EVOLUTION & DEVELOPMENT 6:5, 325 –335 (2004)
Dynamics and function of intron sequences of the wingless gene during
the evolution of the Drosophila genus
J. Costas,1 P. S. Pereira, C. P. Vieira, S. Pinho,2 J. Vieira, and F. Casares,2
Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua do Campo Alegre 823, Porto 4150-180,
Portugal
Author for correspondence (email: fcasfer@cabd.upo.es)
1
Present address: Unidade de Medicina Molecular, Complexo Hospitalario Universitario de Compostela, Rúa Choupana s/n, Edificio Consultas, E15706 Santiago
de Compostela (A Coruña), Spain.
2
Present address: Centro Andaluz de Biologı́a del Desarrollo (CABD), Universidad Pablo de Olavide, Ctra. de Utrera Km.1, Sevilla 41013, Spain.
SUMMARY To understand the function and evolution of
genes with complex patterns of expression, such as the
Drosophila wingless gene, it is essential to know how their
transcription is regulated. However, extracting the relevant
regulatory information from a genome is still a complex task.
We used a combination of comparative genomics and
functional approaches to identify putative regulatory
sequences in two introns (1 and 3) of the wingless gene and
to infer their evolution. Comparison of the sequences obtained
from several Drosophila species revealed colinear and well-
conserved sequence blocks in both introns. Drosophila
willistoni showed a rate of evolution, in both introns, faster
than expected from its phylogenetic position. Intron 3
appeared to be composed of two separate modules, one of
them lost in the willistoni group. We tested whether sequence
INTRODUCTION
The development and function of cells and organisms depend
on the dynamic regulation of gene expression, a process con-
trolled by sequences lying in vast noncoding regions of their
genomes. Although new computational methods have been
developed to identify cis-regulatory regions from primary se-
quence data (reviewed in Markstein and Levine 2002), the
phylogenetic footprint method (Tagle et al. 1988), based on
the conservation of specific sequences in homologous regions
of distantly related species, is still one of the most useful ap-
proaches. The idea underlying this approach is that functional
regions evolve more slowly than nonfunctional ones, due to a
higher selective constraint. If the species under comparison
show appropriate degree of divergence, only functional se-
quences will remain similar. This method, although pointing
to functional sequences, does not give information regarding
what sort of function that may be (regulation of gene tran-
scription, small noncoding RNA transcription units, sequences
& BLACKWELL PUBLISHING, INC.
conservation in noncoding regions is a reliable indicator of
regulatory function and, if this function is conserved, by
analyzing D. melanogaster transgenic reporter lines harboring
intron 3 sequences from D. melanogaster (Sophophora
subgenus) and the species from the Drosophila subgenus
presenting the most divergent sequence, D. americana. The
analysis indicated that intron 3 contains pupal enhancers
conserved during the evolution of the genus, despite the fact
that only 30% of the D. melanogaster intron 3 sequences lie in
conserved blocks. Additional analysis of D. melanogaster
transgenic reporter lines harboring intron 3 sequences from
D. willistoni revealed the absence of an abdomen-specific
expression pattern, probably due to the above-mentioned loss
of a regulatory module in this species.
involved in chromatin structure, etc.). Nevertheless, it has
been shown that phylogenetic footprints are good indicators
of regulatory elements, both in Drosophila and in other spe-
cies (Hardison et al. 1997; Bergman et al. 2002). The use of
several species, diverging at different periods of time, allows
the identification of sequences subject to different degrees of
selective constraint (Duret and Bucher 1997; Hardison 2000;
Bergman and Kreitman 2001).
Our current knowledge of noncoding DNA evolution is
very limited and based on sparse data. Several characteristics
of these regions indicate that they are freer to evolve than
coding ones (Carroll 2000; Stern 2000). Thus, enhancer regions
of genes with complex patterns of expression are generally
organized into separate modules, that is, segments of cis-reg-
ulatory DNA that regulate subsets of the overall expression
pattern (Arnone and Davidson 1997). Individual modules can
evolve independently of each other, and mutations in each
module may have weak or no pleiotropic effects (Carroll
2000; Stern 2000). Individual modules contain multiple
325
326 EVOLUTION & DEVELOPMENT Vol. 6, No. 5, September^October 2004
binding sites for a few transcription factors, most of them
subject to weak selection. This leads to a high turnover of
binding sites over time (Dermitzakis and Clark 2002; Costas
et al. 2003). Nevertheless, this turnover may have no func-
tional consequence if the enhancer evolves by stabilizing se-
lection acting over the entire enhancer module. Under this
model, mutations in binding sites with weak contribution to
the enhancer’s function may be compensated by new muta-
tions that restore the optimal level of expression, leading to
the divergence of enhancer sequences while their function (i.e.,
gene expression) remains conserved (Ludwig et al. 1998,
2000). Nevertheless, changes in cis-regulatory sequences con-
trolling developmental genes may also lead to the origin of
morphological novelties (Averof and Patel 1997; Stern 1998;
Kopp et al. 2000; Sucena and Stern 2000).
