1 Department of Biology, University of Rochester, Rochester, NY 14627, USA
2 Department of Biomedical Genetics, University of Rochester Medical Center,
Rochester, NY 14642, USA
Authors for correspondence (e-mail:
willis_li{at}urmc.rochester.edu
and
Robert.Fleming{at}trincoll.edu)
Accepted 20 October 2003
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SUMMARY |
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Key words: Drosophila, Serrate, Notch, Apterous, Wing development
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Introduction |
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The Drosophila wing imaginal disc has provided one of the best
experimental systems for studying the general principles of organogenesis.
Patterning of the Drosophila wing imaginal disc is coordinated by
organizers localized in the dorsoventral (DV) and anteroposterior (AP)
boundaries (reviewed by Brook et al.,
1996). Long-range signaling molecules, Wingless (Wg; the
vertebrate homolog of which is Wnt) and Decapentaplegic (Dpp; the vertebrate
homolog of which is Tgfß), are induced by N signaling in the DV
organizer, and by Hh signaling in the AP organizer, respectively. Wg and Dpp
form gradients to regulate target genes in a dose-dependent fashion, thereby
providing correct spatial information during development
(Entchev et al., 2000
;
Strigini and Cohen, 2000
;
Teleman and Cohen, 2000
). The
organizers formed between the DV and the AP compartments also serve as
barriers to prevent cells of different compartments from intermingling, thus
preventing disorganized pattern formation
(Milan et al., 2001
). Both the
N and Hh signaling pathways play important roles in building up compartment
barriers (Dahmann and Basler,
2000
; Micchelli and Blair,
1999
; Rauskolb et al.,
1999
).
Apterous (Ap) is an essential selector protein in Drosophila wing
development. It is expressed in the dorsal compartment of the wing disc,
thereby conferring dorsal identity
(Diaz-Benjumea and Cohen,
1993). Chicken Lmx1, a protein similar to Ap, is also expressed in
the dorsal part of the limb bud. Lack of Lmx1 or mouse Lmx1b leads to double
ventral limbs, suggesting a conserved function of Ap homologs in specifying
dorsal appendage development (Chen et al.,
1998
; Riddle et al.,
1995
; Vogel et al.,
1995
). In addition to its ability to specify dorsal identity, Ap
is also required for growth and DV compartmentalization in flies, where it
functions upstream of the N pathway, as N pathway activation is sufficient to
rescue the growth defect of Ap mutants
(Milan and Cohen, 1999a
;
O'Keefe and Thomas, 2001
).
Although it is generally agreed that N signaling plays an important role in DV
compartmentalization, other unidentified molecules downstream of Ap may also
participate (reviewed by Irvine and
Rauskolb, 2001
).
The ligands Serrate (Ser) and Delta (Dl) activate the N pathway at the
developing DV boundary of the Drosophila wing. This activation is
mediated by Fringe (Fng), which is expressed in the dorsal compartment, and
which glycosylates N to inhibit its responsiveness to Ser dorsally, while
potentiating its ability to respond to Dl in ventral cells.
(Fleming et al., 1997;
Moloney et al., 2000
;
Rauskolb et al., 1999
).
Indeed, the Ser-Fng-N signaling pathway is evolutionarily conserved in
appendage development between insects and vertebrates
(Rodriguez-Esteban et al.,
1997
). The vertebrate homologs of Ser, Fng and N are important for
the outgrowth of the limb bud, as indicated by both functional analysis and
their expression patterns in the apical ectodermal ridge (AER), a structure
similar to the Drosophila DV border
(Rodriguez-Esteban et al.,
1997
). Molecules involved in the establishment of proximodistal
(PD) and AP polarities are also highly conserved, suggesting that arthropod
and vertebrate appendages may use similar genetic circuitry to control their
outgrowth (Shubin et al.,
1997
).
