Regulation of Body Pigmentation by the
Abdominal-B Hox Protein and Its
Gain and Loss in Drosophila Evolution
Sangyun Jeong,1Antonis Rokas,2and Sean B. Carroll1,*
1Howard Hughes Medical Institute and Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Drive,
Madison, WI 53706, USA
2Microbial Genome Analysis and Annotation, The Broad Institute of MIT and Harvard, 7 Cambridge Center,
Cambridge, MA 02141, USA
events underlying trait modification have not
been elucidated. Pigmentation of the posterior
male abdomen is a recently acquired trait in
the Drosophila melanogaster lineage. Here, we
directly activates expression of the yellow pig-
mentation gene in posterior segments. ABD-B
regulation of pigmentation evolved through the
ulatory element of the yellow gene of a common
ancestor of sexually dimorphic species. Within
the melanogaster species group, male-specific
pigmentation has subsequently been lost by at
least three different mechanisms, including the
mutational inactivation of a key ABD-B binding
site in one lineage. These results demonstrate
how Hox regulation of traits and target genes
is gained and lost at the species level and have
general implications for the evolution of body
form at higher taxonomic levels.
Many animal bodies are constructed of serially homolo-
gous parts, such as segments, somites, vertebrae, and
appendages. In the course of evolution, the number and
morphology of these structures have undergone dramatic
Understanding the genetic regulatory mechanisms that
govern the differentiation of serially homologous parts is
central to understanding both the development and the
evolution of animal forms.
several types of serially homologous structures (reviewed
in Carroll et al., 2005). A large body of evidence has im-
plicated changes in the regulation of Hox genes (Warren
et al., 1994; Averof and Akam, 1995; Burke et al., 1995;
Averof and Patel, 1997; Stern, 1998; Belting et al., 1998;
Telford and Thomas,1998;CohnandTickle, 1999;Abzha-
nov et al., 1999; Mahfooz et al., 2004) and of the target
genes they control (Warren et al., 1994; Carroll et al.,
1995; Weatherbee et al., 1999; Lewis et al., 2000;
Tomoyasu et al., 2005) in the evolution of animal diversity.
However, despite their prominent roles in development
and evolution, knowledge of the scope and mechanisms
crucial respects (Mahaffey, 2005; Pearson et al., 2005).
First, while Drosophila melanogaster is the best studied
model species, only a modest number of direct Hox-regu-
lated target genes have been identified (Pearson et al.,
2005). Second, nearly all identified target genes encode
signaling proteins or transcription factors that act by regu-
lating the expression of other genes (Pearson et al., 2005).
Third, for some Hox proteins, such as Abdominal-B, no
direct target genes have been characterized. And finally,
despite the inference that the sets of target genes regu-
lated by individual Hox proteins have diverged between
homologous structures (Warren et al., 1994; Carroll,
1995; Weatherbee et al., 1999), the evolutionary gain or
loss of Hox regulation of individual genes has not been
directly demonstrated at the molecular level.
Understanding how Hox-regulated target genes and
traits evolverequires both the identificationof direct target
lution of the Hox-target interaction can be reconstructed.
Toward this end, we have analyzed the development and
evolutionofa Hox-regulated traitinDrosophila.InD.mela-
nogaster, the male has fully pigmented tergites in the fifth
and sixth abdominal segments (A5 and A6), whereas the
female’s tergites have only a narrow pigment stripe (Fig-
ures 1B and 1C). This sexually dimorphic pigmentation
pattern is controlled by a genetic regulatory circuit involv-
ing the Hox gene Abd-B. Loss-of-function mutations of
Cell 125, 1387–1399, June 30, 2006 ª2006 Elsevier Inc. 1387
gain-of-function alleles, such as Abd-BMcp, cause the ex-
pansion of pigmentation to the A4 segment (Celniker
et al., 1990; Hopmann et al., 1995; Figure 1E) or even to
tion factors BAB1 and BAB2 (Couderc et al., 2002), and
doublesex (dsx), which encodes a transcription factor
with a male- (DSXm) and a female-specific (DSXf) isoform
(Burtis, 1993), function to repress male-type pigmentation
in the female abdomen. Loss of these gene functions
causesectopicpigmentationof thefemaleA5 andA6seg-
ments (Baker and Ridge, 1980; Couderc et al., 2002;
Figure 1D). The sexually dimorphic pigment pattern de-
pends upon regulatory interactions among the Abd-B,
bab, and dsx genes (Kopp et al., 2000). Genetic analyses
suggest that ABD-B has dual functions in promoting
male-specific pigmentation: It appears to positively regu-
late the melanin pattern, and it represses the expression
of both bab genes in the A5 and A6 segments. In females,
the repressive action of ABD-B on bab is overcome by the
DSXfprotein, which promotes sufficient bab expression to
suppress posterior pigmentation (Kopp et al., 2000).
