1 Department of Biological Sciences, Stanford University, Stanford, CA 94305,
USA
2 Stanford Genome Technology Center, 855 California Avenue, Palo Alto, CA 94306,
USA
Author for correspondence (e-mail:
bbaker{at}pmgm2.stanford.edu)
Accepted 15 January 2004
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SUMMARY |
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Key words: Drosophila, Sex determination, Microarray, Somatic, Reproduction
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Introduction |
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Drosophila melanogaster is a powerful model system in which to
acquire an understanding of the sex-specific physiology of adult somatic
tissues, because we have a thorough understanding at the molecular-genetic
level of the regulatory hierarchy that controls somatic sexual differentiation
(Fig. 1) (reviewed by
Cline and Meyer, 1996;
Baker et al., 2001
;
Christiansen et al., 2002
).
There have been significant advances in understanding how the actions of
DSXF and DSXM, terminal transcription factors in the
hierarchy encoded by the doublesex (dsx) gene, are
integrated with other key developmental hierarchies to achieve sex-specific
patterns of growth, morphogenesis and differentiation (reviewed by
Christiansen et al., 2002
).
However, we have relatively little knowledge of the genes that are
sex-differentially deployed in adults through the action of the two final
genes in the hierarchy, dsx and fruitless (fru),
which encodes (among several isoforms) a male-specific transcription factor
hereafter referred to as FRUM.
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Here, we identify genes that are expressed sex-differentially in somatic
tissues of adults and regulated by the sex hierarchy. Using arrays that assay
approximately one-third (4040) of Drosophila genes, we analyzed
adults mutant for the regulatory genes transformer (tra),
dsx and fru (Fig.
1). To select a small number of such genes for further study, we
chose a conservative approach. Stringent statistical analysis of these data,
combined with data from wild-type adults and adults that lack germline tissue
(Arbeitman et al., 2002),
identified 63 genes that are sex-differentially expressed in the adult soma
and regulated by the somatic sex hierarchy. Additional selection criteria, and
validation by RNA blot analysis, defined a set of 11 genes for further
characterization. In situ hybridization revealed that sex-differential
expression of all 11 genes is confined to the internal genitalia. Analysis of
the regulation of these genes revealed that the sex hierarchy functions during
development to specify their adult expression patterns, and that dsx
probably functions in diverse ways to set their activities.
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Materials and methods |
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Microarray experiments
Microarray production, data acquisition and analysis were performed as
described in (Arbeitman et al.,
2002). Each hybridization was a comparison of one RNA sample to a
reference sample that was comprised of RNA derived from whole animals
representing all stages of the life-cycle. Normalization was calculated so
that the average ratio of signals from the experimental and reference sample
equalled one. Analyses were performed on log-transformed ratio values. In all
cases, chromosomal males and females were sampled separately. The wild-type
data set and tudor data set were described previously
(Arbeitman et al., 2002
); each
time point was sampled in duplicate. The wild-type data set includes 0- to
24-hour-, 3-day-, 5-day-, 10-day-, 15-day-, 20-day-, 25-day- and 30-day-old
adults. The tudor data set includes 0- to 24-hour- and 5-day-old
adults. Microarray experiments on sex determination mutants
tra1/Df(3L)st-j7 (XX),
fru4-40/frup1 (XY) and
DsxD/dsxm+r15 (XX) were performed with four
(tra and fru) or five (DsxD) replicates.
Microarray experiments performed on dsx null mutants
(dsxm+r15/dsxd+r3) were sampled in
duplicate.
Forced-choice statistical model
The expression levels of a gene in four experimental conditions were
compared to determine whether they were more consistent with control by
dsx or by fru. Each log-transformed data value for a gene
was set as xij, where i=1,2,3 or 4 (for wild-type
males, fru males, tud females and XX
dsxD pseudomales, respectively) and j=1,...,
ni replicates for the given genotype. If a gene is
controlled by dsx, its expression level is expected not to differ
between wild-type males and fru males; if it is controlled by
fru, its expression level is expected not to differ between
tud females and dsxD pseudomales.
