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.
morerapidly evolvingcharacters.Because ofthemajorin-
fluence thatthe comparative study of Hox genesand Hox-
regulated structures has had to date in evolutionary devel-
opmental biology, the demonstration of the evolution of
related Drosophila species should help address concerns
about the extrapolation of microevolutionary processes to
pigmentation and the molecular mechanisms of the evolu-
tionary gain and loss of Hox target regulation among
closely related species, the very sort of process that has
been proposed to explain the large-scale divergence of
homologous structures over much greater evolutionary
distances (Warren et al., 1994; Weatherbee et al., 1999;
Drosophila Strains and Culturing
We used the CantonSstrain as the wild-type form of D. melanogaster.
The babAr07allele is null for both bab1 and bab2 genes (Couderc et al.,
of ABD-B proteins in the A4 segment (Duncan, 1987). D. santomea, D.
subobscura, andD. kikkawai were obtained from J.Coyne,N. Gompel,
and J. David, respectively. D. biarmipes and D. bipectinata were ob-
tained from the Tucson Stock Center. The D. melanogaster yw strain
was used as the P element transformation host. All flies were reared
according to standard procedures.
Abdominal Cuticle Preparations
Flies (3–4 days old) were killed in ethanol and incubated in 10% KOH
solution for 1 hr at 65ºC. Adult abdominal cuticles were cut along the
dorsal midline and mounted with a drop of Hoyer’s medium (Ash-
burner, 1989). The cuticles were flattened with a coverslip and incu-
bated for 3 hr at 65ºC.
Reporter Constructs of D. melanogaster
All reporter plasmids for transgenic flies were made by polymerase
chain reaction (PCR) and/or restriction-enzyme-based strategies. For
the wb and body constructs, a 2.6 kb fragment located between
?2869 and ?269 and a 1.6 kb fragment located between ?1867 and
?269 from the transcription start site (+1), respectively, were sub-
cloned into the pH-Stinger vector carrying nuclear EGFP (Figure 1A
and Figure 2A; Barolo et al., 2004). Serially deleted constructs for six
subregions of the body element (BED1–BED6) and seven overlapping
constructs were made as represented in Figure 2. Each subregion is
indicated in Figure S3.
Imaging EGFP Signal
Forimaging EGFPexpressioninthepupal abdomens, pupae(85–90 hr
after puparium formation) were dissected from their pupal cases and
mounted in Halocarbon 700 oil. Oil-saturated pupae were imaged on
a Bio-Rad MRC 1024 confocal microscope.
Protein Production and DNA Binding Assays
The DNA sequence encoding the ABD-B HD was amplified by PCR
from an Abd-B cDNA plasmid (a gift from W. McGinnis) and the follow-
ing EcoRI or XhoI site-containing primers: ABD-B HD forward primer,
50-CGGAATTCGTGTCCGTCCGGAAAAAGCGCA-30; reverse primer,
50-CGCTCGAGTCAGTTGTTGTTGTTCTGCTGATTG-30. Amplified pro-
ducts were subcloned into the pGEX-4T-1 expression vector (Pharma-
cia Biotech, Inc.) and then verified by sequencing. The purification of
recombinant proteins was performed as described previously (Fran-
gioni and Neel, 1993). The final concentrations of N-laurylsarcosine
and Triton X-100 in STE buffer were 1.5% and 2.0% respectively. The
purified ABD-B HD proteins were used to conduct DNase I footprinting
experiments as described previously (Hersh and Carroll, 2005), except
that the protein-DNA binding reaction was carried out in 13 footprint
buffer (25 mM HEPES [pH 7.9], 50 mM KCl, 0.5 mM DTT, 6.25 mM
MgCl2, 0.025 mM EDTA, 8.5% glycerol). For EMSA assays, forward
and reverse oligonucleotides were annealed and end labeled with
Klenow enzyme and [a-32P]dNTP. Binding reactions were performed
in 13 footprint buffer containing 20 mg/ ml poly(dI-dC) at 4ºC for 30
min. DNA-protein complexes were analyzed by electrophoresis on
a 5% nondenaturing gel. Forward and reverse oligonucleotides used
for EMSA are summarized in Table S2.
Cloning and Mutagenesis of Orthologous yellow Enhancer
Genomic DNA was purified from D. santomea, D. bipectinata, and D.
kikkawai using the QIAamp DNA Mini Kit (QIAGEN). The orthologous
body enhancer elements were amplified by PCR with the following
degenerate primers: forward primer, 50-AKCGTKWGKCAATTATGCC
ARAGAG-30; reverse primer, 50-TAKCGCTCCTGVAGYTTGTAGGC-30.