In the genus Drosophila, many species show differ-
ences in pigmentation traits. Pigmentation of the posterior
male abdomen is a trait found in many members of the
melanogaster species group but not in several other major
groups. The dimorphic regulation of bab expression is
closely correlated with dimorphic pigmentation (Kopp
et al., 2000) as well other pigmentation patterns (Gompel
and Carroll, 2003). It is not known, however, which regula-
tory interactions among Abd-B, bab, dsx, and pigmenta-
tion genes are direct and which are indirect.
Here, we show through biochemical and transgenic
analyses that ABD-B directly activates the expression of
the yellow gene in the male A5 and A6 segments through
binding sites in a specific cis-regulatory element (CRE) of
the yellow gene. Furthermore, we demonstrate that ABD-
B regulation of the yellow gene evolved in a common an-
cestor of the melanogaster species group and that muta-
tional inactivation of a key ABD-B binding site occurred
within the D. kikkawai lineage that has lost dimorphic pig-
mentation. These results demonstrate that Hox proteins
do directly regulate terminal phenotypes and that the
gain and loss of Hox regulation occur at the level of indi-
vidual species through modifications of CREs.
The cis-Regulatory Region Controlling yellow
Expression in the Abdomen
The Yellow protein is expressed in the pupal epidermis in
a striped pattern near the A/P compartment boundary in
all abdominal segments of developing females, in seg-
ments A1–A4 of developing males, and in a broad intense
pattern throughout the anterior of segments A5 and A6 of
males. This pattern foreshadows the melanin pattern of
the adult flies (Wittkopp et al., 2002a). In order to dissect
the regulation of yellow expression in the abdomen, we
first identified cis-regulatory sequences necessary for ac-
curate gene expression.
Genetic analysis has identified a region of the yellow lo-
cus required for gene function in the abdomen (Geyer and
Corces, 1987), and molecular studies have identified dif-
ferent regions of the yellow gene that govern expression
in several structures, including the body, wings, and bris-
viously described 1.4 kb CRE (Wittkopp et al., 2002b),
domen: The contrast between expression in the more an-
terior portion of each segment and the stripe near the A/P
compartment boundary was diminished, and the higher
levels of expression in male segments A5 and A6 was
less pronounced (data not shown). To determine whether
additional sequences flanking this CRE could contribute
to the fidelity of reporter-gene expression, we tested the
activity of a 2.6 kb fragment located 269 bp from the 50
end of the yellow transcription unit (Figure 1A). This region
includes some regulatory sequences that contribute to
gene expression in the wing. This element, termed the
wing/body (wb) element, drove robust, sexually dimorphic
expression of enhanced green fluorescent protein (EGFP)
in the pupal abdomen and a sharp, striped pattern in the
(Figures 1F and 1G), thus recapitulating the native Yellow
If the sexually dimorphic pattern of the wb element is
regulated by Abd-B and bab, its expression should be
modified differently in mutants for these genes. In females
with one mutant copy of the bab locus, the activity of the
wbelement wasfully derepressed inA6and partially dere-
pressed in A5, which correlated with the male-type adult
pigmentation pattern of the same genotype (Figures 1D
and 1H). In addition, the wb element responded to the
Abd-BMcpgain-of-function allele—male-specific expres-
sion of EGFP was expanded to the A4 segment in mutant
element and, hence, the yellow gene are transcriptionally
regulated, directly or indirectly, by both Abd-B and bab
Identification of an ABD-B-Responsive Element
In order to determine whether regulation of yellow might
be direct, we sought to identify regions within the wb ele-
ment that were ABD-B-responsive. We first determined
whether smaller regions of the wb element were sufficient
to drive sexually dimorphic expression of a reporter gene.
A 1.6 kb portion of the wb element lacking the 1.0 kb 50-
most sequence that contains the wing CRE (Geyer and
Corces, 1987; Wittkopp et al., 2002b; Gompel et al.,
2005) drove elevated EGFP expression throughout the
A1–A4 segments as well as robust expression in A5 and
A6 in males (Figures 2A and 2B). We shall refer to this
1.6 kb element as the body CRE.
1388 Cell 125, 1387–1399, June 30, 2006 ª2006 Elsevier Inc.
We then made a series of deletion constructs for six
subregions of the body CRE (BED1–BED6) to determine
which subregion (or subregions) was necessary or suffi-
cient to drive sexually dimorphic expression (Figure 2A).
