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First published online 14 March 2007
doi: 10.1242/dev.001578
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1 Department of Biological Science and Technology, Tokyo University of Science,
2641 Yamazaki, Noda, Chiba 278-8510, Japan.
2 Okazaki Institute for Integrative Bioscience, National Institute for Basic
Biology, National Institutes of Natural Sciences, Higashiyama, Myodaiji,
Okazaki 444-8787, Japan.
3 Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa,
Nagoya 464-8601, Japan.
* Author for correspondence (e-mail: matsuno{at}rs.noda.tus.ac.jp)
Accepted 6 February 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila melanogaster, Drosophila virilis, rhomboid, mirror, gurken, Broad-Complex, EGFR signaling, cis-regulatory element, trans-regulatory landscape, Evolution, Eggshell, Dorsal appendage
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Davidson, 2001;
Simpson, 2002). Although
differences in the expression patterns of genes with instructive roles in
morphogenesis have been demonstrated in various species
(Averof and Patel, 1997;
Gompel and Carroll, 2003;
Shapiro et al., 2004;
Simpson et al., 1999;
Stern, 1998), few studies have
addressed the molecular mechanisms underlying the diversification of these
gene expression patterns (Belting et al.,
1998; Gompel et al.,
2005; Wittkopp et al.,
2002). The dorsal appendages (DAs) are specialized respiratory
structures on the drosophilid eggshell
(Hinton, 1969;
Spradling, 1993a). The
mechanisms of their morphogenesis have been extensively studied in
Drosophila melanogaster, whose eggshell has two DAs
(Fig. 1B)
(Berg, 2005). There is
remarkable diversity in the DA morphology among species, including their size,
shape and number, although these characteristics are stereotyped within each
species (Kambysellis and Craddock,
1997; Patterson and Stone,
1952; Throckmorton,
1962). Thus, comparative studies of gene expression patterns
during DA formation are useful for understanding the mechanisms underlying the
evolutionary diversification of gene expression
(Barkai and Shilo, 2002;
Derheimer et al., 2004;
James and Berg, 2003;
Nakamura and Matsuno, 2003;
Peri et al., 1999;
Perrimon and Duffy, 1998).
The egg chamber of Drosophila consists of a single oocyte and 15
nurse cells surrounded by a layer of somatic follicle cells
(Margolis and Spradling, 1995;
Spradling, 1993a). In D.
melanogaster, the DA primordia arise from a subset of follicle cells that
are specified by epidermal growth factor receptor (EGFR) signaling
(Nilson and Schupbach, 1999;
Wasserman and Freeman, 1998).
Gurken (Grk) is a transforming growth factor-
-like protein and an
oocyte-specific ligand for EGFR that localizes to the dorsal-anterior end of
the oocyte and is presented to the overlying follicle cells
(Neuman-Silberberg and Schupbach,
1993; Nilson and Schupbach,
1999; Wasserman and Freeman,
1998). Grk induces the expression of rhomboid
(rho), which encodes a serine protease, in a single population of
dorsal anterior follicle cells (Lee et
al., 2001; Ruohola-Baker et
al., 1993; Urban et al.,
2001), and the Decapentaplegic signaling pathway helps limit the
expression of rho in these cells
(Peri and Roth, 2000). In
these follicle cells, Rho processes Spitz, a transmembrane ligand for EGFR, to
a secreted and active form, which in turn amplifies EGFR signaling in the
follicle cells (Sapir et al.,
1998; Schweitzer et al.,
1995; Wasserman and Freeman,
1998). The high EGFR signaling activity triggers the expression of
argos at the dorsal anterior midline and subsequently establishes a
negative feedback loop, resolving the single peak of EGFR signaling into twin
peaks (Wasserman and Freeman,
1998). Consequently, a single DA primordium is formed at each
region of peak EGFR signaling activity
(Wasserman and Freeman,
1998).