The genus Drosophila constitutes a very useful model for
the identification and functional annotation of cis-regulatory
sequences using phylogenetic footprinting. The availability of
genome sequences from two different species (D. melanogaster
and D. pseudoobscura) represents a major source for sequence
comparisons. In addition, there are standard protocols for the
analysis of Drosophila gene expression (Sullivan et al. 2000;
Arbeitman et al. 2002; Arnosti 2003). Finally, the genus is
extremely diverse, and both the phylogenetic relationships
between the different species and the evolutionary history of
major representatives of the main lineages are well known
(Ashburner 1989; Powell 1997).
A particularly interesting set of genes comprises those with
complex patterns of expression. As a general rule, their reg-
ulation is controlled by several modules, sometimes spread
over large chromosomal regions (Davidson 2001). One such
gene is the Drosophila wingless (wg) gene. wg is one of the best
characterized members of the Wnt family of genes, which
encode secreted signaling molecules that regulate a wide
variety of processes during animal development (Wodarz and
Nusse 1998; Hlsken and Behrens 2000). wg is required for the
embryonic segmentation and the specification and growth of
most larval and adult organs (http://flybase.bio.indiana.edu/
allied-data/lk/interactive-fly/segment/wingles1.htm). Despite
the complex pattern of wg expression, required for the
regulation of all these processes, it is surprising how little is
known about the cis-regulatory elements controlling this
expression. About 5 kb of sequences just upstream of the first
wg exon suffice to drive the expression of a LacZ reporter
gene in a wg-like pattern during embryogenesis (Von Ohlen
and Hooper 1997; Lessing and Nusse 1998). Another up-
stream element is sufficient to drive reporter gene expression
in the proximal wing (outer hinge region) and the wing mar-
gin (Neumann and Cohen 1996a). Furthermore, the muta-
tions wgSp and wg1
16 indicate the existence of regulatory
sequences in the 30 region of wg affecting the notum and
antenna (Sp; Neumann and Cohen 1996b) and most larval
tissues (wg1
16; Baker 1988).
Two transcripts have been found for the D. melanogaster
wg gene. The long transcript comprises five exons, whereas the
short one only four. The 30 end of the first intron of the longer
transcript is part of the 50 UTR of the shorter transcript
(Release 3.1 of the D. melanogaster genome; Fig. 1A). Al-
though intron 4 is only 133 bp long, the other three introns of
the longer transcript are larger than 1 kb. Bearing in mind
that regulatory sequences are often found within intron se-
quences (Kassis 1990; Haenlin et al. 1994; Bachmann and
Knust 1998), a detailed analysis of wg intron regions could
help understand how wg expression is regulated.
In this study, we applied the phylogenetic footprint ap-
proach to identify regulatory regions within the introns 1 and
3 of the wg gene and to infer the evolutionary dynamics of
these regions. To this goal, we sequenced these introns from
several Drosophila species, both from the Sophophora and the
Drosophila subgenus. Our results revealed the existence of
several well-conserved blocks in both introns as well as a
putative modular structure in intron 3. In vivo analysis using
transgenic flies indicates that intron 3 contains a pupal-
specific regulatory element sufficient to drive transcription in
several fly tissues. Interestingly, D. willistoni presents a faster
rate of evolution in both introns and seems to lack one of the
two putative modules from intron 3. Further in vivo analysis
suggests that the module lost in D. willistoni harbors an
abdomen-specific enhancer.
MATERIALS AND METHODS
Fly stocks
wg-Z is a reporter insertion in the CyO balancer chromosome that
faithfully reproduces wg pattern of expression during development
(Kassis et al. 1992). w1118 (http://flybase.bio.indiana.edu/) was used
for transgenic strain generation. Other Drosophila stocks were ob-
tained from the Tucson Drosophila Species Stock Center (Tucson,
AZ, USA), except species from the virilis group, which were gifts
from A. Hoikkala (D. montana), E. Lozovskaya (D. virilis), and
B. McAllister (D. americana). The stock numbers and the phylo-
genetic relationships of the species used are shown in Fig. 1B.