Only BED3 males lost the male-specific expression of
EGFP in the A5 and A6 segments, demonstrating an es-
sential role of this region (Figures 2A and 2C). To deter-
mine whether the BE3 cis-regulatory region is also suffi-
cient for the response to ABD-B, we made a set of
constructs that contained BE3 and/or adjacent regions
and examined reporter expression in the pupal abdomen
(Figure 2A). All five BE3-containing constructs, including
BE1–3, BE2–3, BE3, BE3–4, and BE3–6, express EGFP
in a male-specific pattern at varying levels and respond
to the Abd-BMcpallele (right column in Figure 2A; Figures
2D–2I). The BE3 region alone drove weak reporter expres-
strong expression (Figure 2H) that was responsive to Abd-
is required for the response to ABD-B and that the BE3–6
region contains the cis-regulatory sequences required for
robust male-specific activation of the yellow gene.
Figure 1. Expression of the yellow Pigmentation Gene Is Transcriptionally Modulated by Abd-B and bab
(A) Organization of the D. melanogaster yellow locus. Arrow indicates the position of transcription initiation. The boundaries of the wing/body CRE are
indicated. The exons are represented by solid black rectangles. Below this map, the wing/body (wb) construct is depicted as a solid bar.
(B–E) Abdominal cuticles are displayed with the dorsal tergites to the right. Segments A4, A5, and A6 are indicated.
(B) Abdominal tergites of D. melanogaster females display a posterior black stripe widened at the dorsal midline of each segment.
(C) Abdominal tergites of D. melanogaster males exhibit male-specific pigmentation of the entire posterior two segments as well as pigmentation
stripes in the A1 to A4 segments as in the female.
(D) D. melanogaster babAr07/+ females display male-type pigmentation in A5 and A6 segments.
(E) Ectopic activity of Abd-B in D. melanogaster Abd-BMcp/+ males leads to the expansion of posterior male pigmentation into the A4 segment.
(F–I) Confocal images of the dorsal abdomen of transgenic pupae expressing the nuclear EGFP reporter protein (green).
(F) Six abdominal segments of a wb/+ female express EGFP in a striped pattern corresponding to the adult female pigmentation pattern (B).
(G)Inwb/+males,thenon-sex-specific stripedexpressionaswellasrobustmale-specificexpressionofEGFPcorrespondprecisely totheadultmale
pigmentation pattern (C).
(H) The posterior two segments of a wb/babAr07female robustly express EGFP in male-specific pattern, reflecting ectopic pigmentation of the same
(I) In wb/Abd-BMcpmales, ectopic expression of EGFP in the A4 segment is consistent with the pigmentation pattern of the same genotype (E).
Cell 125, 1387–1399, June 30, 2006 ª2006 Elsevier Inc. 1389
Figure 2. Identification of a Male-Specific Regulatory Element in the yellow body CRE
(A) Summary of reporter constructs and their activities. The 1.6 kb D. melanogaster body CRE is divided into six subregions, BE1 to BE6, from which
aseriesofdeletion constructs werederived.Intherightcolumn, thesymbol +or?represents thepresenceorabsenceofmale-specificexpression of
EGFP in the A5 and A6 segments. Superscript ‘‘a’’ indicates weak expression of the reporter.
(B) The wild-type body CRE drives elevated reporter expression throughout the A1–A4 segments as well as in segments A5 and A6.
(C) In BED3 males, male-specific pigmentation is lost; the segmental striped expression of EGFP is retained.
(D) In BE1–3 males, reporter expression is very similar to that of the intact body CRE reporter (B).
(E) In BE2–3 males, there is weak expression of EGFP in A5 and A6.
(F) In BE3 males, reporter expression is male specific but very weak.
(G) In BE3–4 males, there is a low level of EGFP expression in A5 and A6.
(H) In BE3–6/+ males, there is robust expression of EGFP in a male-specific pattern.
(I) In BE3–6/Abd-BMcpmales, male-specific expression of EGFP fully extends to the A4 segment.
