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Chance caught on the wing:
cis-regulatory evolution and the origin
of pigment patterns in Drosophila
Nicolas Gompel*†, Benjamin Prud’homme*, Patricia J. Wittkopp†, Victoria A. Kassner & Sean B. Carroll
1
Howard Hughes Medical Institute and Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison, Wisconsin 53706, USA
* These authors contributed equally to this work
† Present addresses: Department of Zoology, Cambridge University, Downing Street, Cambridge CB2 3EJ, UK (N.G.); Department of Molecular Biology and Genetics, 227 Biotechnology Building, Cornell University,
Ithaca, New York 14853, USA (P.J.W.)
...........................................................................................................................................................................................................................
The gain, loss or modification of morphological traits is generally associated with changes in gene regulation during development.
However, the molecular bases underlyi ng these evolutionary changes have remained elusive. Here we identify one of the molecular
mechanisms that contributes to the evolutionary gain of a male-specific wing pigmentation spot in Drosophila biarmipes,a
species closely related to Drosophila melanogaster. We show that the evolution of this spot involved modifications of an ancestral
cis-regulatory element of the yellow pigmentation gene. This element has gained multiple binding sites for transcription factors
that are deeply conserved components of the regulatory landscape controlling wing development, including the selector protein
Engrailed. The evolutionary stability of components of regulatory landscapes, which can be co-opted by chance mutations in
cis-regulatory elements, might explain the repeated evolution of similar morphological patterns, such as wing pigmentation
patterns in flies.
The evolution of new morphological features is due predominantly
to modifications of spatial patterns of gene expression. Changes in
the expression of a particular gene can result from alterations either
in its cis-regulatory sequences or in the deployment and function of
the trans-acting transcription factors that control it, or both.
Understanding the evolution of new morphological traits thus
requires both the identification of genes that control trait formation
and the elucidation of the cis- and trans-modifications that account
for gene expression differences.
Evolution of cis-regulatory elements has been proposed to be a
major source of morphological diversification because mutations in
regulatory elements can produce discrete tissue-specific expression
pattern changes while avoiding deleterious pleiotropic effects
1–3
.In
the best-studied cases of gene expression changes underlying
morphological divergence, cis-regulatory modifications have been
proposed
4–6
, occasionally suggested by genetic evidence
7–10
, but have
only rarely been formally demonstrated
11
or analysed at the mol-
ecular level
12,13
. It is currently not known whether the evolution of
new morphological traits occurs largely through the modification of
pre-existing cis-regulatory elements or from the generation of new
elements; neither is it understood how many or what kinds of
modifications are required for a regulatory element to drive a novel
pattern.
To address these issues, we have analysed the evolution of a
conspicuous male-specific wing pigmentation pattern in Drosophila
biarmipes, a species closely related to Drosophila melanogaster
14
(Fig. 1). Wing pigmentation patterns in insects are highly diversified
and have various biological functions including mimicry, camou-
flage, thermoregulation, and mate selection
15
.InD. biarm ipes, the
sexually dimorphic wing pattern is associated with a courtship
behaviour in which males display their wings conspicuously to the
females, suggesting a function for this spot in mate choice
16,17
. This
wing spot has evolved recently in some species of the D. melanoga-
ster group, such as D. biarmipes, and it is absent from close outgroup
species such as D. pseudoobscura
17
(Fig. 1).
Formation of wing pigmentation results from the conversion of
melanin precursors diffusing from the veins into pigment deposits
at specific positions along the wing, wherever converting proteins
are present
18
. The product of the yellow (y) gene is required for the
production of black pigments, and the distribution of its product
prefigures adult pigmentation patterns
11,19
. The Yellow protein is
expressed uniformly at low levels throughout the developing wings
of D. melanogaster and D. pseudoobscura, where it imparts a low
overall level of melanic pigmentation. In contrast, in D. biarmipes,
in addition to the low, uniform expression, Yellow protein is highly
expressed in an anterior distal spot
19
(Fig. 1). This tight correlation
between a novel Yellow expression pattern and a novel pigmentation
Figure 1 Expression of the Yellow protein prefigures adult wing pigmentation. The
conspicuous spot of dark pigmentation present at the tip of the male wing of Drosophila
biarmipes (left) is a new trait evolved among species of the Drosophila melanogaster
group
14,46
(about 15 Myr of divergence; divergence time is 60–80 Myr for the family
Drosophilidae
30
), superimposed on the ancestral pattern of uniform grey shading and
darker veins found both in D. melanogaster and in D. pseudoobscura, a species from the
sister D. obscura group (25 Myr of divergence
29,30
). In all three species the male pupal
distribution of Yellow in the wing, revealed by a specific antibody (right), foreshadows the
adult pigmentation.