D. virilis, a species that diverged from D. melanogaster
40-60 million years ago, has four DAs (Fig.
1A,C) (Powell,
1997). We previously showed that rho is expressed
differently between D. melanogaster and D. virilis
(Fig. 1D,E). At stage 10A,
rho is expressed in D. melanogaster in a dorsal anterior
saddle-shaped zone that includes the midline
(Fig. 1D), but in D.
virilis there are two dorsal-lateral domains
(Fig. 1E). At stage 10B,
rho expression is refined into two L-shaped stripes in D.
melanogaster (Fig. 1D);
the pattern in D. virilis becomes a V-shaped stripe with its apex
missing (Fig. 1E). Finally, the
rho expression is maintained in two regions of follicle cells where
the two DAs will form in D. melanogaster at stage 12
(Fig. 1D), whereas in D.
virilis, rho expression is restricted to four domains corresponding to
the positions of the future DAs (Fig.
1E). Thus, changes in the regulation of rho expression
could be responsible for the divergence in DA number
(Nakamura and Matsuno, 2003).
In this study, we investigated the mechanisms by which rho expression
diverged during the evolution of D. melanogaster and D.
virilis. Our results suggest that divergence of the trans-acting
landscape regulating the rho expression probably had a crucial role
in the evolution of different DA numbers in these two species.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was used
as a pole cell transplantation host.
Immunohistochemistry
Immunofluorescent staining of ovaries was performed as described
(James and Berg, 2003), except
that Block Ace (Dainippon Pharmaceutical) was used as a blocking reagent. The
following primary antibodies were used: rabbit anti-green fluorescent protein
(GFP) (1:1000, MBL), mouse anti-ß-galactosidase (Gal) (1:500, Promega)
and rabbit anti-Broad-Complex (BRC) core (1:2000)
(Dequier et al., 2001). Alexa
Fluor-488-conjugated antirabbit immunoglobulin G (IgG) (1:400, Molecular
Probes) and Cy3-conjugated anti-mouse IgG (1:400, Rockland) were used as
secondary antibodies. Immunofluorescent staining of embryos was performed as
described (Hayashi et al.,
2004). Embryos were stained with rabbit anti-Vasa (1:500) and
mouse anti-GFP (1:500, Wako Pure Chemicals) antibodies, and then treated with
Alexa Fluor-568-conjugated anti-rabbit IgG (1:500, Molecular Probes) and Alexa
Fluor-488-conjugated anti-mouse IgG (1:500, Molecular Probes).
In situ hybridization
The RNA probes were labeled with digoxigenin (Roche), and in situ
hybridization was performed as described
(Wasserman and Freeman,
1998).
Cloning of mirror (mirr)
Partial DNA fragments of mirr for in situ hybridization were
amplified using genomic DNAs from Canton-S and D.
virilis (#15010-1051.87) as template DNAs by PCR. The primers were:
Mirr_FW, 5'-GATATGATGACCGACC-3' and Mirr_RV,
5'-CCTATAAGCTCTGATTGC-3'.
Cloning of the D. virilis 5' rho regulatory DNA
The DNA probe of the D. virilis rho cDNA was labeled with
digoxigenin (DIG DNA labeling Mix, Roche) by random-primed labeling
(Nakamura and Matsuno, 2003).
A D. virilis phage genomic library (gift of T. C. Kaufman, Indiana
University, Bloomington, IN) was screened with this probe using standard
conditions (Sambrook and Russel,
2001). Twenty-one overlapping phage clones that hybridized with
the D. virilis rho probe were isolated. The three largest genomic
fragments isolated from these phage clones were digested with SalI
and subcloned into the SalI site of pBluescript KS(-). These
subclones were sequenced, aligned and ligated. Finally, a genomic DNA fragment
12-kb upstream of the start site of the D. virilis rho cDNA was
obtained (GenBank accession number AB278158).