First, the within-group mean square (MS) was calculated assuming the gene
was under dsx control. Three means were calculated:
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Then the sum of squared deviations of each data point from its respective
mean was calculated and divided by the degrees of freedom:
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The MS, assuming fru control, was calculated in the same way,
except that genotypes were expected to have the same expression level:
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The MSs were then compared using an F test with the appropriate degrees of freedom.
RNA blot analyses
Total RNA was isolated with Trizol (Invitrogen), followed by RNeasy
(Qiagen) or poly(A)+ isolation using Poly-ATtract (Promega). Blots were
prepared from a Northern Max kit (Ambion). Radiolabeled RNA probes made with
Strip-EZ kit (Ambion) were used at approximately 1-7x106
cpm/ml of hybridization solution. Blots were typically hybridized overnight at
68°C. Bound probes were visualized by phosphorimager (Molecular
Dynamics).
Frozen section in situ hybridization
Frozen section in situ analysis was performed as described by Goodwin et
al. (Goodwin et al.,
2000).
Whole-mount in situ hybridization
Whole-mount in situ analysis was performed as described by Tautz and
Pfeifle (Tautz and Pfeifle,
1989), but with more extensive washes. Probes were made using the
DIG RNA kit (Roche) and hydrolyzed to
200 base pair fragments.
Anti-digoxigenin FAB fragments conjugated to alkaline phosphatase (Roche),
diluted 1:2000, were used.
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Results and Discussion |
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We identified genes that display sex-differential expression using data
from seven of the eight wild-type adult time points. To maintain the
independence of subsequent statistical tests, data from one time point, 0- to
24-hours, were reserved for subsequent comparisons. Here, 1,576 out of 4,040
genes showed significant sex-differential expression: 897 and 679 with higher
expression in females and males, respectively (P<0.001 in a
two-way ANOVA with sex and developmental time as main effects). We next used
three separate one-tailed Student's t-tests to determine which of
these 1,576 genes were likely to be sex-differentially expressed in the soma
due to regulation by tra (see Fig.
1). All t-tests were carried out in duplicate, using both
standard deviation and a global standard deviation
(Jiang et al., 2001); genes
had to pass both tests. The combination of the three tests was expected to
yield a low false-positive rate.
The first and second t-tests asked which of the 1,576 genes were
sex-differentially expressed in somatic tissue and downstream of tra,
by comparing data from tud male and female animals
(P<0.05), and from wild-type female and XX; tra/tra
(hereafter called XX tra) individuals (P<0.05),
respectively. We found 147 genes to be sex-differentially expressed in the
soma and regulated by tra: 91 had higher expression levels in females
and 56 had higher expression levels in males. We
(Arbeitman et al., 2002)
previously employed a test similar to the first t-test, but the final
set of genes was limited to those with a greater than 2-fold expression
difference, a criterion not applied here. The third test here required that,
when a gene was expressed in both the soma and the germline, the germline
expression was minor. We compared expression in wild-type females or males
with that in same-sex tud animals, to identify genes that did not
display significant differences in expression (P>0.2). Although
this test re-uses data, it is purely conservative because it can only exclude
genes from consideration. Of the 147 genes above, 73 genes passed this test,
37 and 36 with higher expression in females and males, respectively.
We confirmed the identity of the 73 genes by sequencing both ends of the cDNAs used to produce the microarray elements. We found ten cDNAs to be chimeric and removed these from further consideration, which left 63 genes that appeared to be expressed sex-differentially in somatic tissues and regulated by tra: 29 with higher expression in females and 34 with higher expression in males. Hereafter, we refer to these genes as female genes or male genes, respectively.
To compare the developmental expression profiles of these 63 genes, we generated a hierarchical cluster, which groups genes on the basis of similarities in their expression profiles (Fig. 2). The cluster included expression data from wild-type animals throughout development, and adult data from 0- to 24-hour and 5-day tud adults, 0- to 24-hour XX tra and XX dsxD pseudomales, dsx null intersexual animals (XX and XY) and fru males that lack transcripts encoding FRUM. Examination of the cluster showed that transcripts from about half of the female genes were present during the earliest stages of embryogenesis, suggesting that they are maternally contributed to the embryo, whereas the other half had their onset of expression during later zygotic stages. Many of the male genes were expressed before the adult stage, but their highest levels of expression were during adult life.