articles
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pattern prompted us to ask whether regulatory evolution at the y
locus underlies the novel distribution of the Yellow protein in
D. biarmipes, or whether this is due to changes in trans-acting
regulators of y.
Regulatory changes in cis to the yellow locus
To test whether the observed differences in Yellow expression
between D. bi armipes and D. melanogaster (Fig. 1) are due to
changes at the y locus, we transformed D. melanogaster with green
fluorescent protein (GFP)-reporter constructs containing non-
coding DNA from the D. biarmipes y (y
bia
) locus. If relevant
evolutionary changes have occurred in cis, then the reporter gene
might be regulated in D. melanogaster in a manner similar to the
native y gene in D. biarmipes. If, however, the changes have occurred
in trans, the D. biarmipes y regulatory element might drive reporter
expression similar to that of the y gene in D. melanogaster (that is,
uniformly). We found that D. melanogaster transgenic flies carrying
the entire 5
0
region (8 kilobases; Fig. 2a) of the y
bia
gene (5
0
y
bia
)
express GFP in the pupal wings in a pattern similar to the native
D. biarmipes Yellow expression (Fig. 2b). Low levels of GFP are
uniformly distributed across the wing, and higher levels of GFP are
confined to the distal part of the anterior compartment. This result
shows that the transcription factors deployed in the developing
wing of D. melanogaster recognize y
bia
cis-regulatory sequences.
Furthermore, the D. biarmipes-like expression pattern in a
D. melanogaster trans-regulatory context shows that evolutionary
changes in Yellow expression involve primarily cis-regulatory modi-
fications at the y locus, which presumably entail the gain (or loss) of
binding sites for transcription factors.
The 5
0
y
bia
element does not recapitulate the precise restriction of
the native spot of Yellow expression; higher levels of reporter protein
expression extend along the proximal–distal axis, indicating that
additional regulatory differences exist between D. biarmipes and
D. melanogaster. Additional reporter constructs suggest that these
differences are trans effects or are due to cis-acting elements located
outside the region we have tested. The unique intron of the
D. biarmipes y gene does contain another cis-regulatory element
(for all developing sensory bristles) but has no activity in the wing
other than in these sense organs. Furthermore, a transgene contain-
ing the 5
0
non-coding, 5
0
untranslated region, first exon, intron and
second exon sequences (partial locus, Fig. 2a) is expressed in a
similar pattern to that of the 5
0
y
bia
element, indicating that the
differences in y expression are not due to any of these sequences
(data not shown).
Having localized major regulatory differences to the y
bia
5
0
region,wenextinvestigatedwhetherthenovelcis-regulatory
activity of the y
bia
region arose in a pre-existing regulatory element
or evolved de novo in the D. biarmipes lineage.
Figure 2 Cis-regulatory changes at the yellow locus are responsible for species-specific
differences in Yellow distribution. a, The organization of the y locus is similar in Drosophila
melanogaster, Drosophila biarmipes and D. pseudoobscura. b, The entire 5
0
region of
D. biarmipes y, comprising sequences between the coding sequences of y and the closest
predicted gene (CG3777), is sufficient to drive reporter GFP expression in D. melanogaster
at a time and in a pattern similar to those of y expression in native D. biarmipes. The y
bia
intron does not drive wing expression other than in the marginal sensory bristles, and the
partial locus drives expression in a pattern similar to the entire 5
0
region of y
bia
(not
shown). Black boxes, coding sequence; grey boxes, fragments analysed in transgenic
constructs.