Transgene construction for rho enhancer analysis
A genomic fragment covering the 2.2-kb upstream region of the D.
melanogaster rho gene was generated by PCR using the D.
melanogaster genomic P1 clone DS02734 (GenBank accession number AC004343)
as a template (Kimmerly et al.,
1996), and subcloned into the BamHI site of
pCaSpeR-NLSlacZ (gift of C. Thummel, Howard Hughes Medical Institute,
University of Utah, Salt Lake City, UT). The primers were: rho2.2-FW,
5'-CGGGATCCCGCAAGCTTTTCCTCTGCTC-3' and
rho2.2-RV, 5'-CGGGATCCCGTTCTCTGCTTGCACCCAC-3.
(Restriction enzyme cloning sites are underlined.)
The 2.2-kb upstream fragment of the D. melanogaster rho gene and fragments containing the 4.2- and 12-kb upstream regions of the D. virilis rho gene were subcloned into the BamHI and KpnI/BamHI sites of pSL[hsp27mp-NLS-EGFP] (T.N., unpublished), respectively. The resulting constructs were digested with AscI, and each insert was cloned into the AscI site of pBac[3xP3-DsRedaf].
Transformation of D. melanogaster and D. virilis
Germline transformation of the P-element CaSpeR vectors was performed
according to a standard protocol
(Spradling, 1993b). For
transformation with the piggyBac vector, piggyBac constructs
were co-injected into the eggs of D. melanogaster yw and D.
virilis w with phsp-pBac, and flies were screened for 3xP3-DsRed
expression under a fluorescence stereoscopic microscope (gift of E. A. Wimmer,
Georg August University, Göttingen, Germany). For each transgene, at
least three independent insertions were isolated and characterized.
Pole cell transplantation
Pole cell transplantation was performed as described
(Kobayashi et al., 1996), with
the following modifications. As a marker for the D. melanogaster
germline, we used an EGFP-vasa construct that expresses GFP
specifically and continuously in the germline throughout the life cycle
(Sano et al., 2002). We
transplanted pole cells from D. virilis (#15010-1051.0) embryos
[200-250 minutes after egg laying (AEL) (25°C)] into D.
melanogaster embryos [100-150 minutes AEL (25°C)] carrying the
EGFP-vasa. The donor embryos were at the cellular blastodermal
stage.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Ip et al., 1992;
Sapir et al., 1998;
Ward and Berg, 2005). This
rho enhancer, termed Dmel rho2.2, is sufficient to drive
the expression of a reporter gene in the developing follicle cells
(Dorman et al., 2004;
Sapir et al., 1998;
Ward and Berg, 2005). At stage
10B, Dmel rho2.2 is activated in L-shaped stripes on either side of
the midline (Dorman et al.,
2004; Sapir et al.,
1998; Ward and Berg,
2005). This expression pattern resembles that of endogenous
rho, which is essential for DA formation, at the same stage
(Fig. 1D and
Fig. 3C)
(Dorman et al., 2004;
Sapir et al., 1998).
We speculated that a similar cis-regulatory element should exist in the
D. virilis rho gene. Therefore, we isolated a 12-kb genomic region
upstream of the D. virilis rho cDNA start site (GenBank accession
number AB278158). To test whether the 12-kb fragment or a 4.2-kb upstream
fragment derived from it contained a regulatory sequence that governs
rho expression in the follicle cells, we analyzed the activity of
these fragments in D. virilis using piggyBac- mediated
transgenesis (Handler, 2002).
When placed upstream of a GFP reporter gene, the 12- and 4.2-kb upstream
fragments drove reporter expression in the follicle cells of D.
virilis during DA formation in essentially the same manner
(Fig. 2 and data not shown).