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Microarray data for four genes identified exclusively in the previous study
indicate that those four genes may be sex-differentially expressed in somatic
tissues, but not regulated by tra. On sequencing the ends of these
genes to confirm their identities, two were found to be from contaminated
sources and were not considered further. One remaining gene, more highly
expressed in males, was found to be RNA on the X 1 (roX1),
which is involved in the male-specific process of dosage compensation and is
known not to be regulated by tra (reviewed by
Meller and Kuroda, 2002). The
other remaining gene, more highly expressed in females, was CG9709, which
encodes Acox57D-d (an enzyme expressed in the peroxisome). CG9709 was not
further validated by RNA blot analysis and we do not have the statistical
power to say with high confidence that it is not controlled by
tra.
Selecting genes for further study
We next chose a subset of 11 of the 63 genes for in-depth analysis, based
on whether the gene: (1) showed a large difference in expression between the
sexes; (2) had biology of interest to us; and (3) showed strong evidence of
regulation by dsx or fru. All 11 genes met the first
criterion, showing expression in one sex with little or no expression in the
other sex by RNA blot (Fig. 3
and Table 2). Only two showed a
<3-fold difference between tud females and males by microarray
analysis (see Table 2): midline fasciclin (mfas), which encodes a signal transduction
protein, and paired (prd), which encodes a transcription factor. The
published information on these genes, and their likely regulatory roles,
favored their inclusion. Other genes considered biologically interesting were
those that suggested ties to existing knowledge of Drosophila
reproductive biology, or that were homologs of mammalian genes involved in
disease processes (see next section).
To identify genes by the third criterion we used two approaches. First,
t-tests were used to compare microarray data between wild-type
females and XX dsxD pseudomales, or wild-type and
fru males. Second, because of low sample sizes, and to avoid
identifying genes based on strain-specific differences, a statistical model
was developed that predicted either dsx or fru regulation
for each gene (see Materials and methods and
Table 2). As both approaches
reanalyze data used previously, the results are heuristic. Of the 11 genes
ultimately selected, 10 were significant for regulation by dsx
according to one or both approaches. The exception was the male gene
mfas, which the forced-choice model found (though not with
significance) more likely to be regulated by fru, and which is known
to function in the central nervous system
(Hu et al., 1998), as do the
male-specific fru products. Having chosen a set of 11 genes, two
female and nine male, we proceeded to determine their patterns of expression
by in situ hybridization.
Tissue-specific adult expression
We determined the expression patterns of the 11 genes by in situ
hybridization to frozen sections and to wholemounts of dissected tissue from
5-day-old wild-type adults. In all cases, the direction of sex-differential
expression agreed with both microarray and RNA blot analyses, and was due to
expression in the sex-specific tissues of the internal genitalia. Of the 11
genes, nine were expressed exclusively in one sex or the other, and two were
expressed in both sexes with overall expression higher in males. Two genes, in
addition to sex-differential expression in the internal genitalia, were
expressed sex-nonspecifically in tissues outside the genitalia.
Female genes
The internal reproductive system of females consists of ovaries, oviducts,
three sperm storage organs (the sperm receptacle and the paired spermathecae),
paired parovaria (also known as female accessory glands), the uterus and
vagina (Miller, 1950)
(Fig. 4A). Spermathecae store
sperm, but have not been well studied at the molecular level. Among the
insects in which parovaria have been studied, functions of their secretions
vary, and, in other Diptera, include antibacterial action
(Marchini et al., 1991
) and
increased sperm penetration of the egg
(Leopold and Degrugillier,
1973
). Spermathecae and parovaria are somatically derived,
surrounded by fat body and connected by short ducts to the uterus.
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CG7777 was expressed in both somatic and germline tissues
(Fig. 4C, part II), as
predicted by RNA blot analysis (Fig.