Amplification and sequencing
DNA for amplification was extracted from single flies as in Gloor
and Engel (1992). Intron 1 of wg was amplified from D. erecta,
D. willistoni, and D. virilis using the forward primer 5 0 -ATCTT
CACTCCTCCGCTC-3 0 , located in a 50 upstream region con-
served between D. melanogaster and D. virilis (block ‘‘In’’ in Les-
sing and Nusse 1998) and the reverse primer 50 -AATGTTG
TTGGGTTCGCC-30 , complementary to the 50 end of exon 2,
based on comparisons among sequences from D. melanogaster
(GenBank AC003617), Gryllus bimaculatus (GenBank AC:
AB044713), and Bombyx mori (GenBank AC: D14169.1). Ampli-
fication of intron 3 of wg from D. americana was done using the
forward primer 5 0 -GCGGGATTGGGAGTGGGG-3 0 and the re-
verse primer 50 -CAGTCGCATCCAGCAGGTCTT-30 , located in
Costas et al. Evolution and function of wingless introns 327

Fig. 1. (A) Map of the Drosophila melanogaster wingless (wg) gene. (B) Phylogenetic relationships of the species analyzed in this work. (A)
The two wg transcripts, wg-RA and wg-RB, are indicated. The white bars indicate the 50 and 3 0 UTRs (only the 30 UTR is labeled). The
coordinates of the boundaries of the wg-RA transcript: 7,299,392 . . . 7,308,486[1]. The four introns are labeled (Int 1 to 4), and their lengths
in kilobases (kb) (except for Int 4) are indicated below. The wgspd
flg (Neumann and Cohen 1996a) and the embryonic regulatory regions
(Von Ohlen and Hooper 1997; Lessing and Nusse 1998) are indicated. The genomic deletions causing other wg mutations (wg1
15, wg1
16,
and wg1
17) are also indicated as gray bars. The exact breakpoints in some mutants are not well defined (discontinuous lines). (B) Numbers
represent the stock reference from the Tucson Drosophila Species Stock Center. Approximate divergence times were taken from Ashburner
(1989), Russo et al. (1995), and Nurminksy et al. (1996).

the flanking exons, based also on comparisons between sequences
from D. melanogaster, G. bimaculatus, and B. mori. After se-
quencing intron 3 from D. americana, we designed new primers
based on comparisons of this intron between D. melanogaster and
D. americana. The new primers (forward primer, 50 -GATTCGGG
TTCAAGTTCTC-30 ; reverse primer, 5 0 -GCACTCCTGTCG
CATCTC-3 0 ) were used to amplify intron 3 of wg from
D. melanogaster, D. erecta, D. willistoni, D. virilis, D. hydei, and
D. montana. The 5 0 end of intron 3 from D. nebulosa was amplified
using the same forward primer and the reverse primer
50 -GAAGGAGCCCATAACAAGT-30 , complementary to the
second conserved block from the 5 0 end of intron 3 between
D. willistoni and D. melanogaster (Fig. 2).
Polymerase chain reaction products were gel extracted using the
QIAquick gel extraction kit (Qiagen, Valencia, CA, USA) cloned
into pCR4-TOPO vectors (Invitrogen, Carlsbad, CA, USA) and
sequenced (both strands) in an ABI310 automatic sequencer
(Applied Biosystems, Foster City, CA, USA) using internal prim-
ers. To avoid sequence errors due to possible polymerase chain
reaction misincorporations, at least two independent products were
sequenced for each intron and species, except for D. nebulosa, to
confirm differences in conserved blocks. Sequences were submitted
to GenBank under accession numbers AY380303 to AY380312.
Analysis of sequences
Introns 1 and 3 of D. pseudoobscura were obtained from the
Drosophila Genome Project at Baylor College of Medicine (http://
www.hgsc.bcm.tmc.edu/projects/drosophila/) after their identifica-
tion by BLAST search using the corresponding introns of D.
melanogaster as input. The sequences are located on contigs 1969
(positions 21014–23192) and 1893 (positions 14971–16574),
respectively, from the Whole Genome Assembly as of 13 January
2003.
Conserved noncoding sequences in pairwise comparisons be-
tween D. melanogaster and each one of the other species were
identified with the help of VISTA server (Mayor et al. 2000), with
window size of 10 bp with an identity of 90% (as in Bergman et al.
2002).
DnaSP v3.53 (Rozas and Rozas 1999) was used for the analysis
of nucleotide diversity (i.e., the average number of nucleotide dif-
ferences per site) along each intron length, by a sliding windows
approach, using a window size of 150 bp and a step size of 1 bp.