Therefore, we used the 4.2-kb fragment in the following studies, because a
shorter fragment is preferable for generating transgenic lines efficiently in
D. virilis and D. melanogaster. At stage 10B, Dvir
rho4.2 drove reporter expression in a pattern identical to the
distribution of endogenous D. virilis rho mRNA
(Fig. 2A,C). At stage 12, Dvir
rho4.2 also drove GFP expression in the four domains that
subsequently form the four DAs (Fig.
2D); this pattern was also similar to that of endogenous
rho at the same stage (Fig.
2B). We concluded that Dvir rho4.2 consists of an
enhancer orthologous to Dmel rho2.2.
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;
Tzolovsky et al., 1999). The
juxtaposition of rho-expressing and BR-C-expressing cells is required
for DA formation in D. melanogaster
(Ward and Berg, 2005). In
cells expressing rho, Spitz is converted into an active and secreted
ligand for EGFR (Lee et al.,
2001; Urban et al.,
2001), so that EGFR signaling is activated and triggers the
expression of BRC in adjacent cells
(Schweitzer et al., 1995;
Ward and Berg, 2005). In
D. virilis and D. melanogaster, the rho and BR-C
expression domains are mutually exclusive at stage 10B
(Nakamura and Matsuno, 2003),
indicating that the genetic cascade involving rho and BR-C is
conserved between these species. To test whether Dvir rho4.2
carries sufficient information to restrict its activation anterior to the
BR-C-expressing domain, we analyzed the expression of Dvir
rho4.2-GFP and BR-C in D. virilis. Dvir rho4.2
was activated in a single row of cells anterior and adjacent to
BR-C-expressing cells (Fig.
2E-G). These results suggested that Dvir rho4.2
contains sufficient information for comparative studies with Dmel
rho2.2 in these two species.
Changes in the trans-regulatory landscape could cause the evolutionary divergence in rho expression
Phylogenetic analyses indicated that four-DA eggs are ancestral compared
with two-DA eggs (Fig. 1A)
(Powell, 1997;
Throckmorton, 1962). This
suggests that changes occurred in the trans-acting landscape regulating the
cis-regulatory elements of rho and/or in these cis-regulatory
elements themselves during the evolution from the four- to two-DA eggs. We
assumed that if divergence of the trans-regulatory landscape was wholly
responsible for the species-specific expression patterns of rho, the
exogenous rho enhancers would adopt the same expression pattern as
the endogenous rho expression (i.e. in D. virilis, Dmel
rho2.2 should be activated in a similar pattern to Dvir
rho4.2). By contrast, if evolutionary modifications of the
cis-regulatory elements were responsible for the divergence in rho
expression, the activation pattern of Dvir rho4.2 and Dmel
rho2.2 in the heterologous species should match the expression
pattern of the endogenous rho in their species of origin (i.e. their
homologous species).
To address this issue, we introduced the Dmel rho2.2-GFP
reporter construct into D. virilis. As reported previously, Dmel
rho2.2 was activated in L-shaped domains at stages 10B and 12 in
D. melanogaster (Dorman et al.,
2004). Interestingly, Dmel rho2.2 was activated in the
V-shape with its apex missing in D. virilis at stage 10B
(Fig. 3A). This pattern was
very similar to the expression patterns of Dvir rho4.2-GFP and the
endogenous rho in D. virilis at this stage. Furthermore, at
stage 12, Dmel rho2.2 drove expression in a pattern resembling that
of Dvir rho4.2-GFP and endogenous rho in D.
virilis, which was significantly different from the activation pattern of
the Dmel rho2.2 in its homologous species, D.
melanogaster (Fig.
3B). These results suggested that the Dmel rho2.2
enhancer could respond to the heterologous trans-regulatory landscape in a
manner similar to the endogenous rho enhancer in D. virilis
during DA formation. Given these results, we speculated that diversification
in the trans-regulatory landscape, rather than modification of the cis-acting
elements, was mostly responsible for the differences in rho
expression patterns during DA formation in these two species.