Here, we report the identification of a Drosophila wing enhancer at the Ser locus, which can be sequentially activated by selector and multiple signaling molecules during wing development. We show that Ser is temporally regulated by Ap, N, Wg and Egfr signals, and that the Ser enhancer can serve as a direct integration module for this selector protein and the extracellular signaling molecules. Our results suggest a possible mechanism by which selector(s) and signaling pathway(s) act in a sequential fashion to control the outgrowth of arthropod and vertebrate appendages.
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Materials and methods |
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In situ hybridization, immunostaining and X-Gal staining
In situ hybridization was performed as described by Fleming et al. (Fleming
et al., 1990) with modifications, including the use of a
digoxigenin-UTP-labeled Ser RNA probe (Boehringer Mannheim), omission
of proteinase K treatment, and a hybridization temperature of 55°C. The
following primary antibodies were used: monoclonal mouse anti-ßgal
(1:1000, Promega), rabbit anti-ßgal (1:4000, Cappel), rat anti-Ser
(1:1000, provided by K. Irvine) and monoclonal mouse anti-Dl Mab202 (1:250,
provided by M. Muskavitch). The following secondary antibodies were used: goat
anti-mouse TR, donkey anti-mouse TR, donkey anti-rabbit Cy5 (1:250, Jackson
Immunological Laboratories) and goat anti-mouse HRP (1:250, Promega). All
discs were dissected in PBS, fixed in 4% paraformaldehyde/PBS for 10 minutes,
and rinsed four times in PBT (0.3% Triton X-100/PBS). They were then incubated
at 4°C overnight with primary antibodies in 5% normal goat or donkey
serum/PBT (depending on the choice of secondary antibodies). The discs were
washed three times in PBT for 20 minutes, and then incubated at room
temperature for 2 hours, or at 4°C overnight, with secondary antibodies in
5% normal goat or donkey serum/PBT. The discs were then washed three times in
PBT for 20 minutes, further dissected, and mounted in 2%DABCO/70% glycerol.
All steps were performed at room temperature except those mentioned
specifically. HRP detection was performed by standard protocols. Fluorescent
images were obtained using a Leica confocal microscope. X-Gal staining was
performed as described (O'Kane,
1998) with the following modifications. Larvae were dissected in
PBS, fixed in 4% paraformaldehyde/PBS for 3 minutes and stained in an
Eppendorf Thermomixer at 600 rpm. Ser-lacZ middle and late third
instar discs were stained for 12 minutes at 37°C; Ser-lacZ early
third instar discs, all (mAp)Ser-lacZ and (mdTCF)Ser-lacZ
discs were stained at 37°C overnight.
Genetics and phenotypic examination
The Gal4/UAS system (Brand and Perrimon,
1993) was used for the following experiments. To rescue the
BdG/Ser+r83k mutant wing phenotype, we crossed
transgenic flies carrying constructs 1-7 in a BdG/TM6B, Tb
background to UAS-Ser/UAS-Ser;
Ser+r83k/Ser+r83k flies. Experimental flies
died in the late pupal stage, and the adult wings were dissected out of
non-Tubby pupae and examined. To study expression patterns of constructs 1 to
7, the following fly stocks were used: UAS-nuc-lacZ,
UAS-lacZ, UAS-GFP and ap-lacZ. To study constructs
8 and 10, the following fly stocks were used: dpp-Gal4, ptc-Gal4, en-Gal4
(e116e), UAS-ChAp (Milan and
Cohen, 1999b
), UAS-dLMO
(Milan and Cohen, 1999b
),
UAS-Ni (provided by S. Artavanis-Tsakonas),
UAS-armS10 (Pai et
al., 1997
) and UAS-DN-TCF
(van de Wetering et al.,
1997
). Ectopic expression was also achieved using a flip-out
technique under control of the actin promoter
(Ito et al., 1997
).
