küntzel and Marilyn
Menotti-Raymond for communicating unpublished results, and Angie Crowley of San Joaquin
Valley Fisheries for collecting and organizing founder animals. This work was supported by
funds from the National Institutes of Health, and a Stanford Graduate Fellowship (to L.Z.H.).
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Table 1. Hair type distribution and pigmentary phenotypes
Animal Guard Hairs Awls/Auchenes Zigzags
Golden
(AW/AW; Sly+/Sly+)
Black (2%) Black (4%)
Banded (8%)
White (2%)
Banded (84%)a
Yellow
(a/a; SlyTo/Y)
Dark-tippedb
(2%)
Dark-tippedb (10%)
Dark-tippedb (88%)
a Black tip and base, wide yellow band
b Darkened tip (mixed pigment types); pale yellow shaft; white base
23
Table 2. Phenotype of F3 progeny from Black x Tortoiseshell intercrossa
Phenotype Sex No. progeny
Black Male 39
Yellow Male 33
Tortoiseshell Male 0
Black Female 46
Yellow Female 0
Tortoiseshell Female 37
a Based on cross as depicted in Figure 2A and described in the text.
24
Table 3. Genetic markersa
Marker Name Type Primersb Coordinatesc
DBarX111 2(Rbm10) GTCCTGAAAGGCCCGTGT GAACCAAAGGCCAAAACTGA
20212996-20214673
DBarX 51 1 GGAGGCTTAGCTTACCCCTTT ACGTGAAATTGTGTGCGTGT
40781605-40782207
DBarX 52 1 CACATGAGCTCTGTTGCATCT AAAGTCACCCTCATGGGAAG
53759564-53760159
DBarX 112 2(Bgn) GACCACAACAAAATCCAGGCb
CTCTCACCTGGAGGAGCTTGb 70738334-70738684
DBarX 113 2(Tbl1x) GGACGCTCACACAGGAGAAGb
GTCCTTGGAAGGTTTTGACTGb 74895226-74895824
DBarX 114 2(CNS) CCAGCTTTGGTCATTTGACAGb
AGGGACTTGGAAAGCTCACAb 84947982-84948629
DBarX 53 1 AAGGCAATGGCTTCATTGTT TGCTTCTGACCTTTGAGCAG
129415830-12941667
DBarX 54 1 CCACCTCTGTGAGCACATTC TGTCTAAATGTGTTTCTCACACTCC
131291877-131292275
DBarX 55 1 ATGCACCCCTCCAGTTCTGC TGGATCTGCAGTAATGTGCAGT
136812895-136813493
DBarX 56 1 GAGCGTCCCGGGAGCTCCTT CCACCTCTTACAGGAAGCC
147389767-147390410
DBarX 115d 2(CNS) ATTGCCTGCCAAATCACACTb
TGATTAAGTGACCCCAGAAATCb 156649400-156649723
DBarX 57 1 TCCATTGTTATTCAAGGAAGGAA GCTTTTGTGCAGATTCCCAG
158286467-158286797
DBarX 119d 2(CNS) AGGTGATGTCAGCGGCTCTb
TCTTTCCAGAACTCATCCCCb 158595286-158595666
DBarX 116 2(CNS) GTCGATTAGCATTGGCATTTb
AAAGCATAGCACACACAAGGAb 162665068-162665558
DBarX 118d 2(Prps2) GCTCATTGGTCAGCCAATCT CCATGTGCTTTGTGATTCCA
163801537-163803099
DBarX 117 2(CNS) GAACAGCAAGGAGGAAATTGb
CCTGAAGCCTCCCACTCCb 164867514-164867994
a Marker names correspond to those on Figures 2 and 3; types “1” and “2” refer to SSLP and
SNP markers, respectively, the latter followed either by the gene name or CNS (conserved non-
coding sequence) depending on how the primers were derived. Oligonucleotide primers were
25
originally designed from mouse genome sequence, but unless otherwise stated (see footnote 3),
new primers were generated from the amplified hamster sequence and used for subsequent
genotyping.