3B). CG7777 mRNA was detected in spermathecae and oviducts
(Fig. 4C, part II, parts e,f),
and in egg chambers at most stages. In stages 8 through 10, expression was
strong in nurse cells (germline origin)
(Fig. 4C, part II, part c). By
stages 13 and 14, when the chorion is being secreted by follicle cells
(somatic origin), expression was seen in follicle cells most strongly
in those of the dorsal appendage (Fig.
4C, part II, part d). In tud animals, CG7777 message was
apparent in spermathecae, and in the peritoneal or epithelial sheath of
immature ovaries or ovarioles, respectively
(Fig. 4C, part II, part g).
CG7777 is predicted to encode an aquaporin, a member of the MIP (Major
Intrinsic Protein) family of transmembrane transporters (reviewed by
Verkman, 2002), and is most
similar to aquaporins 1 and 4 (Blastp, e value 1e-35) in humans.
Male genes
The internal male reproductive system includes the testes, seminal
vesicles, accessory glands, ejaculatory duct and ejaculatory bulb (or sperm
pump) (Bairati, 1968)
(Fig. 5A). Secretions from the
male accessory glands perform many functions in the mated female, including
decreasing her receptivity to mating, increasing ovulation and egg production,
and enhancing sperm survival and storage (reviewed by
Wolfner, 2002
). Sperm,
accessory gland secretions and ejaculatory duct secretions are propelled
forward from the ejaculatory duct to the ejaculatory bulb, which adds to the
propulsion of seminal fluid and contributes secretions of its own
(Bairati, 1968
). Five genes
were expressed exclusively in one organ and we describe these first.
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CG18284 was strongly expressed in the main cells and at the junction of the
accessory glands to the ejaculatory duct
(Fig. 5C, part I, parts a-c).
CG18284 is similar to several lipases, including, in Drosophila, Lip1
and Lip3 (Blastp, e value 3e-63 and 2e-62, respectively).
Lip1 expression is not sex-differential in adults and may be a
digestive enzyme, whereas Lip3 is not expressed in the adult
(Pistillo et al., 1998).
Previous studies have reported strong triacylglycerol lipase activity in
accessory glands, and transfer of this activity to mated females
(Smith et al., 1994
). Six
lipases in the D. melanogaster genome have been identified through
homology searches using D. simulans accessory gland ESTs
(Swanson et al., 2001
). Two of
these, CG17101 and CG17097, are quite similar to CG18284 (Blastp, e value
2.1e-190 and 1.1e-111, respectively), and all three map to the same
chromosomal region (2L,
31F5). Swanson et al. suggest that lipases
contributed by the male to the mated female might be involved in lipid
nutrient metabolism or in fusion of spermatazoa and eggs
(Swanson et al., 2001
).
CG17022 was strongly expressed in the accessory gland (Fig. 5C, part I, part d). Its predicted protein is serine-rich, with no significant similarities to known proteins.
CG17843 (Fig. 5C, part I,
part e) is conserved in the two canonical domains of the recently proposed
QSOX family of sulfhydryl oxidases and has been named dmQSOX2 (reviewed by
Thorpe et al., 2002). In many
organisms, QSOX are known to localize to intracellular and extracellular
spaces of secretory tissues, where they are thought to catalyze and maintain
the formation of disulfide bonds in secreted proteins. In addition, they may
participate in forming the extracellular matrix of these tissues.
Interestingly, sulfhydryl oxidase message has been found in the male rat
genital tract, where proposed functions include protecting spermatozoa from
microbial or sulfhydryl degradation
(Benayoun et al., 2001
).
Accessory gland expression of CG17843 is consistent with these suggested
functions.
CG8708 is expressed strongly and exclusively in the epithelium of the
anterior ejaculatory duct (Fig.