articles
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Evolution of a wing-specific cis-regulatory element
In D. melanogaster, analysis of the y regulatory region has revealed
that an 800-base-pair (bp) element located 1 kilobase upstream of
the transcription start site, named the w ing
mel
element (Fig. 2a), is
sufficient to drive gene expression throughout the pupal wing
(Fig. 3b) and is necessary for adult wing pigmentation
11,20,21
.We
hypothesized that functional modifications of this element might
account for differences in Yellow expression between the wings of
D. melanogaster and D. biarmipes. There is strong sequence con-
servation of this portion of the y locus between the two
species (Fig. 3a and Supplementary Fig. 1). We transformed
D. melanogaster with a GFP-reporter construct containing a 920-
bp fragment from D. biarmipes orthologous to the D. melanogaster
wing element (wing
mel
), termed wing
bia
(Fig. 2a). This fragment
drives a reporter pattern resembling that driven by the 5
0
y
bia
element, with slightly less contrast between the levels of overall
expression in the wing and in the anterior distal area (not shown). A
larger fragment encompassing wing
bia
,namedwing
bia large
(1,542 bp; Fig. 2a) drives a reporter pattern identical to the 5
0
y
bia
element (Fig. 3b). These results indicate that the sequences required
for the strong anterior-distal activation of Yellow expression in
D. biarmipes pupal wings are located within and immediately
adjacent to a wing-specific cis-regulatory element that is ortholo-
gous to the wing
mel
element.
To determine whether the novel wing
bia
sequences evolved within
an ancestral w ing cis-regulatory element, we examined D. pseudo-
obscura, an outgroup species that belongs to a clade generally devoid
of wing pigmentation patterns other than the grey (light black)
homogeneous shading (Fig. 1). Phylogenetic character reconstruc-
tion suggests that the pigmentation spot was present in the common
ancestor of D. biarmipes and D. melanogaster and has been lost in the
D. melanogaster lineage
22,23
. There is substantial sequence conserva-
tion at the y gene between D. biarmipes and D. pseudoobscura (Fig. 3a
and Supplementary Fig. 1), which allowed us to identify a region in
D. pseudoobscura that is orthologous to the wing
bia
element, named
wing
pse
(724 bp). This wing
pse
element drives ubiquitous wing
expression (Fig. 3b), demonstrating that a functional wing element
is ancestral to the D. melanogaster/D. biarmipes lineage and that
sequences within and/or adjacent to this element were modified to
control high levels of expression in the anterior distal part of the
wing in D. biarmipes .
To understand the organization of the wing
bia
element and to
localize its novel functional sequences, we further dissected the
wing
bia
element. We found that the sequences necessary for the
anterior distal expression are separable from those controlling the
general wing expression in D. biarmipes. Two complementary, non-
overlapping sequences of the wing
bia
element, right
bia
and left
bia
(Fig. 2a), drive respectively ubiquitous expression throughout the
wing blade and strong activation in the anterior distal area of the
wing (Fig. 3b) (as do the complementary subfragments from
the wing
bia large
element, right
bia large
(not shown) and left
bia large
;
Fig. 4b). A similar dissection clearly separates two wing-specific
complementary functions in D. pseudoobscura (ubiquitous
expression, and expression around the veins) but yields non-
functional elements in D. melanogaster (Fig. 3b). These results
indicate that sites in both regions of the w ing element are required
for its function in D. melanogaster and D. pseudoobscura, and that
some or all of the novel sequences in D. biarmipes responsible for the
specific anterior distal wing expression of Yellow are located in
the left
bia large
element, hereafter referred to as the spot element. The
distinct and robust activities of the two parts of the wing
bia
element
raise the possibility that the wing element has been subfunctiona-
lized in D. biarmipes into two elements controlling expression
throughout the wing and in the spot, respectively.
Figure 3 The cis-regulatory sequences governing spot formation evolved in the context of
an ancestral wing enhancer. a, Conservation of the wing element sequence between
D. biarmipes (bia) and D. melanogaster (mel)orD. pseudoobscura (pse) determined by
Vista
47
with a 10-base-pair window length; only conservation above 75% is shown as
solid boxes. Arrows show the boundaries of the left and right fragments. b, Reporter
expression driven by the orthologous wing elements and its subfragments left and right
(columns) of D. melanogaster (top), D. biarmipes (middle; the wing
bia large
element is
shown) and D. pseudoobscura (bottom), all expressed in D. melanogaster. The ubiquitous
expression driven by the outgroup species wing
pse
element (expression is present in vein
cells at a lower levels comparable to those in left
pse
) shows that the sequences
responsible for the spot pattern in D. biarmipes have evolved in the context of an ancestral
wing regulatory element. The sequences controlling the spot pattern are separable from
those controlling general expression in D. biarmipes (left and right). Note that the posterior
boundary of activity of the left
bia
construct lies near or at the anterior–posterior
compartment boundary.