Transgenic flies
P-element LacZ reporter vectors for intron 3 of D. melanogaster,
D. americana, and D. willistoni were constructed by subcloning
328 EVOLUTION & DEVELOPMENT Vol. 6, No. 5, September^October 2004

Fig. 2. Blocks of 49 bp present in all species or in all but one for intron 1 (A) and intron 3 (B). Black boxes, blocks common to all species;
white boxes, blocks absent from Drosophila virilis; gray boxes, blocks absent from D. willistoni; gray boxes with an asterisk, blocks absent
from D. pseudoobscura. The sign ‘‘ ^ ’’ splits two adjacent blocks. Brackets above intron 3 represent the main putative regions of the intron.

the introns from pCR4-TOPO vectors (see above) into pCAB70
(Bachmann and Knust 1998). Introns were removed by digestion
with EcoRI and placed in the same orientation into EcoRI-digested
pCAB70 (in front of the hsp70 minimal promoter followed by the
lacZ gene and an SV40 trailer).
P-element-based germline transformation was performed using
standard methods. Several independent lines were established. At
least three independent lines for each construct were analyzed.
Histochemical staining with X-Gal and microscopic
preparation of specimens
Embryo and larval stainings were performed as described in Cap-
devila and Guerrero (1994) and pupal staining as in Hama et al.
(1990). Stained and dissected pieces were mounted in Hoyer’s/lactic
acid (1:1) medium and the slides incubated overnight at 801C
before microscopic examination. Embryos, larvae, and pupae of
the recipient D. melanogaster strain (w1118) showed no endogenous
X-Gal reactivity. pCAB70-derived transgenics showed background
X-Gal reactivity in the larval anterior spiracles (weak) and in a
dorsomedial longitudinal row of large positive cells in the pupal
abdomen.
RESULTS
Sequence analysis of the wg introns 1 and 3
We sequenced introns 1 and 3 of the wg gene from D. erecta,
D. willistoni, and D. virilis. The corresponding sequences from
D. pseudoobscura were obtained from the Drosophila Genome
Project at Baylor College of Medicine. Because our extended
analyses focused on intron 3, we also sequenced intron 3 from
D. hydei, D. montana, and D. americana (approximate diver-
gence times and phylogenetic relationships shown in
Fig. 1B).
We searched for conserved blocks of at least 10 bp with
a homology of 90% in pairwise comparisons between D.
melanogaster and each of the other species, corresponding
sequences, using the VISTA alignment tool (as in Bergman
et al. 2002) (Table 1). There were a total of 287 nucleotide
positions in intron 1 and 212 in intron 3 lying within conserved
blocks common to all species, representing 17.1% and 18.0%
of the D. melanogaster introns 1 and 3, respectively. This last
value reached 19.7% when we repeated the analysis of intron
3 using only the subset of species analyzed in intron 1.
Costas et al. Evolution and function of wingless introns 329

Table 1. Summary of the results from VISTA pairwise comparisons

Total Length No. of Average Length Median Sum of Block Identity in Longest Drosophila
Species (bp) Blocks (SD) Length Length (bp) Blocks (%) Block (bp) melanogaster1 (%)
Intron 1
D. erecta 1640 20 73.20 (68.26) 53 1464 96.9 239 87.25
D. pseudoobscura 2179 37 21.62 (16.27) 17 800 96.0 84 47.68
D. willistoni 2210 25 18.72 (9.81) 13 468 96.3 49 27.89
D. virilis 1857 29 16.96 (9.63) 15 492 96.4 45 29.32
Intron 3
D. erecta 1155 12 82.58 (105.03) 31 991 96.5 369 84.13
D. pseudoobscura 1604 23 23.00 (19.83) 14 529 95.7 98 44.91
D. willistoni 869 9 30.11 (19.10) 27 271 97.8 71 23.00
D. hydei 1181 16 23.12 (18.58) 14 370 96.2 80 31.41
D. montana 1406 16 23.87 (15.38) 17.5 382 95.8 65 32.43
D. virilis 1378 14 25.07 (16.17) 22 351 95.7 65 29.80
D. americana 1385 13 24.92 (15.65) 20 324 96.6 65 27.50
1
Percentage of DNA in conserved blocks in regard to the total length of the intron in D. melanogaster (intron 1, 1678 bp; intron 3, 1178 bp).

The average percentage of identity of conserved blocks was
very high and quite similar in different species (495.5% in all
comparisons), suggesting the existence of very few neutral
nucleotide positions within blocks. The longest stretch of
consecutive conserved nucleotides defined a common block
of 32 bp in the case of intron 1 and up to 62 bp (65 bp using
the same subset of species as in intron 1) in intron 3. Figure 2
shows the location of all common blocks of at least 10 bp as
well as the 12 blocks of at least 10 bp common to all but one
species. Interestingly, D. willistoni lost four consecutive blocks
at the beginning of intron 3. To test whether these four blocks
were also absent from other species of the D. willistoni group,
we amplified the 50 end of intron 3 from D. nebulosa, a dis-
tantly related species from this group. Sequencing of the
amplicon revealed that D. nebulosa also lacks these blocks.