hs-flp was used to generate the random clones; heat shock was
performed in a water bath (30 minutes at 37°C) in late second to early
third instar. The clones were marked by the presence of GFP. To study
Ser mRNA expression, the following stocks were used:
w1118, UAS-rho*
(Xiao et al., 1996
) and
ve1vn1 (rho1vn1)
(de Celis et al., 1997
). For
separating third instar larvae into early, middle and late stages, second
instar larvae (with closed openings at the end of anterior spiracles) were
collected and transferred to apple juice plates. The third instar larvae were
selected and transferred to new apple juice plates every hour, and staged (as
hours after the second/third instar (L2/L3) molt) by measuring the incubation
time of the third instar larvae at 25°C. The beginning of the third instar
was characterized by the presence of finger-like anterior spiracles, and
molting (Bodenstein, 1994
).
Each period of the third instar early, middle and late lasts for about 24
hours (0-24 hours, 25-48 hours and 49-72 hours after the L2/L3 molt,
respectively).
DNase I footprinting and electrophoretic mobility shift assays
Footprinting assays were performed using the Core Footprinting System
(Promega) with a minor modification in preparation of the probes. The 794 bp
Ser minimal enhancer was divided into two overlapping fragments (75
bp overlap) by PCR, and cloned into pBluescript II SK+ (Stratagene). To
generate single-end-labeled probes, DNA fragments were amplified by PCR with
5'-phosphorylated T7 or T3 primers. Only one unphosphorylated 5'
end of the PCR DNA fragments could be labeled with [-32P]ATP
by T4 DNA polynucleotide kinase. DNA sequencing products were labeled with
-35S-ATP using Thermo Sequenase Cycle Sequencing Kit
(Amersham Pharmacia Biotech). 6xHis-tagged Ap
LIM
(Benveniste et al., 1998
) and
6xHis-tagged dTCF-HMG domain (Halfon
et al., 2000
) were purified using QIAexpressionist (Qiagen).
GST-Su(H) was purified, and electrophoretic mobility shift assays were
performed as described by Bailey and Pasakony
(Bailey and Pasakony, 1995
).
The proteins were dialyzed and recovered in 27.5 mM HEPES (pH 7.5), 55 mM KCl
and 5.5 mM MgCl2. 1.1 mM DTT was also present in the GST-Su(H)
protein mixture. One-tenth volume of glycerol was added, and aliquots were
frozen in liquid nitrogen and stored at 80°C.
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Results |
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To study Ser gene regulation during wing development, we first
identified wing regulatory elements in the Ser gene by attaching
various 5' and 3' flanking sequences to a yeast Gal4
gene, and then tested their ability to rescue the
BdG/Ser+r83k mutant wing phenotype by using the
Gal4/UAS system to direct expression of a Ser cDNA
(Fig. 1A)
(Brand and Perrimon, 1993). As
indicated by rescue efficiencies shown in
Fig. 1A, constructs 1, 2 and 4,
containing sequences located far from the coding region, either 5' or
3', showed no rescue of the mutant phenotype. Construct 3, containing
7.4 kb of the 5' UTR and putative promoter sequences, was able to
partially rescue the BdG/Ser+r83k mutant wing
phenotype from the posterior end up to L2
(Fig. 1D). We also examined the
expression patterns of a UAS-nuc-lacZ or UAS-lacZ reporter
gene driven by constructs 1-4. Consistent with the rescue experiments,
constructs 1 and 2 showed little or no expression in the wing disc
(Fig. 1H; data not shown).