5C, part II, parts a-d). CG8708 is similar to a class of
ß3-galactosyltransferases in a variety of organisms (Blastp, e value
1e-73 in C. elegans and mouse, for example). The putative human
ortholog, which shows 41.3% similarity to CG8708, has been identified as core
1 UDP-galactose:N-acetylgalactosamine-alpha-R ß1,3-galactosyltransferase
(core 1 ß3-Gal-T) (Ju et al.,
2002a), which catalyzes the last step in the formation of the core
1 structure a precursor for many membrane-bound and secreted
mucin-type glycoproteins. In rat, the shorter of two transcripts for core 1
ß3-Gal-T was previously found in the testis only; humans have two
transcripts as well (Ju et al.,
2002a
; Ju et al.,
2002b
). We also found two transcript size classes of CG8708 on RNA
blots (Fig. 3E). Both sizes
were detected in the male soma and a trace amount of the shorter transcript
was also found in the female soma. Frozen section in situ analysis detected
sex-nonspecific expression of CG8708 in the salivary glands of adults
(Fig. 5C, part II, part e), and
of a stage 13 embryo found within an adult female
(Fig. 5C, part II, part f),
suggesting a developmental role.
In the ejaculatory bulb, CG2858 was highly expressed but in variable
patterns between the horns of each lobe and in streaks of cells extending up
the sides of each lobe (Fig.
5C, part III, parts c-e). CG2858 contains a male sterility (MS2)
domain (Blastp, expect 3e-56, 196 residue domain, 100% aligned), first
identified in Arabidopsis thaliana
(Aarts et al., 1997) and now
known in many organisms. In the jojoba seed, MS2 has been characterized as a
fatty acyl-coA reductase that is involved in the biosynthesis of wax storage
lipids (Metz et al., 2000
).
Drosophila ejaculatory bulb secretions contribute substantially to
the formation of the waxy mating plug in the mated female
(Bairati and Perotti, 1970
;
Lung and Wolfner, 2001
). These
secretions include Protein Ejaculatory Bulb-melanogaster (PEB-me; PEB
FlyBase), which may contribute to the mating plug structure
(Lung and Wolfner, 2001
), and
the lipid cis-vaccenyl acetate (Brieger
and Butterworth, 1970
). The discovery of a putative MS2 protein
suggests identification of an enzymatic contributor to the waxy mating plug
structure.
Two genes, CG12558 and CG9519, were detected in both the accessory glands
and ejaculatory bulb (Fig. 5C,
part IV). CG12558 (Fig. 5C, part IV, parts a-d) encodes a predicted endopeptidase with a trypsin-like
serine protease domain (Blastp, expect 4e-12, 88% aligned). To date, nine
putative proteases have been identified among Drosophila male
accessory gland proteins (reviewed by
Wolfner, 2002). Lack of
identity to these proteases suggests CG12558 is the tenth such protein.
CG9519 (Fig. 5C, part IV,
part e) contains a conserved choline dehydrogenase domain (Blastp, expect
1e-94, 542 residue domain, 98.5% aligned). Although CG9519 is annotated as a
choline dehydrogenase (The FlyBase
Consortium, 2003) and is similar to human choline dehydrogenase
(5e-76), it also shares a high degree of identity with Drosophila
glucose dehydrogenase (GLD) (Blastp, e value e-102). GLD has previously been
shown to be regulated by the sex hierarchy
(Feng et al., 1991
) and
transferred to females during mating
(Cavener and MacIntyre, 1983
).
However, the pattern of localization we found for CG9519 differs from that of
GLD in D. melanogaster; GLD is present only in the ejaculatory duct
of males and is also expressed in the female reproductive tract
(Cox-Foster et al., 1990
;
Feng et al., 1991
).
mfas encodes a protein containing fasciclin and ß-Ig-H3
domains, which are thought to mediate cell adhesion
(Hu et al., 1998).
mfas transcript was detected in both sexes (although at higher levels
in males than females) according to both microarray and RNA blot analyses, and
was detected in the internal genitalia of both sexes. In males, mfas
transcript is strongly expressed in a region of each testis extending
approximately one tenth its length from the proximal end, and in a small area
around the testis attachment site on the seminal vesicle
(Fig. 5C, part V, part c), in
both main and secondary cells of the accessory glands, at the junction with
the anterior ejaculatory duct (Fig.