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Multiple sites evolved in the wing spo t element
In principle, the evolution of the spot pattern could arise through
gaining binding sites for a single transcription factor that is
expressed precisely in the cells that form the spot pattern. Alter-
natively, the spot pattern could result from the evolution of a
combination of binding sites for multiple activators, as well as
potential repressors that might restrict expression to this area.
Resolution of these possibilities bears on the general question of
the number of steps involved in the evolution of new patterns of
gene expression and cis-regulatory element function.
To distinguish between these possibilities, we derived a series of
reporter gene constructs with smaller portions of the 675-bp spot
element. A 196-bp construct (335–530; Fig. 4a) retained activity in
the anterior distal region of the wing, although we noted that
reporter expression now extended into the posterior compartment
(Fig. 4d). This suggested that one or more sites critical for activation
Figure 4 The spot element evolved through the acquisition of sites for both activators and
repressors. a, Schematic of the 675-base-pair spot element showing the boundaries of
deletion constructs and the location of identified binding sites. b, d–f, Expression of GFP
driven by the spot element (b) and related constructs. c, The anterior border of expression
of the selector gene engrailed abuts the spot element expression domain. d, A 196-base-
pair element drives the spot pattern but is derepressed in the posterior compartment.
e, Deletion of bp 425–453 abolishes activity of the spot element, indicating that sites
required for activation lie within this region. f, Disruption of two characterized Engrailed
binding sites from the spot element derepresses reporter expression in the posterior
compartment. g, The two candidate sites are bound specifically by the Engrailed protein
in vitro. Increasing amounts of Engrailed homeodomain–GST fusion protein (0.25–5 nM)
specifically shift labelled DNA oligonucleotides representing native sequences containing
putative binding sites (left part of each gel) but not sequences in which Engrailed sites
have been mutated (underlined in the sequences, right part of each gel). Addition of anti-
GST antibody supershifts complexes. Addition of specific (spe.) or non-specific (non-spe.)
unlabelled competitor DNA (þ, 50 ng; þþ, 500 ng) reveals the specificity of the
formation of complexes. Supershift and competition experiments were performed in the
presence of 5.0 nM protein.
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resided in this 196-bp element and that one or more sites necessary
for the restriction of expression from the posterior compartment
resided outside it.
To localize further sequences required for activation of the spot
element, we constructed a series of small deletions spanning the
length of the 196-bp element (Fig. 4a, e, and data not shown). We
found that a fragment lacking the internal sequences from bp 425 to
453 completely lacked reporter expression (Fig. 4e). This indicates
that sequences required for activation in the spot are located within
or overlap with bp 425–453. Together, these results indicate that
sites for both at least one activator and one repressor have evolved in
the spot element.
Direct regulation of the spot element by Engrail ed
We next sought to identify potential trans-acting factors that
regulate the spot element in D. biarmipes. The conspicuous posterior
boundary of gene expression observed with the left
bia
and spot
elements (Figs 3b and 4b) is reminiscent of the compartment
boundary of the wing
24
defined by the anterior border of expression
of the selector transcription factor Engrailed
25
(Fig.4c).The
posterior expansion of GFP expression in the deletion constructs
shown in Fig. 4d would be consistent with posterior repression of
the spot element by Engrailed.
To test whether Engrailed might be a direct regulator of the
wing
bia
element, we searched the spot sequence for putative
Engrailed binding sites
26
and identified several candidate sites.