Construct 3 was mostly expressed in the dorsal compartment, preferentially in
the posterior region (Fig. 1I), also in line with the rescue experiment. Although construct 4, containing 8 kb
of the 3' end of the Ser transcript and flanking region was
expressed in wing discs, its expression pattern was less defined and did not
completely recapitulate the endogenous Ser pattern
(Fig. 1J,
Fig. 2). Thus, constructs 1-4,
containing individual regulatory regions of 20 kb 5' and 8 kb 3'
flanking sequences, are not sufficient to fully rescue the
BdG/Ser+r83k mutant wing phenotype.
|
To determine a minimal sequence requirement in the 8 kb of the 3' flanking region, two smaller enhancer fragments, 4 kb and 2.7 kb, respectively, were combined with the 7.4 kb 5' flanking sequence to make constructs 6 and 7. They were then tested for their ability to rescue the BdG/Ser+r83k wing phenotype and to direct lacZ expression in wing discs. Constructs 6 and 7 were able to rescue the BdG/Ser+r83k wing phenotype as well as construct 5 (Fig. 1F); their expression patterns were also indistinguishable from that of construct 5 (data not shown). These results suggest that regulatory elements important for correct Ser expression during wing development reside in the 2.7 kb 3' flanking region. This hypothesis was confirmed by a fusion of the 2.7 kb sequence and a lacZ reporter gene (construct 8), which recapitulated Ser expression patterns in wing discs (Fig. 5F). We refer to construct 8 as the Ser wing enhancer.
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|
Ap regulates Ser expression in early third instar
Ser is expressed in the dorsal compartment during the early stages
of wing disc development (Fig.
2E). This expression pattern is identical to that of the selector
gene of the dorsal compartment, ap, which encodes a homeodomain
transcription factor (Cohen et al.,
1992). It has been hypothesized that early Ser expression
in the dorsal compartment is under the direct control of Ap
(Irvine and Vogt, 1997
).
However, no direct evidence has been shown to support this hypothesis. To
determine whether Ser is a direct target gene of Ap, we tested
whether the 794 bp Ser minimal wing enhancer is regulated by Ap using
both in vivo and in vitro methods. Construct 10, Ser-lacZ containing
the 794 bp Ser minimal enhancer, is expressed in a stripe in the
dorsal compartment flanking the DV boundary at 24 hours after the L2/L3 molt
in early third instar (Fig.
3A,B). We expressed a constitutively active form of Ap (ChAp)
(Milan and Cohen, 1999b
) using
the Gal4/UAS system and examined Ser-lacZ expression. When we used
Dpp-Gal4 to drive ChAp expression at the anteroposterior (AP) boundary, we
found ectopic Ser-lacZ expression in the ventral wing regions along
the AP boundary, overlapping dpp-Gal4 expression in early and late third
instar (Fig. 3D,E,G,H). This
indicated that Ap was sufficient to activate Ser expression, probably
cell-autonomously. To determine whether Ap function is necessary for
Ser expression, we expressed an Ap antagonist, dLMO
(Milan and Cohen, 1999b
), in
cells along the AP boundary, using a patched (ptc) promoter. This led
to the loss of Ser-lacZ expression in the early third instar and
partial reduction of Ser-lacZ in the late third instar
(Fig. 3J,K,N,O), suggesting
that Ap is required in vivo for Ser expression in the dorsal
compartment.
To test whether early Ser expression can be directly regulated by
Ap, we used DNaseI footprinting analysis to determine the interaction sites
between the 794 bp DNA sequence and Ap. A total of 14 protected Ap binding
sites were detected spanning the 794 bp element
(Fig. 3R-V,
Fig. 7A). The binding of Ap to
this Ser minimal wing enhancer is sequence specific with two major
binding sequences, TAATNN and CAATNN (Fig.
3W). The TAATNN consensus sequence matches the six-nucleotide
consensus binding sequence for homeodomain proteins
(Gehring et al., 1994). There
is also the non-canonical CAATNN consensus sequence derived from the aligned
sequences, which matches the consensus binding sites for some homeodomain
proteins, such as murine S8 (de Jong et
al., 1993
). The existence of four CAATNN sites suggests that Ap
may bind the CAATNN sequences specifically, in addition to the canonical
TAATNN sites.
|
A positive-feedback loop through the N pathway regulates Ser expression in mid third instar
Ser is expressed along the DV boundary in the mid third instar. It
has been shown that a constitutively active N expressed under control of the
ptc promoter causes ectopic Ser expression along the AP
border (Panin et al., 1997).