5C, part V, parts d and b, respectively), and in the ejaculatory
bulb in a pattern similar to that described for CG2858
(Fig. 5C, part V, part e). In
females, prominent signal was detected in follicle cells that surround oocytes
of stage 9 and 10 egg chambers (Fig.
4C, part III, part c), until later stages when these cells are
sloughed off (Fig. 4C, part
III, part d). Weak signal was seen in parovaria of some individuals
(Fig. 4C, part III, part e),
and in fat tissue surrounding the spermathecae
(Fig. 4C, part III, part f).
The expression of mfas in a region of the gonad containing mature
germline cells in both sexes, combined with its predicted role in cell
adhesion, raises the possibility that it may function to mediate
germline/somatic interactions prior to release of the sperm or egg into the
seminal vesicle or oviduct, respectively. In addition, mfas was
expressed in the central nervous system (CNS;
Fig. 4C, part III, parts g,h);
previous analyses demonstrated that mfas is expressed on the midline
neurons and glia in the embryo (Hu et al.,
1998
). At the level of resolution of our analysis, we are unable
to detect expression differences between males and females in the CNS.
Finally, in males we detected prd in the accessory glands and, at
low levels, in the testes (Fig.
5C, part VI). In females, prd is strongly expressed in
the fat body surrounding the spermathecae
(Fig. 4C, part IV), although
this expression appears variable among individuals. Localization of
prd to male accessory glands had previously been documented
(Bertuccioli et al., 1996),
but expression in the testes and female fat body had not been reported.
prd encodes a transcription factor in the Pax homeodomain gene
family. In addition to its role in activation of segment-polarity genes,
prd is involved in the development of adult male accessory glands and
in the regulation of at least three accessory gland proteins
(Xue and Noll, 2002
).
Identification of a gene, outside our derived data set, with a sex-specific developmental expression profile
Any set of criteria designed to identify genes from microarray studies may
exclude some genes of legitimate interest. Here, for example, visual
inspection of the adult microarray data
(Fig. 5B), and subsequent in
situ analysis, identified CG6788, expressed only during the 0- to 24-hour
stage of adult male life. This gene was not identified above because the first
ANOVA test used to identify genes did not use the 0-24 hour time point. CG6788
encodes a predicted cell adhesion protein that contains a fibrinogen domain
(Blastp, expect 4e-59, 215 residues, 92.1% aligned) with similarity to
angiopoietin-like 1 precursor (Blastp, expect 3e-37). This gene is expressed
in the male ejaculatory bulb at the 0- to 24-hour stage
(Fig. 5C, part VII), but not in
5-day-old males. Expression was observed in a striped pattern in the
epithelial cells that border each lobe. The timing of CG6788 expression
suggests that it might play a role in ejaculatory bulb development rather than
a physiological role.
On the nature of sex-differential gene expression in adults
Previous microarray studies in Drosophila showed that about half
the genome is sex-differentially deployed
(Jin et al., 2001;
Arbeitman et al., 2002
;
Parisi et al., 2003
;
Ranz et al., 2003
). In C.
elegans, a smaller percentage of the genome is so deployed (12%)
(Jiang et al., 2001
). Our
results indicate that, in Drosophila, less than 1.5% of the genome is
sex-differentially regulated by tra in the soma; however, because our
statistical approach is conservative, this is likely to be an underestimate.
In C. elegans, the fraction of the genome sex-differentially deployed
in somatic tissue is also small [based on Jiang et al., the low estimate is
3-4% (Jiang et al., 2001
)].
Thus in two very different animals it appears that sex-differential
transcription in the adult is largely due to germline expression, with a much
smaller set of genes sex-differentially expressed in the soma.
Our 63-gene set was defined as those genes whose somatic sex-differential expression is downstream of tra. We used a forced-choice statistical model to determine whether dsx or fru was more likely to regulate each gene in our subset and found that, for 58 genes in our 63-gene set, and for 10 of the 11 genes in the group chosen for further study, sex-differential expression is more likely to be a consequence of dsx activity than fru (Table 2). This indicates that the approach taken was very successful in identifying genes sex-differentially expressed as a consequence of dsx action, but detected few, if any, genes regulated by fru.