Curiously, D. willistoni presented a faster evolutionary rate
than expected from the known phylogenetic relationships of
the species in the case of both introns (Table 1).
We found an uneven distribution of common blocks with-
in intron 3, with blocks grouped at the 5 0 and 3 0 ends of the
intron, in some species (Fig. 2). This, together with the loss of
consecutive blocks from D. willistoni, suggested the existence
of two independent modules within this intron, comprising
the 5 0 and the 30 blocks, respectively (therefore the 50 one
would be absent in D. willistoni) (Fig. 2). If blocks indeed
formed modules, we would expect that each of the modules
evolved at a different rate according to its own constraints.
The degree of conservation of the two different modules in
pairwise comparisons between D. melanogaster and each one
of the other species revealed that the putative module I
evolved faster than the putative module II in all cases but
D. pseudoobscura, where this situation is inverted (Table 2). In

Table 2. Conservation of different regions from intron 3

Module I Module II
Intermodules
Species Length % Drosophila melanogaster1 Length Length % Drosophila melanogaster1
Drosophila erecta 236 90.7 227 519 98.5
D. pseudoobscura 294 65.6 623 555 57.8
D. willistoni2 F F F 548 46.9
D. hydei 172 39.1 441 448 46.8
D. montana 222 40.5 610 429 47.3
D. virilis 206 37.7 564 466 47.3
D. americana 227 37.2 585 432 45.1
1
Percentage of conserved blocks in regard to the total length of the modules in D. melanogaster (module I, 215 bp; module II, 528 bp). This
percentage is not calculated for the intermodules region (231 bp in D. melanogaster) because of the lack of homology.
2
The length of module I and the intermodule region is not computed because of the lack of blocks defining the limits of these regions in D. willistoni.
330 EVOLUTION & DEVELOPMENT Vol. 6, No. 5, September^October 2004

A sliding windows approach along this intron also showed a

contrasting level of divergence in the different regions of in-
tron 3 (Fig. 3). Furthermore, in agreement with this modular
structure, the distance separating conserved blocks within
modules (interblocks distance) was less variable (more con-
strained) than the distance separating the putative modules
(intermodules distance) as measured by the coefficient of var-
iation (CV 5 100  SD/mean). Thus, the CV was higher for
the intermodule region (37.18%) than for the interblock dis-
tances (22.97% and 18.14% for the putative modules I and II,
respectively), although this mainly might be due to the re-
duction of the intermodule length only in the D. melanogaster
subgroup.

Functional analysis of wg intron 3

Fig. 3. Variation of nucleotide diversity along intron 3 in
Drosophila melanogaster–D. erecta comparisons (A) and D. virilis–
D. americana–D. montana (B) using a sliding windows approach.
Location of the two putative modules are represented by thick
lines under the x-axis.
the case of the more closely related species (D. melanogaster–
D. erecta and D. virilis–D. americana–D. montana), whose
degree of divergence is lower than 10%, it was possible to
accurately align all the sequences along the total intron length.
The previous analyses allowed us to pose several hypotheses
that we then tested using wg intron 3, as follows.
First, the sequence conservation within wg introns indicates a
cis-regulatory role of these introns in wg expression. We tested
this point by generating D. melanogaster transgenic strains
carrying the melanogaster intron 3 linked to a reporter LacZ
gene (Dmel Int3-Z). Analysis of these strains revealed that
intron 3 contains enhancers active in the abdomen and gen-
italia of pupae (while being inactive during embryonic and
larval development; data not shown) in patterns reminiscent
of wg expression (Fig. 4). Thus, we detected a posterior stripe
in the terguites (weaker in abdominal [A] segments A1 and A6
and segment A7 in females) and a patch in the sternites (with
the exception of A1) (Fig. 4, A–D) and in the vaginal plates of
the female and in the clasper in the male (Fig. 4, H–K). Dmel
Int3-Z was also expressed in a patch of cells in the notum in a
position similar to the anterior-most stretch of the wg me-
sonotal stripe (Fig. 4, E and F) and, ectopically, in the wing

Fig. 4. Patterns of expression in late pupae driven by wg intron 3 from different species, detected by X-Gal histochemical staining. Solid
arrows indicate pattern elements shared with the wg-Z reporter, empty arrows pattern element losses, and red arrowheads point to novel
expression domains not shared with wg-Z. (A–D) Abdominal patterns of expression of the wg-Z (A) and of the Dmel Int3-Z (B), Dwil Int3-Z
(C), and Dame Int3-Z (D) flies. te, terguites; st, sternites; pl, pleura. In the dorsal abdomen, Dmel Int3-Z (B) and Dame Int3-Z (D) show a
posterior tergal stripe (arrow, te), although somewhat thinner and extending more laterally than the wg-Z stripe (A; arrow, te). This
posterior stripe is lost in Dwil Int3-Z (C; empty arrow, te). Asterisk indicates transformation vector background staining (probably in
oenocytes). In the ventral abdomen, Dmel Int3-Z (B) and Dame Int3-Z (inset in D) show a patch of staining in sternites A2 to A6 (and to
A7 in females), similar to the expression driven by wg-Z (A; st, arrow). This expression is also lost in Dwil Int3-Z (C; st, empty arrow). All
three species’ Int3-Z drive expression in the pleura (lateral abdomen) (B–D; pl, red arrowhead), which is not seen in wg-Z individuals (A, pl).