Two of these sites, clustered within 43 bp, are specific to the
D. biarmipes s pot element (absent from D. melanoga ster and
D. pseudoobscura elements; Supplementary Fig. 1) and one site is
located outside the 196-bp construct that exhibits some reporter
expression in the posterior compartment (Fig. 4d). Gel-shift experi-
ments on the native and mutated versions of these two sites showed
that Engrailed binds specifically to them in vitro (Fig. 4g). Disrup-
tions of these two Engrailed binding sites in the context of the spot
element result in the specific derepression of reporter gene
expression in the posterior compartment (Fig. 4f). These results
show that the selector protein Engrailed directly represses the
expression of the y gene in the posterior compartment of the wing
and is one of the inputs that shapes the contours of the wing spot in
D. biarmipes.
Multistep and multigenic evolution of the spot
Although multiple cis-regulatory modifications at the y locus have
produced a profound evolutionary change in Yellow protein
expression, it is important to ascertain whether changes in this
one gene are sufficient for the evolution of the physical trait or
whether additional evolutionary events are required. We have found
that changes at y alone are not sufficient to create a pigmentation
spot.
D. mela nogaster y mutants carrying the D. biarmipes y gene
(Fig. 2a, partial locus) recover only their species-specific pigment
patterns; no wing spot is generated (not shown). Additional loci
must therefore be involved.
The formation of pigment patterns is a multigenic process, and
evolution at other pigmentation loci could also contribute to
pattern evolution
18,27,28
.ThemalespotofD. biarmipes is also
associated with the localized downregulation of the melanin-
inhibiting product of the ebony (e) gene during wing development
19
(Fig. 5c), in a pattern that is approximately the inverse of Yellow
expression. This suggests that, at least, both the repression of e and
the activation of y are necessary for the formation of a dark spot.
Consistent with this hypothesis is the observation that in
D. melanogaster e mutants carrying the y
bia
partial locus transgene
(Fig. 2a), a slight darkening is observed specifically in the anterior
area of the wing where yellow is strongly expressed (data not
shown). However, this darkening is not comparable to the intense
pigmentation spot of D. biarmipes. Changes in the expression of
other pigmentation genes must also be involved. Furthermore, we
have not been able to test whether changes in the trans-acting
regulatory network of D. biarmipes might also contribute to the
unique patterns of gene expression in the area of the wing spot.
Taken together, these results indicate that the evolution of the novel
pigmentation pattern of D. biarmipes required changes at multiple
loci.
To determine whether the inverse regulation of expression of y
and e is a general mechanism for the evolution of novel wing
pigmentation patterns, we examined the expression of these pro-
teins in D. guttifera, a species that separated from the D. melano-
gaster lineage about 40 Myr ago
29
. This species has independently
evolved a strikingly different and more complex wing pigmentation
pattern (Fig. 5a). We found that the pattern of expression of the two
proteins also exhibits an inverse relationship with higher levels of
Yellow (Fig. 5b) and lower levels of Ebony (Fig. 5d) in the pupal
wing where the eventual adult pigmentation spots will form. This
indicates that the evolution of both y and e expression is involved in
the formation and evolution of novel wing pigmentation patterns in
drosophilids.
Chance caught on the wing: novelty by co-option
In drosophilid flies, the shape of the wings and the pattern of
Figure 5 Concerted changes in the expression of Yellow and Ebony underlie the evolution
of novel wing patterns. a, The distant species D. guttifera (a member of the D. quinaria
group
48
) has evolved a complex pattern of dark spots located at the intersection of wing
veins and where campaniform sensilla form. The grey shading is also reinforced in some
interveins. b, The pupal distribution of Yellow also prefigures this adult pattern. c,In
Drosophila biarmipes, the spatial repression of Ebony is also associated with the formation
of the adult male spot of pigmentation
19
. d, This repression of Ebony associated with
pigmentation patterns seems to be general, because it is also seen in D. guttifera, where
the adult spots will form.
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© 2005 Nature Publishing Group
venation have not changed much over 60–80 Myr of evolution
30,31
.
Their development and patterning are largely understood in
D. melanogaster and the regulatory proteins involved are con-
served
32
. One such protein, the selector protein Engrailed, is a
deeply conserved feature of the compartmental organization of
arthropod segments and appendages. In the Drosophila wing,
Engrailed is part of the regulatory circuit that sequentially organizes
the patterning of the anterior–posterior axis
33
. Here we have shown
that the activity of this transcription factor has been co-opted to
control a feature of the novel wing pigmentation pattern in
D. biarmipes through the evolution of specific binding sites within,
or in the immediate vicinity of, a wing-specific regulatory element
of the y gene. Because the expression driven by the spot element is
also spatially modulated in D. melanogaster, this indicates that other
conserved components of the wing trans-regulatory landscape (that
is, one or more activators) have similarly been co-opted by the
evolution of binding sites within the y wing element.