Given our potential to detect genes sex-differentially deployed in any
somatic tissue of adults, it is striking that all 11 genes whose expression
patterns we determined were expressed in internal genital tissues. Although we
have only assayed 11 out of 63 potential genes here, the results of previous,
smaller scale screens are consistent with our results (reviewed by
Wolfner, 1988). The tissues
that comprise the internal genitalia are small; the largest is the male
accessory gland, which has about 1050 cells
(Bertram et al., 1992
). Thus
our experiments could readily detect sex-differential gene expression in a
very small fraction of adult cells, with the qualification that, to the degree
the tissues of the internal genitalia are producing large amounts of a limited
number of transcripts, this sensitivity may be less impressive than it
seems.
Why, then, as we know that cells of other parts of adults are sexually
dimorphic, did whole organism studies not detect genes expressed
sex-differentially in these tissues? Whole-animal assays are probably missing
these genes because they are sex-differentially expressed at low levels in
relatively few cells, or sex-specifically in one tissue and
sex-nonspecifically in other tissues. These considerations are supported by a
comparison of the genes identified by SAGE analysis as sex-differentially
expressed in the head (Fujii and Amrein,
2002) with our data set. Of the 19 genes identified as having
significant sex-differential expression levels in head tissue, five were in
our array (Cypd4d21, CG4979, CG11458, CG7433, CG5288). Cypd4d21 showed
significant sex-differential expression, in the same direction, in both
studies. By contrast, CG7433 expression was significantly higher in male head
tissue, but significantly lower in male whole bodies in our study. We did not
detect a sex difference in the expression levels of the other three genes.
These considerations probably explain why genes differentially expressed as a
consequence of fru activity were not identified.
Regulation by the sex hierarchy of sex-differentially expressed somatic genes
Temporal regulation of sex-differential adult gene expression by the hierarchy
We next examined when sex hierarchy regulation of the 11 selected genes
occurs. There are two known mechanisms by which sex-differential gene
expression in adults is generated: (1) the sex hierarchy actively regulates
gene expression in adults, as is the case for Yolk protein 1
(Yp1) (Belote et al.,
1985); or (2) the hierarchy functions earlier in development to
specify which sex-specific adult tissues will be formed but does not regulate
gene expression in those tissues in the adult (reviewed by
Christiansen et al., 2002
). We
used temperature-sensitive tra2 alleles, which allowed us to switch
between the male and female mode of splicing dsx
(Nagoshi et al., 1988
). In
chromosomally XX tra-2ts animals, female development
occurs at the permissive temperature (16°C), whereas male development
occurs at the non-permissive temperature (29°C). Animals were raised at
one temperature, collected 0-24 hours after eclosion and maintained at their
original temperature for one more day. We then switched half of each group to
the other temperature (16°C to 29°C, or 29°C to 16°C). All
animals were maintained for three more days, and RNA was then extracted. Under
these conditions, expression of Yp1 (the positive control,
Fig. 6C) responded to
temperature shifts as expected (Belote et
al., 1985
); the Yp1 transcript was reduced when animals
were switched from 16°C to 29°C and induced when animals were switched
from 29°C to 16°C. By contrast, expression of the 11 other genes
analyzed did not change substantially over the three days following the
temperature shifts (Fig. 6).
Thus, sex-differential expression of all 11 genes is the consequence of the
developmental action of the sex hierarchy and is independent of the hierarchy
during adult stages.
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Modes of dsx regulation of sex-differentially expressed genes
As suggested by our microarray data, the 11 genes expressed in internal
genital organs are almost certainly regulated by dsx rather than by
fru, as dsx is known to regulate development of these
tissues and FRUM is not expressed within the internal genitalia.
The DSXM and DSXF proteins are known to have both
positive and negative roles (Baker and
Ridge, 1980). We therefore sought to understand the manner in
which the DSX proteins regulate these genes. For the two genes expressed in
multiple tissues in adults, we carried out in situ hybridizations to XX and XY
dsx null individuals to assess the mode of dsx regulation in
individual organs. For the five genes that are expressed in one organ of the
internal genitalia and nowhere else in adults, we used microarray data from
dsx null individuals. The results of these analyses are suggestive of
multiple modes of dsx regulation.