However, it is not clear whether the N pathway directly regulates Ser
through its downstream transcription factor, Suppressor of Hairless [Su(H)]
(reviewed by Artavanis-Tsakonas et al.,
1999
). To test whether the Ser enhancer is directly
regulated by the N pathway, we first tested the responsiveness of the
Ser wing enhancer (in construct 8, which is identical to Construct 10
in terms of expression patterns and levels; see
Fig. 1) to N signaling. Using
the flip-out system (Ito et al.,
1997
), we generated random clones expressing constitutively active
N (Ni) in the wing disc. As shown in
Fig. 4A-D, the ectopic
expression of construct 8 Ser-lacZ was detected in the clones
expressing constitutively active N. Thus, the Ser enhancer contains
cis elements responsive to the N pathway.
|
To test whether the two Su(H)-binding sites were functional in vivo, we
synthesized a mutant Ser-lacZ construct, (mSu(H))Ser-lacZ,
carrying mutations in two nucleotides of both Su(H)-binding consensus
sequences (RTGRGAR to RTARAAR)
(Nellesen et al., 1999). This
construct showed significantly reduced activity in the wing disc in mid third
instar (Fig. 4I), as compared
with the Ser-lacZ disc at the same stage
(Fig. 4H). These data show that
at least two Su(H)-binding elements are involved in determining the activity
of the Ser minimal wing enhancer in vivo. We conclude that N
signaling directly regulates Ser gene expression by binding of Su(H)
to the Ser minimal wing enhancer.
Wg signaling regulates Ser expression in late third instar
In late third instar, Ser is expressed in cells flanking the DV
boundary. It has been shown that Wg signaling can regulate Ser
expression in these flanking cells (de
Celis and Bray, 1997;
Micchelli et al., 1997
).
However, it is not known how Wg signaling controls Ser expression at
the molecular level. To assess the possibility that Ser may be
directly regulated by the Wg pathway through the Ser wing enhancer,
we first tested whether construct 8 Ser-lacZ responds to the
Wg pathway. Using the flip-out system, we found that a constitutively active
component of the Wg pathway, ArmadilloS10 (ArmS10)
(Pai et al., 1997
), can
upregulate Ser-lacZ expression cell autonomously in the wing
pouch territory, which is consistent with a previous study demonstrating that
Wg signaling induces Ser expression in that area, but not in the
thorax or hinge (Fig. 5A-D)
(de Celis and Bray, 1997
).
This result demonstrated that Wg signaling is sufficient to upregulate
Ser enhancer expression. To further test whether Wg signaling is
required for Ser-lacZ expression, we expressed a suppressor
of Wg signaling, dominant-negative TCF (DN-TCF), in the posterior wing
compartment, driven by the engrailed (en) promoter.
Expression of DN-TCF greatly diminished Ser-lacZ expression
in posterior cells of the ventral compartment, and significantly reduced
Ser-lacZ levels in the posterior dorsal compartment, as
compared with wild-type Ser-lacZ expression
(Fig. 5F,G). Thus, Wg signaling
is necessary for expression of the Ser enhancer in cells flanking the
DV boundary. Taken together, we conclude that Wg signaling contributes to
activation of the Ser enhancer in these cells.
To test whether Ser enhancer expression could be directly
regulated by Wg signaling through its downstream transcription factor dTCF, we
performed DNase I footprinting to look for binding sites for dTCF-HMG (DNA
binding domain) (Halfon et al.,
2000). dTCF-HMG is able to bind nine sites within the 794 bp Ser
enhancer. Three of these sites conform to a class of canonical dTCF binding
sites, CCTTTGATCTT. Interestingly, consistent with a recent report
(Lee and Frasch, 2000
), we
also found that four other sites match a motif bound by HMG proteins. There
are two non-canonical binding sites, which do not conform to either the dTCF
or HMG canonical class (Fig. 5L
and Fig. 6A).
|
Ser is regulated by the Egfr pathway in presumptive wing veins
Ser is expressed in presumptive wing veins in late third instar,
as well as at the pupal stage (Fig.