(E–G) Patterns of expression in the notum of wg-Z (E) and of Dmel Int3-Z (F) and Dwil Int3-Z (G) flies. Dmel Int3-Z (F) shares only the
anterior-most stretch (solid arrow) but not the rest (open arrow) of the wg-Z longitudinal stripes (E, arrows). No expression above
background is seen in the nota of Dwil Int3-Z (G) or Dame Int3-Z (not shown) transgenic flies (open arrows mark the putative position of
the wg stripe). (H–K) Patterns of expression of wg-Z and Int3-Z in the terminalia. (Int-3 from the three species drives identical expression in
the terminalia, so only Dmel Int3-Z stainings are shown). (H and I) wg-Z is expressed in the claspers (cl) and genital arch (ga) of the males
(also in the spermatic pump, asterisk; H) and in the dorsal part of the vaginal plates (vp) of the female (I), as well as in the analia (an) of both
sexes (distal anal plates and hindgut; H, I [Chen and Baker 1997; Sánchez et al. 1997]). Dmel Int3-Z is expressed in the claspers of the males
(J; cl, arrow) but not in the genital arch (J; ga, open arrow) or in internal genitalia (spermatic pump; not shown). In the females (K), Dmel
Int3-Z is expressed in the vaginal plates, both dorsally (arrow) and ventrally (red arrowhead). No expression is detected in the analia in
either sex (an, open arrows in J and K).
Costas et al. Evolution and function of wingless introns 331
332 EVOLUTION & DEVELOPMENT Vol. 6, No. 5, September^October 2004
blade and the abdominal pleura and weakly in the head cap-
sule (Fig. 4B and not shown).
Second, the regulatory function of the intron is preserved
during the evolution of the Drosophila genus (with the excep-
tion of the willistoni group), despite 30% of overall sequence
conservation in intron 3 across our sample. To test this point,
we chose D. americana intron 3, whose sequence is the least
conserved when compared with D. melanogaster (with the
exception of D. willistoni; Table 1), to generate D. melano-
gaster transgenic lines harboring the D. americana intron 3
reporter construct (Dame Int3-Z). The pattern of expression
driven by this intron was very similar to that of Dmel Int3-Z
flies, such as the expression in the dorsal and ventral abdo-
men, in the female and male genitalia, and the pleura (Fig. 4).
Small differences can be noted though, like a weaker pleural
expression, the loss of the mesonotal patch (Fig. 4D and
not shown), and a novel expression in the Wheeler’s organ
(not shown). Therefore, the enhancer functions contained
within intron 3 seem to be essentially conserved across the
Drosophila genus.
Third, the lack of module I of intron 3 in D. willistoni has
functional relevance. We expect the loss of one module to lead
to discrete changes, rather than to a global effect, in the pat-
tern of expression. The additional or lost pattern elements
would indicate the specific regulatory function contributed by
that module. To test this hypothesis, we generated D. mela-
nogaster transgenic reporter lines now containing the D.
willistoni intron 3 (Dwil Int3-Z). The pattern driven by this
construct was clearly different from those from D. melano-
gaster and D. americana intron 3 in that it loses expression in
the dorsal and ventral abdomen (Fig. 4C, with the exception
of sternite A7 in females [not shown], which shows expres-
sion) while retaining some specific pattern elements, such as
the expression in the genitalia and the ectopic expression in
pleura and wing blade (Fig. 4C and not shown). Therefore,
these results suggest that module I, which is lost from D.
willistoni intron 3, contains cis-regulatory elements responsi-
ble for dorsal and ventral abdominal expression. Module II is
most probably responsible for the genitalia expression as well
as for the ectopic expression in pleura and wing blade.
DISCUSSION
We applied the phylogenetic footprint approach to identify
novel regulatory regions within the introns of the wg gene and
to infer the evolutionary dynamics of these novel regions.