These findings suggest a general means by which novel expression
patterns and characters can arise (Fig. 6a). Specifically, the random
mutation of ancestral cis-regulatory elements (including point and
insertional mutations) generates potential binding sites. If and
when these sites can be recognized by transcription factors
expressed in cells in which the ancestral element is active, the
pattern or level of gene expression may be modified (Fig. 6a), in a
manner similar to the mechanism of gene co-option demonstrated
by the vertebrate crystallin genes
34
. The patterns of expression of the
eligible transcription factors are initially cryptic with respect to the
target gene or trait, but these cryptic ‘prepatterns’ are revealed once
functional binding sites have evolved in target genes. In this sense,
and in this example, evolution is precisely a matter, as Jacques
Monod put it, of ‘chance caught on the wing’
35,36
.
This model has two specific implications for the evolution of
novel wing patterns. First, it explains how the observed diversity of
wing pigmentation patterns might result from combinations of the
numerous transcription factors expressed in the wing. Each of these
combinations might constitute a distinct prepattern for pigmenta-
tion genes such as y or e, provided that the corresponding binding
sites evolve in the proper cis-regulatory context. For instance, some
of the spots on the wing of D. guttifera surround the sensory organs
located on the veins, which form at similar positions in most
drosophilids. This raises the possibility that transcription factors
involved in the positioning of these landmark organs have been co-
opted to change y or e regulation in D. guttifera. Second, this model
might explain the widespread repeated evolution of strikingly
similar pigmentation patterns observed in distantly related species
(for instance, pigmentation patterns similar to those studied here
have evolved independently in other dipterans; Fig. 6b). The
evolutionary stability of the trans-regulatory landscape in droso-
philid wings, reflected by the strong conservation of the wing shape
and venation pattern in the family, suggests that similar pigmenta-
tion patterns might arise in parallel through the repeated evolution
of binding sites for the same transcription factors in cis-regulatory
regions of pigmentation genes. A
Methods
Fly stock and maintenance
Flies were bred at 25 8C on Wheeler–Clayton
37
or cornmeal
38
medium. Constructs were
transformed into D. melanogaster yw mutants as described previously
39,40
. The CantonS
strain was used as wild-type D. melanogaster. Drosophila pseudoobscura, Drosophila
biarmipes and Drosophila guttifera stocks were obtained from the Tucson stock centre
(stock numbers 14011-0121.94, 14023-0361.01 and 15130-1971.10, respectively). All
mature D. biar mipes males of this stock exhibited the wing spot. The en-Gal4 and
UAS–GFP stocks were obtained from the Bloomington Drosophila stock centre.
Immunochemistry
Pupal wings (70 h after puparium formation), still attached to the fly, were allowed to
unfold in water after removal of the pupal cuticle. Flies were transferred to phosphate-
buffered saline (PBS), in which the wings were cut off with a razor blade. Wings were fixed
flat for 15 min between a slide and a coverslip in 4% formaldehyde PBT (PBS containing
0.03% Triton X-100), transferred on ice to a scintillation vial in the fixing solution for a
further 15 min, sonicated briefly in the fixative with a Branson 200 ultrasonic cleaner, fixed
for a further 30 min, washed with PBT, blocked for 1 h in PBT containing 1% bovine serum
albumin, stained with a rat anti-yellow or a rabbit anti-ebony primary antibody
19
and
revealed respectively with a fluorescein isothiocyanate (FITC)-conjugated anti-rat
antibody or FITC-conjugated anti-rabbit IgG antibody (Jackson Immunoresearch).
Cloning
The D. biarmipes y locus sequence was amplified by direct and inverse polymerase chain
reaction (PCR; details are available from the authors on request). The entire 5
0
region was
amplified by PCR with primers designed in the coding sequences of y and the closest gene
upstream of y in D. melanogaster (CG3777; ref. 41). All y fragments for reporter constructs
were cloned into a customized version of the P-based transformation vector
42
from which
one of the two gypsy insulators had been removed and a new polylinker had been added.