Degenerate PCRproductsweresubcloned intothepGEM-TEasyvec-
tor (Promega), and five clones from four independent PCR reactions
were sequenced. Based on sequence alignment using DIALIGN,
each orthologous body element was subcloned into pH-Stinger vector
in a multistep process. For the sub body construct, the PCR reaction
was performed with a D. subobscura genomic plasmid (Wittkopp
et al., 2002b) and the following XbaI or BamHI site-containing primers:
forward primer, 50-GCTCTAGATCCGAAACAAGTGCAATTTCT-30; re-
verse primer, 50-CGGGATCCTTGCTCTCTGAGGCAGTTTTT-30. Am-
plified product was subcloned into pH-Stinger vector and verified by
sequencing. Mutations of the native body elements shown in Figures
6C–6E were generated by PCR. For all constructs, a minimum of
four independent lines were examined.
Supplemental Data include Supplementary Experimental Procedures,
Supplementary References, three figures, and two tables and can be
found with this article online at http://www.cell.com/cgi/content/full/
We thank W. McGinnis and P. Wittkopp for DNA clones; J. Coyne, N.
Gompel, and J. David for stocks; B. Hersh for technical comments; K.
Vaccaro for technical assistance; and B. Prud’homme and T. Williams
for suggestions on the manuscript. S.J. was supported by the Korea
Science and Engineering Foundation, and the work was supported
by the Howard Hughes Medical Institute (S.B.C).
Received: February 6, 2006
Revised: March 24, 2006
Accepted: April 25, 2006
Published: June 29, 2006
Abzhanov, A., Popadic, A., and Kaufman, T.C. (1999). Chelicerate Hox
genes and the homology of arthropod segments. Evol. Dev. 1, 77–89.
Ashburner, M. (1989). Drosophila: A Laboratory Manual (Cold Spring
Harbor, NY, USA: Cold Spring Harbor Laboratory Press).
Averof, M., and Akam, M. (1995). Hox genes and the diversification of
insect and crustacean body plans. Nature 376, 420–423.
Averof, M., and Patel, N.H. (1997). Crustacean appendage evolution
associated with changes in Hox gene expression. Nature 388, 682–
1398 Cell 125, 1387–1399, June 30, 2006 ª2006 Elsevier Inc.
Baker, B.S., and Ridge, K.A. (1980). Sex and the single cell. I. On the
action of major loci affecting sex determination in Drosophila mela-
nogaster. Genetics 94, 383–423.
Barolo, S., Castro, B., and Posakony, J.W. (2004). New Drosophila
transgenic reporters: insulated P-element vectors expressing fast-
maturing RFP. Biotechniques 36, 436–440, 442.
Belting, H.G., Shashikant, C.S., and Ruddle, F.H. (1998). Modification
of expression and cis-regulation of Hoxc8 in the evolution of diverged
axial morphology. Proc. Natl. Acad. Sci. USA 95, 2355–2360.
Burke, A.C., Nelson, C.E., Morgan, B.A., and Tabin, C. (1995). Hox
genes and the evolution of vertebrate axial morphology. Development
Burtis, K.C. (1993). The regulation of sex determination and sexually
dimorphic differentiation in Drosophila. Curr. Opin. Cell Biol. 5, 1006–
Carbone, M.A., Llopart, A., deAngelis, M., Coyne, J.A., and Mackay,
T.F. (2005). Quantitative trait loci affecting the difference in pigmenta-
tion between Drosophila yakuba and D. santomea. Genetics 171, 211–
Carroll, S.B. (1995). Homeotic genes and the evolution of arthropods
and chordates. Nature 376, 479–485.
Carroll, S.B. (2000). Endless forms: the evolution of gene regulation
and morphological diversity. Cell 101, 577–580.
Carroll, S.B., Weatherbee, S.D., and Langeland, J.A. (1995). Homeotic
genes and the regulation and evolution of insect wing number. Nature
Carroll, S.B., Grenier, J.K., and Weatherbee, S.D. (2005). From DNA to
Diversity: Molecular Genetics and the Evolution of Animal Design,
Third Edition (Malden, MA, USA: Blackwell Science Press).
Proc. Natl. Acad. Sci. USA 90, 1566–1570.
Celniker, S.E., Sharma, S., Keelan, D.J., and Lewis, E.B. (1990). The
molecular genetics of the bithorax complex of Drosophila: cis-regula-
tion in the Abdominal-B domain. EMBO J. 9, 4277–4286.
and axial patterning in snakes. Nature 399, 474–479.
Couderc, J.L., Godt, D., Zollman, S., Chen, J., Li, M., Tiong, S., Cram-
ton, S.E., Sahut-Barnola, I., and Laski, F.A. (2002). The bric ? a brac
locus consists of two paralogous genes encoding BTB/POZ domain
proteins and acts as a homeotic and morphogenetic regulator of
imaginal development in Drosophila. Development 129, 2419–2433.