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Evolution of the cis-regulatory elements also contributed to the species-specific activation patterns of the rho enhancers
Our results suggested that the trans-regulatory landscapes that direct
rho expression are evolutionarily divergent between D.
virilis and D. melanogaster. However, we also noted that the
expression domain of the Dvir rho4.2-GFP was slightly expanded
posteriorly compared with that of the Dmel rho2.2-GFP in D.
melanogaster (compare Fig.
3E with Fig. 3C).
This difference became more obvious at stage 12 (compare
Fig. 3F with
Fig. 3D). At this stage, the
Dvir rho4.2-GFP was expressed in a square on either side of the
midline, rather than in the L-shaped pattern
(Fig. 3F).
To study further the differential activation patterns between Dvir rho4.2 and Dmel rho2.2 in D. melanogaster, we introduced Dvir rho4.2-GFP and Dmel rho2.2-ß-Gal simultaneously into D. melanogaster, and observed the expression patterns driven by Dvir rho4.2 and Dmel rho2.2 with anti-GFP and anti-ß-Gal antibody staining, respectively. Both the GFP and ß-Gal products of the reporter constructs carry a nuclear localization signal. Thus, the expression of these reporters was detected as nuclear staining at the single-cell level. The expression of Dvir rho4.2-GFP and Dmel rho2.2-ß-Gal differed temporally and spatially (Fig. 4). When Dmel rho2.2-ß-Gal expression started, early in stage 10B, the Dvir rho4.2-GFP expression was not yet detectable (Fig. 4A-C). This difference was not owing to features of the GFP and ß-Gal reporters (for example, their relative translational efficiencies), because when the GFP and ß-Gal reporters were expressed under the control of the same enhancer, Dmel rho2.2, their expression patterns were essentially identical, temporally and spatially (see Fig. S1 in the supplementary material). Late in stage 10B, both Dvir rho4.2-GFP and Dmel rho2.2-ß-Gal were detectable (Fig. 4D-F). The anterior border of the expression domain of Dvir rho4.2-GFP and Dmel rho2.2-ß-Gal was the same (Fig. 4F). However, whereas Dmel rho2.2-ß-Gal was expressed in a single row of cells (Fig. 4D), Dvir rho4.2-GFP was expressed in one or two extra rows posterior to the single row where both reporters were expressed (Fig. 4E). At stage 12, expression of the Dmel rho2.2-ß-Gal was still restricted to the single row of cells (Fig. 4G), whereas the expression domain of the Dvir rho4.2-GFP had expanded posteriorly (Fig. 4H). These results suggest that the functions of the rho enhancers also evolved between these two species, although both enhancers maintained the ability to respond to the positional information that defines the anterior borders of their activation. However, we also noted that a few of the anterior-most cells expressed Dvir rho4.2-GFP but not Dmel rho2.2-ß-Gal at stage 12 (Fig. 4I), suggesting that some other functions might have also changed between the two enhancers.
Conservation of the Grk signal during evolution of the DA number
During DA patterning, the germline oocyte provides positional information
to the somatic follicle cells through the Grk signal
(Nilson and Schupbach, 1999).
A mathematical model predicts that the distribution and amounts of Grk could
play a key role in determining the number of DAs
(Shvartsman et al., 2002).
Considering that the Grk signal serves as the first cue for the positional
information that defines the expression patterns of rho in the
follicle cells, we proposed the following two hypotheses: first, that some
divergence in the pivotal mechanism for determining the number of DAs occurred
in the Grk signal itself, and second, that the trans-regulatory landscape that
modifies the Grk signal in somatic follicle cells changed.