b All of the type 1 (SSLP) primers represent hamster sequence. However, for 7 of the 9 type 2
(SNP) primers (those indicated with a superscript), oligonucleotides based on the mouse
sequence yielded robust products when amplifying hamster genomic DNA, and we did not
redesign new hamster primers.
c Physical position of amplicons on the mouse X chromosome based on coordinates from the
July 2007 (build 37) release.
d Not included in Figures 2 and 3 because they added no new map information. Genotypes from
DXBar115 and DXBar119 were identical to those obtained with DXBar57; genotypes from
DXBar118 were identical to those obtained with DXBar117.
26
Legends to Figures
Figure 1. Pigmentary phenotypes of hamsters with different Agouti and Sly genotypes. A,
Yellow, black, tortoiseshell, and golden animals as described in the text. Genotypes are inferred
from X-linked inheritance of the tortoiseshell phenotype, and from molecular genetic analysis of
Agouti as described in Figure 4. B, Representative zigzag hairs from golden (Sly+/Sly+; AW/AW)
and yellow (SlyTo/Y; a/a)animals. Upper panels show ~80% of the hair; the lower 20% of a
“golden” hair is black, and the lower 20% of a “yellow” hair is pale yellow with a white base.
(The dimensions of the hair are such that the shaft is difficult to see in photos that contain the
entire length of the hair). Dashed box indicates region of the hairs shown at higher magnification
in the lower panels, and illustrates that the tip of the “yellow” hair contains a mixture of yellow
and black pigment, while the shaft of the “yellow” hair is pale relative to the corresponding
region of a “golden” hair.
Figure 2. Transmission and linkage of Sly. A, Structure of pedigrees used for segregation and
linkage analysis. Open, hatched, and yellow symbols represent black, tortoiseshell, and yellow
animals, respectively, with associated Sly genotype inferred on the basis of coat color phenotype
and sex. As described in the text, 17 such pedigrees yielded 155 F3 progeny, who were then
genotyped for the molecular markers as indicated. B, C, Haplotype segregation diagram, with
grey or yellow indicating whether the chromosome of origin carried Sly+ or SlyTo, respectively,
and haplotypes organized into single (B) and multiple (C) recombinants. Gene order is based on
multiple regression analysis of intermarker distances; minimizing the number of double
crossovers yields an alternative order for the first three markers, DXBar112 – DXBar52 –
27
DXBar51. Recombination frequency (RF) in cM between each marker and Sly is given on the
right together with the number of informative meioses.
Figure 3. Comparative X chromosome maps for mouse, hamster, human, dog, and rat. (A)
Physical position (rounded to the nearest megabase, Mb) is given for mouse, human and dog
markers, and the location of the pseudoautosomal regions (PAR or PAR1) indicated for mouse
and human. As described in the text, the hamster map refers to Xp (the long arm is
heterochromatic) with the position of the centromere based on comparison to molecular
cytogenetic results (KUROIWA et al. 2001a; KUROIWA et al. 2001b; KUROIWA et al. 2001c). A
homology block on human (46 – 54 Mb) and dog (40 – 45 Mb) Xp corresponds to the position of
Sly in the hamster. (B) Position of markers on the hamster X chromosome relative to the rat.
Figure 4. Genetics and epistasis of Agouti and Sly. A, As described in the text, black hamsters
carry a missense mutation that predicts a Cys115Tyr substitution, predicted to disrupt Agouti
function. B, Sly is epistatic to Agouti because SlyTo/SlyTo; a/a animals exhibit the same phenotype
as SlyTo/SlyTo; AW/AW animals.
Figure 5. Comparative genomics of Sly and Orange. A, Representative images of FISH for
mouse BAC probes 1 and 2 hybridized to mouse or hamster chromosomes, as indicated. (BAC
probes 1, 2, and 3 are, respectively, RP24-77C5, RP24-186P1, and RP24-96H13.) B, Diagram of
BAC hybridization results showing that the region homologous to the cat Orange gene lies in the
centromere-proximal region of the mouse X chromosome but the centromere-distal region of the
hamster X chromosome. By contrast, genetic mapping studies place the hamster Sly gene close to
the centromere of Xp. C, Summary of comparative mapping. The position of the cat Orange
mutation is based on work from Schmidt-Küntzel et al.