2G and Fig. 6A). As
Egfr signaling is required for vein development
(Diaz-Benjumea and Garcia-Bellido,
1990; Guichard et al.,
1999
), we analyzed whether Ser expression in provein
cells is regulated by the Egfr pathway. We examined Ser expression in
both gain-of-function (gof) and loss-of-function (lof) Egfr signaling-mutant
backgrounds. First, in a rho gof mutant
(UAS-rho*) (Xiao et
al., 1996
), we observed that Ser appeared to be
ectopically expressed between L3 and L4
(Fig. 6C,D), exactly where
ectopic rho activity was localized (data not shown). We next observed
that Ser expression in the proveins was eliminated in a rho
and vein (vn, encoding a Egfr ligand) double-mutant (Egfr
lof) background, in which vein formation is completely abolished
(Fig. 6E,F)
(de Celis et al., 1997
). These
results suggest that the Egfr pathway may regulate Ser expression
during vein development at the pupal stage.
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Discussion |
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Sequential regulation of Ser by a selector gene and multiple signaling pathways
The results reported here demonstrate that a 794 bp cis-acting regulatory
module in the Ser locus can be temporally regulated by three distinct
mechanisms that are employed for the proper establishment of the DV organizer
during wing development. First, the selector protein Ap directly activates
Ser expression in the dorsal compartment during the early third
instar, which sets up N activation for the next stage. Second, by the middle
of the third instar, the N pathway maintains Ser expression by a
positive-feedback loop along the DV boundary. This feedback loop maintains Ser
and Dl expression, leading to the activation of N signaling at the DV
boundary, which is essential for establishing the DV organizer
(Panin et al., 1997). Third,
at the end of the third instar, as a result of Wg signaling, Ser is
expressed in two stripes flanking the DV boundary, which limits N activation
to the DV border (Fig. 7B). In
addition, we have demonstrated that Ser expression in provein cells
is dependent on input from the Egfr pathway. Our results indicate how
tissue-specific selector and signaling molecules can work sequentially to
achieve a complex developmental process, such as organogenesis, which involves
a complex temporal and spatial regulation of genes. However, our conclusion
that the Ser minimal wing enhancer is sequentially regulated by Ap,
Notch, Wg and Egfr does not exclude the possibility that these
molecules/signaling pathways may cooperate and synergistically stimulate gene
expression at certain stages. In this case, mutations that specifically impair
response to the intended factor would affect Ser-lacZ expression in
other phases of disc development.
Ap regulates Ser directly in early third instar
Here, we provide evidence that Ser is indeed a direct target gene
for Ap, thus forming a link between Ap, which specifies dorsal identity, and
the signaling pathways that organize the DV boundary
(Diaz-Benjumea and Cohen,
1993). Specifically, we show that Ap regulates the Ser
minimal wing enhancer in vivo, and binds the enhancer in vitro through two
major DNA sequences, TAATNN and CAATNN
(Fig. 3W). Ap may regulate the
Drosophila FMRFa neuropeptide gene and a mouse glycoprotein hormone
-subunit gene enhancer by binding to TAATNN sequences
(Benveniste et al., 1998
;
Rincon-Limas et al., 2000
;
Roberson et al., 1994
). Thus,
TAATNN sequences may regulate most, if not all, Ap target genes.