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). In the
D. melanogaster hosts, the D. virilis germline was
recognized by the absence of GFP expression. First, we examined whether the D. virilis pole cells could enter the gonad of the D. melanogaster embryo and whether the D. virilis germline cells could form the chimeric egg chamber in concert with D. melanogaster follicle cells in D. melanogaster ovaries (Fig. 5B-B'',C). The transplanted D. virilis pole cells, which expressed only endogenous Vasa (Fig. 5B'', purple), were incorporated into the gonad of D. melanogaster together with the D. melanogaster pole cells (Fig. 5B''). Furthermore, these germline cells derived from D. virilis formed a chimeric egg chamber together with the D. melanogaster follicle cells (white arrowhead in Fig. 5C), despite their phylogenic divergence. In contrast to the prediction from the mathematical model, we found that all the chimeric eggs (n=16) observed in four host females had two DAs (Fig. 5E), identical to the pattern of D. melanogaster (Fig. 5D).
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).
In addition to its role in DA formation, the Grk signal has an essential role
in the dorsal-ventral patterning of the egg
(Neuman-Silberberg and Schupbach,
1993; Nilson and Schupbach,
1999). When the dorsal-ventral patterning is aberrant in D.
melanogaster, the DAs are shifted with respect to the dorsal midline,
dorsally or ventrally, respectively, with the loss or gain of Grk signaling
(Neuman-Silberberg and Schupbach,
1993; Nilson and Schupbach,
1999; Schupbach,
1987). Therefore, we measured the distance between the roots of
the two DAs in wild-type and chimeric eggshells, both of which were laid by a
single transplanted host female to avoid any other effects, such as
nutritional status. The distances between the two DAs in the wild-type
(48.2±4.6 µm, n=57) and chimeric (49.2±3.6 µm,
n=12) eggshells showed no statistically significant difference
(Fig. 5F,G,
0.5>P>0.2, Student's t-test), suggesting that the
dorsal-ventral patterning occurred normally in the chimeric eggs. It is
unlikely that the two different patterning events, DA formation and
dorsal-ventral patterning, both of which are triggered by Grk signal-dependent
positional information, were both properly compensated for by chance. Thus, we
speculate that the role of Grk in DA patterning is conserved between D.
virilis and D. melanogaster, suggesting that subsequent events,
downstream of the Grk signal, diverged to elicit the different expression of
rho between these two species.
Mirr is a candidate molecule responsible for the divergence of rho expression
In D. melanogaster, mirr, which encodes a homeobox transcription
factor, is expressed in dorsal anterior follicle cells
(Jordan et al., 2000;
Zhao et al., 2000). This
expression pattern depends on the Grk signal, and mirr subsequently
activates the expression of rho during oogenesis
(Jordan et al., 2000;
Jordan et al., 2005;
Zhao et al., 2000). Therefore,
mirr might introduce the evolutionary changes into the landscape of
trans-regulatory factors that are responsible for the divergence of
rho expression in D. virilis and D. melanogaster.
To test this hypothesis, we analyzed the mirr expression patterns in
these two species.
In both species, mirr expression began similarly, at the dorsalanterior region of the follicle cells, at stage 9, prior to the onset of rho expression (Fig. 6A,E). After this stage, the mirr expression followed species-specific patterns, which resembled the rho expression pattern in each species. In D. melanogaster, the expression of mirr was repressed in the dorsal midline at stage 10A (white arrowhead in Fig. 6B) and was elevated in the follicle cells at the anterior boundary (white arrow in Fig. 6B), which corresponded to the anterior row of rho-expressing cells in the L-shaped pattern at stage 10B (Fig. 1D). This anterior expression continued until stage 10B (white arrow in Fig. 6C). By contrast, early in stage 10A in D. virilis, the mirr expression was repressed only at the dorsal-anterior region, not the entire dorsal midline (white arrowhead in Fig. 6F). Late in stage 10A, the region lacking mirr expression expanded and became a triangle (Fig. 6G), which consequently divided the mirrexpressing region into two dorsal-lateral domains that were reminiscent of the rho-expressing domains of D. virilis at stage 10A (Fig. 1E). After stage 10B, the expression of mirr was barely detectable in either species (Fig. 6D,H). The expression of D. virilis mirr was not restricted to the domains expressing rho (Fig. 1E). Therefore, the expression of mirr does not always cause the expression of rho. Nevertheless, our results suggest evolutionary changes in the mirr expression patterns as a candidate mechanism for the divergence of rho expression at stage 10A in D. virilis and D. melanogaster.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Gompel et al., 2005;
Wittkopp et al., 2002). The
gain and loss of cis-acting elements have played central roles in the
divergence of the expression patterns of genes that play crucial roles in the
generation of specific characteristics in different species
(Gompel et al., 2005). In this
study, we investigated the contribution of these two processes to the
evolutionary diversification of DA numbers in D. virilis and D.