A
B
Sly+/Sly+; a/a
Sly+/Sly+; AW/AW
Sly+/Sly+; AW/AW
SlyTo/Sly+; a/a
SlyTo/Y; a/a
SlyTo/Y; a/a
Sly+/Sly+; AW/AW
SlyTo/Y; a/a
DXBar51DXBar112DXBar52DXBar55DXBar54DXBar53DXBar114DXBar113DXBar57DXBar116DXBar117DXBar56SlyDXBar111
DXBar51DXBar112DXBar52DXBar55DXBar54DXBar53DXBar114DXBar113DXBar57DXBar116DXBar117DXBar56SlyDXBar111
41 36 1 5 1 2 1 2 0 2 9 5 3 6 0 1 5 2 3 2 1 3 1 0 3 0 0 1
1 1 1 1 1 1 1
39 ± 4.5 (117)34 ± 6.1 (59)32 ± 5.4 (75)36 ± 4.8 (100) 32 ± 4.4 (112)32 ± 4.3 (116)23 ± 4.3 (97)28 ± 9 (25)15 ± 3.4 (112)11 ± 4.1 (57)11 ± 3.3 (90)4.2 ± 2.1 (95)----5.1 ± 3.5 (39)
A
B
C
Par.
Single recombinant
Multiple recombinant
RF with Sly (n)
SlyTo/Sly+
SlyTo/Sly+Sly+/Sly+
Sly+/Y SlyTo/Sly+
SlyTo/Sly+
Sly+/Y
Sly+/Y
Sly+/Y SlyTo/Y
A
B SyrianHamster
Rat
DXBar111
DXBar51
DXBar112
DXBar113
DXBar114
DXBar53DXBar54DXBar55
DXBar56
DXBar57
DXBar116DXBar117
Mb
1
159
128122120
74
6353484640
12
PAR PAR1
Mb MbcM (intermarker dist.)cen
cen
cen cen
Physical map ofMouse Chr. X
Markername
Genetic map ofHamster Chr. X
Physical map ofHuman Chr. X
152
134
124
10610098
54
46
28
1913119
2.90.3
6.9
0.7
8.1
4.9
4.2
4.1
1.83.0
4.3
5.3
41
20
54
7175
85
129131137
147158163165
DXBar111
DXBar51
DXBar52
DXBar112DXBar113
DXBar114
DXBar53DXBar54DXBar55
DXBar56DXBar57DXBar116DXBar117
Mb
Physical map ofDog Chr. X
124
109
99
847876
45
40
24
131086
Sly
non-agouti
wild-type
Eumelanin(black/brown pigment)
Pheomelanin(red/yellow pigment)
C D P C A S C Q C R F F Human. . . . . . . . . . . . Dog. . . . . F . . . . . . Pig. . . . . . . . . . . . Mouse. . . . . . . . . . . . Syrian hamster (wt). . . . . . Y . . . . . Syrian hamster (non-agouti)
Ser Cys Gln
TCC TGC CAGTCC TAC CAG
Ser Tyr Gln
A
B
AgoutiMc1rSly
109 120
Hamster candidateinterval
(Sex-linked yellow)
Human Chr. X Dog Chr. XCat candidate
interval(Orange)
A B
C
Sly
O
Mb Mb
46
54
120
131
4045
106
96
DXBar111
DXBar56
FCA1464
Rap2117
Mb106
9.6 cM
6 cM
(BAC3)
(BAC2)
(BAC1)
BAC1BAC2BAC3
(BAC3)(BAC2)(BAC1)
(BAC3)(BAC2)(BAC1)
heteroc.
Mouse X/X
Mouse Hamster
Hamster Sly+/Y
cen cenHamster SlyTo/Y
regionhomologous
to Orange
approximatelocation
of Sly