Although this method may detect different types of noncod-
ing conserved sequences, it is generally assumed that most of
these sequences correspond to enhancer elements. Compari-
son of the sequences from introns 1 and 3 of wg between
D. melanogaster and different species of the genus revealed a
pattern of conservation similar to other described regulatory
regions, characterized by the existence of several blocks of
conserved sequences embedded within regions of variable size
lacking any homology (Ludwig et al. 1998; Kim 2001). The
degree of conservation (Table 1) is higher than the reported
average for eight intergenic regions from these species
(D. erecta, 69%; D. pseudoobscura, 28%; D. willistoni, 11%)
(Bergman et al. 2002). In addition, at least in the case of the
D. melanogaster–D. virilis comparison, this divergence is sim-
ilar to the average of 40 known or suspected regulatory se-
quences (27%) (Bergman and Kreitman 2001), suggesting
that these introns perform some regulatory function.
Although, in general, the degree of divergence is in
accordance with the known phylogenetic relationship of the
species, both introns evolve at a faster-than-expected rate in
the lineage leading to D. willistoni. This faster evolutionary
rate has been detected previously in other genomic regions of
this species, affecting both coding and noncoding DNA
(Bergman et al. 2002). This fact indicates the existence of
global mechanisms to explain this faster evolution. A histor-
ically small population size, probably associated with
Pleistocene refugia in the Neotropics, may be one reason
for this increased evolutionary rate (Griffith and Powell 1997).
Alternatively, a shift in the pattern of point mutation affecting
the closely related saltans and willistoni groups may lead to an
accelerated rate of nucleotide substitution in functionally less
constrained regions (Rodriguez-Trelles et al. 1999, 2000). In
agreement with this hypothesis, both introns present a con-
siderablely lower G 1 C content in D. willistoni than in the
remaining species (data not shown).
Comparison of intron 3 sequences allows further inferen-
ces on the structure of this intron. Several features suggest
that intron 3 is composed of two independent modules. First,
it has been shown that conserved noncoding sequences tend
to be spatially clustered with conserved spacing between them,
and this clustering may be used to predict enhancer sequences
(Bergman and Kreitman 2001; Bergman et al. 2002). In
agreement with this fact, the distribution of conserved blocks
within intron 3 is clearly uneven, with a cluster of blocks at
each edge of the intron separated by a region without any
well-conserved homologous sequence (Fig. 2). Furthermore,
the length variation of the intermodules region is higher than
in the interblocks regions within each one of the modules (as
inferred by the CV), which suggests that the intermodules
region is freer to vary in length.
Second, 4 of the 11 conserved blocks are lost in D. willis-
toni and probably in the whole D. willistoni group. These four
blocks are the four common blocks present in the putative
module I (Fig. 2). This is in agreement with the prediction that
in the case of promoters with a modular organization, loss of
a crucial binding site may lead to eventual loss of the entire
module (Wray et al. 2003). In addition, the strong correlation
between the size of introns 1 and 3 in the other four species
with available data (Pearson r 5 0.9941, P 5 0.0059) is lost
Costas et al.
when the D. willistoni data are included (Pearson r 5 0.0566,
P 5 0.9280), in agreement with a deletion of an entire module.
Finally, another prediction by Wray et al. (2003) is that
each independent module evolves at a different rate, depend-
ing on its different functional constraint. Two facts indicate
this may be the case for the different regions of intron 3. On
the one hand, pairwise comparisons between D. melanogaster
and each of the other species reveal a substantial difference in
the proportion of nucleotides found in conserved blocks
within each module (Table 1). Surprisingly, module II is the
one evolving at the lowest rate in all species but one,
D. pseudoobscura. Although there are few data of multiple
species comparison of regulatory regions, this lineage-specific
variation in evolutionary rates seems to be a characteristic of
these regions. Thus, a similar situation has been described for
an enhancer of hairy in Drosophila (Kim 2001). On the other
hand, a sliding windows approach, in the case of the more
closely related species, shows considerable variation in the
degree of divergence along the intron, with an increased
divergence in the middle of it, matching the intermodules
region (Fig. 3), again supporting a modular structure for
intron 3.
Our comparative analysis of the intron 3 sequences has
led us to ask and to test in vivo the following questions:
Is there any enhancer function associated with intron 3? Is
this function conserved in the genus Drosophila? Does the loss
of a putative module in D. willistoni have any functional
implication?