melanogaster. In addition to the importance of cisregulatory elements
demonstrated previously, our findings suggest that the landscape of
trans-regulatory factors could also change and affect morphological divergence
during evolution.
In D. melanogaster, rho expression has an instructive role in
defining the pattern of DA precursor cell formation
(Sapir et al., 1998;
Ward and Berg, 2005). In
addition, we previously demonstrated that the expression patterns of
rho diverged and were correlated with the position and number of DAs
in D. virilis and D. melanogaster
(Nakamura and Matsuno, 2003).
Therefore, in this study, we mostly focused on the enhancers of rho
in these species. To distinguish whether divergence in the trans-regulatory
landscape or the cis-regulatory elements is important for the evolutionary
change in rho expression patterns between D. melanogaster
and D. virilis, we introduced reporter constructs of Dvir
rho4.2 and Dmel rho2.2 into these two species.
Phylogenic analyses of Drosophila species suggest that the four DAs
are an ancestral characteristic, and that the flies with two DAs evolved from
four-DA ancestors (Fig. 1).
Thus, the characteristics of Dmel rho2.2 were probably derived from
the ancestral Dvir rho4.2 enhancer. We found that Dvir
rho4.2 and Dmel rho2.2 adopted the expression pattern of
the endogenous rho of the heterologous species. These results suggest
that Dvir rho4.2 and Dmel rho2.2 did not diverge in
terms of their ability to respond to the trans-acting factors in follicle
cells. Therefore, we speculated that changes in the cis-regulatory elements
from Dvir rho4.2 to Dmel rho2.2 were not the main cause
for divergence in the activation patterns of these enhancers in their
homologous species.
Although the DNA sequences of Dvir rho4.2 and Dmel
rho2.2 diverged drastically, several putative binding sites for
transcription factors, such as ETS, Su(H) and BR-C, were common to both (data
not shown), which could explain the conserved function of the two enhancers.
Recently, it was reported that BR-C represses the activity of Dmel
rho2.2 in a cell-autonomous manner during DA patterning
(Ward et al., 2006), and this
repression allows the enhancer to be activated in the L-shaped region. These
two rho enhancers share five overlapping binding sites for BR-C (data
not shown). Thus, these BR-C-binding sites might serve as cis-regulatory
elements to transmit the conserved functions of these two enhancers. Notch (N)
signaling also regulates Dmel rho2.2
(Ward et al., 2006), and we
found that one binding site for Su(H) is conserved among all six
Drosophila species examined (data not shown). Conservation of the
binding sites for these various transcription factors and their possible
involvement in the evolution of DA patterning suggest that rho
expression is controlled by complex responses to multiple transcription
factors, instead of by a simple EGFR-signal feedback system, which is
consistent with the model proposed by Peri and Roth
(Peri and Roth, 2000).
We identified Mirr as a candidate for the difference in the landscape of
trans-regulatory factors between D. melanogaster and D.
virilis. The distribution of the mirr transcript was
significantly different between these species. mirr induces
rho expression, and regulates N signaling by repressing
fringe, probably thereby regulating rho
(Blair, 2000;
Bruckner et al., 2000;
Moloney et al., 2000).