Duncan, I. (1987). The bithorax complex. Annu. Rev. Genet. 21, 285–
Ekker, S.C., Jackson, D.G., von Kessler, D.P., Sun, B.I., Young, K.E.,
and Beachy, P.A. (1994). The degree of variation in DNA sequence
recognition among four Drosophila homeotic proteins. EMBO J. 13,
Frangioni, J.V., and Neel, B.G. (1993). Solubilization and purification of
enzymatically active glutathione S-transferase (pGEX) fusion proteins.
Anal. Biochem. 210, 179–187.
Garcia-Bellido, A. (1975). Genetic control of wing disc development in
Drosophila. Ciba Found. Symp. 29, 161–178.
Geyer, P.K., and Corces, V.G. (1987). Separate regulatory elements
mental transcription of the yellow locus in Drosophila melanogaster.
Genes Dev. 1, 996–1004.
Gompel, N., and Carroll, S.B. (2003). Genetic mechanisms and con-
straints governing the evolution of correlated traits in drosophilid flies.
Nature 424, 931–935.
Gompel, N., Prud’homme, B., Wittkopp, P.J., Kassner, V.A., and Car-
roll, S.B. (2005). Chance caught on the wing: cis-regulatory evolution
and the origin of pigment patterns in Drosophila. Nature 433, 481–487.
Hersh, B.M., and Carroll, S.B. (2005). Direct regulation of knot gene
expression by Ultrabithorax and the evolution of cis-regulatory ele-
ments in Drosophila. Development 132, 1567–1577.
Hopmann, R., Duncan, D., and Duncan, I. (1995). Transvection in the
pendent interactions in trans. Genetics 139, 815–833.
Kopp, A.,Duncan,I., Godt,D., andCarroll, S.B. (2000). Genetic control
and evolution of sexually dimorphic characters in Drosophila. Nature
Lewis, D.L., DeCamillis, M., and Bennett, R.L. (2000). Distinct roles of
the homeotic genes Ubx and abd-A in beetle embryonic abdominal
appendage development. Proc. Natl. Acad. Sci. USA 97, 4504–4509.
ila santomea. Evolution Int. J. Org. Evolution 56, 2262–2277.
Lohmann, I., McGinnis, N., Bodmer, M., and McGinnis, W. (2002). The
Drosophila Hox gene deformed sculpts head morphology via direct
regulation of the apoptosis activator reaper. Cell 110, 457–466.
Mahaffey, J.W. (2005). Assisting Hox proteins in controlling body form:
are there new lessons from flies (and mammals)? Curr. Opin. Genet.
Dev. 15, 422–429.
Mahfooz, N.S., Li, H., and Popadic, A. (2004). Differential expression
patterns of the hox gene are associated with differential growth of
insect hind legs. Proc. Natl. Acad. Sci. USA 101, 4877–4882.
Pearson, J.C., Lemons, D., and McGinnis, W. (2005). Modulating Hox
Prud’homme, B.,Gompel, N.,Rokas, A.,Kassner, V.A., Williams,T.M.,
Yeh, S., True, J.R., and Carroll, S.B. (2006). Repeated morphological
evolution through cis-regulatory changes in a pleiotropic gene. Nature
Stern, D.L. (1998). A role of Ultrabithorax in morphological differences
between Drosophila species. Nature 396, 463–466.
Stern, D.L. (2000). Evolutionary developmental biology and the prob-
lem of variation. Evolution Int. J. Org. Evolution 54, 1079–1091.
Telford, M.J., and Thomas, R.H. (1998). Expression of homeobox
genes shows chelicerate arthropods retain their deutocerebral seg-
ment. Proc. Natl. Acad. Sci. USA 95, 10671–10675.
Tomoyasu, Y., Wheeler, S.R., and Denell, R.E. (2005). Ultrabithorax is
required for membranous wing identity in the beetle Tribolium casta-
neum. Nature 433, 643–647.
Warren, R.W., Nagy, L., Selegue, J., Gates, J., and Carroll, S. (1994).
Evolution of homeotic gene regulation and function in flies and butter-
flies. Nature 372, 458–461.
Weatherbee, S.D., and Carroll, S.B. (1999). Selector genes and limb
identity in arthropods and vertebrates. Cell 97, 283–286.
Weatherbee, S.D., Nijhout, H.F., Grunert, L.W., Halder, G., Galant, R.,
Selegue, J., and Carroll, S. (1999). Ultrabithorax function in butterfly
wings and the evolution of insect wing patterns. Curr. Biol. 9, 109–115.
Wittkopp, P.J., True, J.R., and Carroll, S.B. (2002a). Reciprocal func-
tions of the Drosophila Yellow and Ebony proteins in the development
and evolution of pigment patterns. Development 129, 1849–1858.
Wittkopp, P.J., Vaccaro, K., and Carroll, S.B. (2002b). Evolution of
yellow gene regulation and pigmentation in Drosophila. Curr. Biol.
Cell 125, 1387–1399, June 30, 2006 ª2006 Elsevier Inc. 1399