The 794 bp Ser minimal wing enhancer is regulated by Ap, and is
expressed in the dorsal compartment of wing and haltere discs at 7.5 hours
after the L2/L3 molt in early third instar
(Fig. 3X1,Y1). The 7.4 kb
5' flanking sequence and the 8 kb 3' flanking sequence can also
direct reporter gene expression in all of the dorsal compartment during wing
development (Fig. 1I,J;
Fig. 2A,B; data not shown). A
9.5 kb Ap cis-response element was also isolated 7.5 kb upstream of the
Ser translational initiation site (it contains most sequences in construct 1
and construct 2, and a 2 kb BamHI/BamHI fragment in between
the two constructs; Fig. 1A),
although it is not clear whether Ap directly regulates this element. Further
dissection of this element into smaller fragments did not succeed in
recapitulating the dorsal anlage expression pattern
(Bachmann and Knust, 1998
).
These results suggest that crosstalk between different cis-elements is
required to regulate Ser dorsal expression, and that there is more
than one Ap response element at the Ser locus
(Fig. 1A,
Fig. 2A,B)
(Bachmann and Knust, 1998
).
Given the importance of Ap-regulated Ser expression, multiple Ap
response elements might be expected. Enhancer redundancy has been observed in
many genes and may have evolved as a protection against loss of gene activity
when mutations occur in regulatory sequences (reviewed by
Arnosti, 2003
).
Around 24 hours after the L2/L3 molt, a transition occurs in Ser
minimal enhancer expression from all dorsal cells to dorsal cells near the DV
boundary [24 hours after the L2/L3 molt is defined as early third instar
because 48-72 hours AEL (after egg laying) is generally taken as the early
third instar, which is equal to 0-24 h after the L2/L3 molt]
(Fig. 3X1-2). During this
transition, Ser expression in dorsal cells flanking the DV boundary
may be regulated by Ap, as well as by the N pathway
(Klein and Arias, 1998;
Klein et al., 2000
). At 24
hours after the L2/L3 molt, (mAp)Ser-lacZ displayed no activity, and
(mSu(H))Ser-lacZ expression was evident in dorsal cells near the DV
boundary (Fig. 3Y2; data not
shown). Although these data suggest that Ap regulates Ser expression
in dorsal cells near the DV boundary, they do not exclude the possibility that
Notch may still be involved in directly regulating Ser expression
during this transition, as Su(H) may still be able to bind to and activate
(mSu(H))Ser-lacZ (also see below).
Notch signaling in the formation of the DV boundary
Activation of N signaling at the nascent DV boundary is essential for the
formation of the DV boundary (de Celis et
al., 1996; Micchelli and
Blair, 1999
; Rauskolb et al.,
1999
; Sturtevant and Bier,
1995
). Ser and Dl are highly expressed at the DV border in
mid-third instar and their expression can be ectopically activated by a
constitutively active form of N, which suggests a positive-feedback loop
between N ligands and the receptor (de
Celis and Bray, 1997
; Panin et
al., 1997
). The activation of such a feedback loop between N and
its ligands is likely to be among the earliest events in the formation of the
DV boundary. Our finding that the Ser wing enhancer is regulated by
the N pathway, and that two Su(H)-binding sites are required for the in vivo
activity of this enhancer in the mid third instar, suggests that N signaling
can directly regulate Ser expression through Su(H). Although these
results are consistent with direct activation of the Ser gene by
Su(H), they do not preclude the possibility that N signaling may regulate
Ser through other transcription factors, possibly downstream of
Su(H). This would explain why (mSu(H))Ser-lacZ showed a significant,
but not dramatic, loss of enhancer activity
(Fig. 4H,I). Alternatively, it
remains possible that Su(H) can still bind to and activate at least one of the
two mutant Su(H) binding sites in (mSu(H))Ser-lacZ.
Wg signaling directly regulates Ser in late third instar
Our in vitro and in vivo results suggest that the regulation of
Ser by Wg signaling occurs directly through dTCF. Using DNase I
footprinting, we found two major classes of dTCF binding sequences: the dTCF
consensus sequence CCTTTGATCTT and the HMG consensus sequence WTTGWW, which
are consistent with previously identified dTCF binding sequences
(Lee and Frasch, 2000;
Riese et al., 1997
;
van de Wetering et al., 1997
).