Although whether or not Mirr function is also involved in the regulation of
rho transcription in D. virilis remains to be tested, it is
conceivable that changes in the expression patterns of mirr may
account, at least in part, for the divergence in the activation patterns of
Dvir rho4.2 and Dmel rho2.2 in D. melanogaster
and D. virilis.
In D. melanogaster, rho is expressed in a saddle-shaped pattern at stage 10A. We analyzed the genomic region within 26.2-kb upstream and 11.8-kb downstream of the transcription initiation site of rho, but failed to identify an enhancer element responsible for this early expression pattern (data not shown). The function of this early rho expression in DA formation has not yet been studied. Therefore, we could not exclude the possibility that an enhancer that regulates the early expression of rho is involved in the diversification of the rho expression pattern. However, we speculate that this early expression of rho does not play a significant role in determining the number of DAs, because D. pseudoobscura and D. melanica have eggs with two DAs, but the saddle-shaped pattern of rho expression was not detected in these species (T.K., Y.N. and K.M., unpublished). Therefore, the subsequent expression of rho is probably what plays a crucial role in determining the DA number.
Changes in cis-acting elements may contribute to the evolutionary divergence in rho expression between D. melanogaster and D. virilis
Our present analysis revealed that the functions of Dvir rho4.2
and Dmel rho2.2 are largely conserved. However, we also found that
Dmel rho2.2 had evolved a novel trait during its diversification
from Dvir rho4.2. In D. melanogaster, both Dvir
rho4.2 and Dmel rho2.2 were activated in the L-shaped
pattern at stage 10B. However, Dvir rho4.2 was activated in one or
two extra rows of cells posterior to the single row of cells where Dmel
rho2.2 was active at this stage. At stage 12, Dvir
rho4.2 was activated much more posteriorly, although Dmel
rho2.2 was still active only in the single row of cells. Given that
Dvir rho4.2 is ancestral to Dmel rho2.2, we speculate
that Dmel rho2.2 lost a cis-acting element capable of being
activated in this posterior region, or gained a cis-acting element that
suppresses its activity in this region at stage 12. Indeed, it is likely that
this posterior activation of rho is an ancestral characteristic,
because the endogenous expression of rho in this region is found in
D. virilis but not D. melanogaster.
The Grk signal may not play a crucial role in the evolution of DA number in D. melanogaster and D. virilis
For the formation of DAs, the patterning of EGFR signaling activity in the
follicle cells plays crucial roles in D. melanogaster
(Wasserman and Freeman, 1998).
Two major events are involved in the regulation of EGFR signaling activity in
these cells. First, Grk specifically localizes to the dorsal anterior part of
the oocyte and activates EGFR in the overlying follicle cells
(Wasserman and Freeman, 1998).
Second, in the follicle cells, positive and negative feedback loops elaborate
the pattern of EGFR signaling activity that ultimately determines the number
of DAs (Wasserman and Freeman,
1998). Thus, the first and second events are germ- and
soma-derived events, respectively.
As predicted from the above model, the intensity of Grk expression and the
width of its expression domain in the oocyte are thought to define the number
of DAs (Shvartsman et al.,
2002). A mathematical study predicted that changes in the amount
and distribution of Grk protein in the oocyte can account for the evolution of
eggshells with zero to four DAs in Drosophila species
(Shvartsman et al., 2002).
However, our experiments involving a chimeric egg chamber suggest that changes
in the follicle cells, but not in the oocyte, have an instructive role in
determining the number of DAs. These results suggest that the change in Grk
signaling did not contribute to the evolution of DA numbers in these species.
This is consistent with a previous finding that the distribution and amount of
grk mRNA do not show a significant difference between D.
melanogaster and D. virilis
(Peri et al., 1999). However,
our results do not exclude the possibility that changes in Grk signaling play
major roles in the diversification of DA numbers during the evolution of other
Drosophila species.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/8/1529/DC1
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ACKNOWLEDGMENTS |
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