1390 Cell 125, 1387–1399, June 30, 2006 ª2006 Elsevier Inc.
Direct Regulation of the yellow Gene by ABD-B
We next examined whether the ABD-B protein directly ac-
tivates the yellow gene through interaction with binding
sites in the body CRE. We systematically searched for
ABD-B binding sites in vitro by DNase I footprinting of all
domain (HD). We identified four sites that were strongly
ing sites were clustered together within the BE3 region
(Figure 3A; BS4, BS5, BS6, and BS7). BS5 contains
tifs (which is the preferred core binding site for ABD-B;
Ekker et al., 1994), but the other two sites do not contain
any potential core sequence for Hox proteins (Ekker
et al., 1994) (Figure 3B). In order to confirm ABD-B binding
were performed using four overlapping oligonucleotides
containing pairs of putative sites. The short sequence be-
binding site sequence in the context of either the BS45 or
the BS67 oligonucleotide (Figure 3B). Analysis of ABD-B
binding to the wild-type and mutated oligonucleotides
indicated that only mutations in BS5 and BS7 reduced
ABD-B binding (Figure 3C). The footprinting of BS4 and
BS6 appears then to be a consequence of ABD-B binding
to the adjacent bona fide BS5 and BS7.
To test whether BS5 and BS7 are required for the func-
tion of the CRE in vivo, we introduced mutations into the
core sequences of both binding sites within the BE3–6
element (KO[ABD-B]; Figure 3B). Disruption of these sites
expression in segments A5 and A6 (compare Figures 3D
and 3E), demonstrating that the yellow gene is directly
regulated by the ABD-B Hox protein.
Evolution of Posterior Pigmentation
in the melanogaster Species Group
expression and make an informative choice of species for
further study, we first considered the evolutionary history
of male-specific pigmentation in Drosophila. The mela-
nogaster species group includes three major clades: the
Oriental lineage (to which D. melanogaster belongs), the
montium subgroup, and the ananassae subgroup (see
Figure S1 in the Supplemental Data available with this arti-
cle online). Ancestral character reconstruction indicates
state in the Oriental lineage (posterior probability of di-
morphically pigmented ancestor 97% ± 2%; Figure S2)
and the melanogaster species group (probability of dimor-
phic ancestor 84% ± 8%; Figure S2). Furthermore, the
male-specific repression of bab expression is widespread
throughout all three clades (Kopp et al., 2000), suggesting
that dimorphic regulation of bab was present in the com-
mon ancestor of the entire melanogaster species group.
members of the obscura, willistoni, and saltans groups, do
male-specific pigmentation appears to have arisen once
in the melanogaster species group, and the absence of
male-specific pigmentation in species such as D. bipecti-
sequent losses of the trait.
Thistraithistoryshouldbereflected inthemolecular ge-
netic architecture of trait formation. The identification of
the ABD-B-regulated body CRE of the yellow gene offers
the opportunity to trace when ABD-B regulation was
Therefore, we selected several potentially informative
species for further study based upon their pigmentation
patterns and phylogenetic relationships (Figure 4). These
included D. biarmipes, another member of the Oriental
lineage that is dimorphically pigmented; D. santomea,
losses of the trait in the Oriental, montium, and ananassae
subgroups, respectively; and D. subobscura, a member of
the obscura group (the sister clade of the melanogaster
species group) that exhibits intense monomorphic pig-
mentation of all abdominal segments.
Evolution of the yellow body CRE and Dimorphic
In order to determine how the function of the body CRE
may have changed during Drosophila evolution, we iso-
lated orthologous body CREs from D. santomea, D. kikka-
wai,D.bipectinata, andD.subobscura;the entire50region
of the D. biarmipes yellow gene, which includes the body
CRE, was isolated previously (Gompel et al., 2005). We
then tested CRE activity when linked to the EGFP reporter
and transformed into D. melanogaster. Reporter expres-
sion driven by the D. biarmipes 50region (50ybia, Figures
5A and 5F) was sexually dimorphic, as we expected if the
mon origin and respond to the same transcription factors.
Surprisingly, the D.santomea (san body, Figures5Band
also drove sexually dimorphic expression of EGFP, even
though in these species abdominal pigmentation is either
monomorphic (D. bipectinata) or absent (D. santomea).
to transcriptional regulatory inputs present in D. mela-
nogaster. Indeed, the expression of the san body and bip
body reporter constructs responded to the Abd-BMcpmu-
tation (Figures 5L and 5M). Based upon the phylogenetic
relationships among these species (Figure 4), the most
parsimonious explanation for the shared responsiveness
of the D. santomea, D. biarmipes, and D. bipectinata
CREs to Abd-B is that the common ancestor of all of
these species possessed an ABD-B-responsive CRE and
therefore, as we inferred above, was likely to have been
However,while D.kikkawai isdescended fromthe same
ancestor, the kik body CRE drives only monomorphic re-
porter expression (Figures 5D and 5I) and does not re-
spond to ectopic Abd-B activity (Figure 5N). Therefore,
Cell 125, 1387–1399, June 30, 2006 ª2006 Elsevier Inc. 1391