To determine how dsx regulated the female gene CG17012 in
spermathecae, we carried out in situ hybridization on wholemounts of internal
genitalia of dsx null and wild-type individuals. Spermathecae are not
recognizable in all dsx mutant individuals
(Hildreth, 1965), so just
those individuals with spermathecae were evaluated. Reduced expression, as
compared with wild-type females, was seen in spermathecae of dsx null
individuals (Fig. 7A,B). The
diminished CG17012 expression in dsx mutants suggests that
DSXF positively regulates its sex-specific expression. The
microarray data for CG17012 are consistent with this conclusion and do not
reveal any effect of DSXM on CG17012 expression. However, another
possible explanation for reduced expression in dsx null individuals
is that dsx mutants are unable to mate. There is evidence for changes
in female behaviors mediated (perhaps at the transcriptional level) by seminal
fluid and sperm transferred during copulation, including a reduction in
receptivity to courtship and enhanced egg laying (reviewed by
Wolfner, 2002
).
|
For the three genes (CG17843, CG17022 and CG18284) expressed exclusively in
the male accessory glands, the microarray results suggest that their
expression is not dependent on DSXM, as comparable high expression
levels are seen in XY wild-type and XY dsx null individuals
(Table 3). Rather,
male-specific expression appears to be the consequence of the negative action
of DSXF in females, as their expression is higher in XX
dsx null individuals than in wild-type females
(Table 3). These observations
are consistent with the previous finding that male-specific development of the
accessory glands is the consequence of DSXF acting in females to
prevent accessory gland formation (Ahmad
and Baker, 2002). Interestingly, expression of these three genes
is significantly higher in XY dsx null than XX dsx null
(P<0.05), but is not significantly different between wild-type
males and fru males, suggesting an additional sex-differential
complexity to their expression.
|
Finally, for CG2858 and CG8708, expressed exclusively in the ejaculatory bulb and ejaculatory duct, respectively, the microarray data suggest another mode of dsx regulation. For both of these genes, expression appears to be lower in XY dsx null animals than in the three genotypes expressing DSXM (XY wild type, XX tra and XX dsxD pseudomales, Table 3), suggesting that DSXM positively regulates their expression. Expression also appears higher in XX dsx null compared with XX wild-type animals, suggesting negative regulation by DSXF in the formation of these tissues in females. This pattern of regulation has not been previously reported for a gene under the control of dsx, although it is the exact converse of how the Yp genes are regulated.
Taken together these findings reveal a rich diversity of dsx
function. In the accessory gland the sole role of dsx revealed to
date is the action of DSXF to prevent the formation of the organ in
females, whereas in the spermathecae, ejaculatory duct and ejaculatory bulb
dsx appears to have two types of functions. First, the hierarchy must
be acting, via dsx, to direct these tissues to an alternative
developmental fate in the inappropriate sex
(Hildreth, 1965;
Keisman et al., 2001
). Second,
as shown here, dsx may also function in the appropriate sex in these
three organs prior to adulthood, and probably during the late larval/early
pupal period (DiBenedetto et al.,
1987
; Chapman and Wolfner,
1988
; Feng et al.,
1991
), to establish the potential for the appropriate patterns of
gene expression.
The sex determination hierarchy in Drosophila is well understood at the molecular-genetic level, but the genes that are sex-differentially regulated by the hierarchy have only begun to be identified. Here, we examined sex-differential gene expression in adults, the stage of the Drosophila life cycle that displays the most striking differences between the sexes. This study adds substantially to our knowledge of the types of genes expressed sex-differentially in somatic tissues, provides molecular entry points for elucidating the functions of reproductive organs of both sexes, and expands our understanding of the timing and mode of gene regulation by the sex hierarchy.
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ACKNOWLEDGMENTS |
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Footnotes |
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These authors contributed equally to this work
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