The pattern of expression of the D. melanogaster intron
3-LacZ reporter indicates that this sequence contains regula-
tory information sufficient to control gene expression during
pupal stages, in dorsal and ventral abdomen, as well as in the
female and male genitalia, in a pattern similar to that of the
endogenous wg. No other abdominal or genital pupal epi-
dermal enhancers have been characterized in the approxi-
mately 11 kb 50 and 10 kb 3 0 of sequence surrounding the wg
transcription unit studied so far (Neumann and Cohen
1996a,b; Lessing and Nusse 1998; J. P. Couso, personal com-
munication; unpublished data). Comparison of this expres-
sion with that of the endogenous wg gene reveals that Int3-Z
also drives expression in ectopic domains. A likely explana-
tion for this fact is that the enhancers contained within intron
3 lack some repressor element(s) lying somewhere else in the
wg gene. Comparison of the lacZ expression driven by
D. melanogaster and D. americana intron 3 (the species with
the least conserved sequence, excluding D. willistoni) reveals a
very similar pattern of expression, indicating that the regu-
latory information within intron 3 has been essentially pre-
served during the evolution of the genus Drosophila, despite
the fact that less than 30% of the D. melanogaster intron 3
sequence lies in blocks conserved with D. americana. These
results are in agreement with the stabilizing selection model
proposed by Ludwig et al. (1998, 2000), in which the function
Evolution and function of wingless introns 333
of an enhancer is preserved despite an important change in
sequence over time.
Finally, we tried to detect any consequence associated with
the loss of the putative module I from D. willistoni by com-
paring the expression pattern driven by its intron 3 with those
of D. melanogaster and D. americana. The willistoni enhancer
fails to drive LacZ expression in the dorsal and ventral
abdominal segments, indicating that module I possesses the
information to trigger gene expression in those regions.
A logical step following our studies is to ask whether the
regulatory changes caused by the loss of module I of intron 3
in the willistoni group are responsible for some willistoni-
specific trait(s) related to wg function in this species. wg is
expressed during pupal stages in dorsal and ventral abdom-
inal histoblasts nests, where it is essential to specify their fates,
so when its function is lacking, both terguites and sternites
develop aberrantly into lateral pleural tissue (Shirras and
Couso 1996; Kopp et al. 1999). Because of the key role of wg
during development, the similar morphology of the abdomen
in Drosophila species, and the high degree of conservation of
developmental processes, we would expect that wg function in
D. melanogaster abdomen to be conserved across the species
used in our study. Although we show here that module I is
likely responsible for driving wg expression precisely in these
tissues, it is very unlikely that it is the only abdominal-specific
wg regulatory region in the willistoni group, because its loss
would therefore be deleterious. A possible explanation for this
problem is that a redundant module I-like function is found
elsewhere, at least in D. willistoniFbut most probably in all
the genusFcompensating for the loss of module I. In fact, the
existence of redundant regulatory regions controlling wg ex-
pression in the wing margin has been previously proposed by
Neumann and Cohen (1996a). If the functional contribution
of module I to the pattern of expression of wg is very small,
such a minor enhancer might have been maintained through
very weak positive selection only in species with large pop-
ulation sizes but lost if the lineage leading to the willistoni
group suffered some population shrinkage along its evolu-
tionary history (Griffith and Powell 1997). It might be then
that the loss of weak-selected regulatory modules may be a
general phenomenon in D. willistoni (Bergman et al. 2002).
Alternatively, module I might be contributing to putative
nondevelopmental functions of wg and its presence or absence
in different Drosophila species responsible for some specific
differences in processes such as behavior, fecundity, or lon-
gevity. Nevertheless, to our knowledge, there is no report to
date of wg participating in those processes.
It is interesting to highlight the limited sensitivity labora-
tory experiments may have when it comes to determining gene
function. Phylogenetic footprinting detects the output of a
‘‘natural experiment’’ involving the entire species on an ev-
olutionary time scale; it points to the existence of a function
but not to its nature. This means that the true function of a
334 EVOLUTION & DEVELOPMENT Vol. 6, No. 5, September^October 2004
regulatory element might not be studied with current labo-
ratory experiments if it is subject to very weak selection, as
proposed previously for the case of gene functions (Tautz
2000).
Acknowledgments
We thank J. P. Couso for communicating results before publication;
P. Rodrigues, E. Sánchez-Herrero, D. Stern, C. Súnkel, and J. R. S.
Whittle for comments on the manuscript; and N. Ribeiro for tech-
nical assistance. This work was supported by grant POCTI/37402
from the Portuguese Fundação para a Ciência e a Tecnologia (FCT)
and an EMBO Young Investigator Award to F. C.; J. C., C. P. V.,
and P. S. P. were recipients of postdoctoral fellowships SFRH/BPD/
7094/2001, SFRH/BPD/5592/2001, and SFRH/BPD/5591/2001,
respectively, from FCT.
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