Fragments from D. melanogaster and D. pseudoobscura were amplified by PCR from
genomic DNA and specific primers designed using available genome sequences
43,44
(see
Supplementary Table 1 for primer sequences).
Biochemistry
The D. melanogaster Engrailed homeodomain sequence was cloned into the glutathione
S-transferase (GST) gene fusion vector pGEX-3X (Amersham Bioscience). The GST
fusion protein was purified by affinity chromatography
45
. DNA probes for electrophoretic
mobility-shift assays were double-stranded oligonucleotides labelled with
32
P by end-
filling in at both ends with the Klenow fragment of DNA polymerase I. Single-stranded
oligonucleotides were annealed at a final concentration of 0.1
m
M in 10 mM Tris-HCl
pH 7.5 containing 0.1 M NaCl and 1 mM EDTA. Sequences of the oligonucleotide pairs
were as follows: native sequences, 5
0
-TTTCCGCCTAATTGATG-3
0
and 5
0
-TTTCATCAAT
TAGGCGG-3
0
,5
0
-TTTTGCCAATCATTTTT-3
0
and 5
0
-TTTAAAAATGATTGGCA-3
0
;
mutated versions, 5
0
-TTTCCGCCTcccTGATG-3
0
and 5
0
-TTTCATCAgggAGGCGG-3
0
,
TTTTGCCgggCATTTTT-3
0
and 5
0
-TTTAAAAATGcccGGCA-3
0
. Labelled probes were
purified with G50 Sephadex beads (Sigma) on chromatography columns (Bio-Rad).
DNA-binding assays, competition experiments and gel migrations were performed with
10–15 fmol of labelled probes (about 10
4
c.p.m.) following a published protocol
26
;they
were pre-run for 0.5 h and run for 1.5 h at 4 8C on 8% native polyacrylamide minigels in
0.5 £ Tris/borate/EDTA buffer pH 8.3. Non-specific competitor consisted of herring
sperm DNA (Sigma) and the specific competitor was as used elsewhere
26
.
Figure 6 Cryptic prepatterns and the evolution of novel gene expression patterns through
the evolution of cis-regulatory sequences. a, The upper panel shows a model of the
conserved landscape of transcriptional regulators that pattern and shape the Drosophila
wing (green and pink represent repressor and activator, respectively). The evolution of
binding sites for a subset of these regulators in the yellow wing cis-regulatory element
(coloured stars) co-opts them to modify yellow expression (lower panel). Combined with
other regulatory changes at other loci, the changes at the y locus result in a novel
pigmentation spot. b, Wing pigmentation patterns similar to D. biarmipes (left) or
D. guttifera (right) evolved independently in other fly families (here Otitidae and
Lauxaniidae).
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Wing imaging
Adult wings were mounted flat in Hoyer’s medium
38
and processed for bright-field
imaging with a 4 £ or 10 £ dry lens on a Zeiss Axiophot microscope equipped with a
Kontron charge-coupled device camera. For all reporter lines, pupal wings 70–90 h after
puparium formation were mounted flat between a slide and a coverslip in PBT, without
fixation, and imaged immediately with an Optiphot confocal microscope (Nikon)
equipped with a 4 £ dry lens and a BioRad 1024 system. Antibody-stained preparations
were mounted in glycerol and imaged.
Received 30 September; accepted 1 December 2004; doi:10.1038/nature03235.
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Acknowledgements We thank J. True, C. E. Nelson, C. M. Walsh and C. T. Hittinger for technical
advice; J. True, S. Blair and members of the Carroll laboratory for discussions; B. L. Williams and
J. Yoder for critical comments on the manuscript; S. Castrezana and T. Markow (Tucson
Drosophila Stock Center) for providing Drosophila stocks; J. P. Gruber for the Euxesta sample; and
S. Barolo for the pH Stinger vector. N.G. was funded by an EMBO long-term postdoctoral
fellowship; B.P. and N.G. are recipients of a Philippe Foundation fellowship. The project was
supported by the Howard Hughes Medical Institute (S.B.C.).
Competing interests statement The authors declare that they have no competing financial
interests.
Correspondence and requests for materials should be addressed to S.B.C. (sbcarrol@wisc.edu).
The D. biarmipes y locus sequence is deposited in GenBank under accession number AY817623.
articles
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