Interestingly, the presence of dTCF/HMG binding sites in the Ser
minimal wing enhancer may explain the crosstalk observed between the 3'
Ser enhancer and the 5' Ser promoter
(Fig. 1). HMG proteins can bend
DNA, and could therefore bring the 3' enhancer close enough to interact
with the transcriptional machinery binding at the 5' promoter (reviewed
by Thomas, 2001
).
In late third instar, Wg signaling is maintained in the DV organizer by the
N pathway (Micchelli et al.,
1997). Wg signaling activates Ser and Dl
expression in the cells flanking the DV boundary, which in turn activates N
signaling to maintain a positive-feedback loop between N and Wg signals
(Fig. 7B)
(de Celis and Bray, 1997
;
Micchelli et al., 1997
).
Because of an autonomous repression effect of N ligands on their receptor,
Ser and Dl expression in the flanking cells also prevents N
signaling from spreading out of the DV border. N signaling then turns off
Ser and Dl expression by inducing cut at the border
(de Celis and Bray, 1997
).
Although the molecular nature of the dominant-negative effects of N ligands,
and the repression of Ser and Dl by N signaling remains unknown, these
mechanisms may play important roles in keeping the boundary sharp
(Micchelli et al., 1997
).
Interestingly, the Ser minimal wing enhancer is also repressed at the
DV border, suggesting that it is possible to study the molecular mechanism of
Ser repression at the border using this 794 bp enhancer.
Regulation of Ser in provein cells by Egfr signaling
We have demonstrated that Ser is expressed in provein cells and
that its expression is regulated by Egfr signaling at the pupal stage. N
signaling also plays an important role in determining vein cell fate
(de Celis et al., 1997;
Huppert et al., 1997
). Our
data on Ser expression in provein cells is consistent with a report
on Ser function during vein development
(Zeng et al., 1998
). Thus, in
addition to its essential role in development of the Drosophila leg
and vertebrate limbs, Egfr/Fgf signaling also plays a role in
Drosophila wing development, suggesting a conserved role of Egfr
signaling in `appendage' development
(Campbell, 2002
;
Diaz-Benjumea and Garcia-Bellido,
1990
; Galindo et al.,
2002
; Guichard et al.,
1999
). Interestingly, the Ser minimal wing enhancer is
expressed in provein cells at both larval and pupal stages (S.-J.Y., W.X.L.
and R.J.F., unpublished). Further investigation of this element may shed light
on how Egfr signaling regulates vein differentiation.
The Ser minimal wing enhancer, an evolutionarily conserved element
Given that the Ser-Fng-N pathway is evolutionarily conserved in appendage
development between insects and vertebrates
(Laufer et al., 1997;
Rodriguez-Esteban et al.,
1997
), the mechanism by which Ser is sequentially
regulated by Ap, N, Wg and Egfr may also be conserved in appendage outgrowth
of other arthropods and vertebrates. Consistent with this hypothesis, the Ap,
Wg/Wnt and Egfr/Fgf pathways are also involved in appendage development in
vertebrates, as well as D. melanogaster
(Kawakami et al., 2001
;
Shubin et al., 1997
). Indeed,
a BLAST search of the Drosophila pseudoobscura genome identified a
putative homolog of the Ser minimal wing enhancer. Interestingly,
this enhancer region is also located less than 1 kb downstream of the putative
D. pseudoobscura Ser 3'UTR. Sequence comparisons between the
Ser minimal wing enhancer from D. melanogaster and the
putative D. pseudoobscura enhancer show a significant degree of
similarity, whereas the similarities in the 5' and 3' flanking
regions are lower (Fig. 7A). Importantly, sequences of putative Ap, Su(H) and dTCF binding sites are highly
conserved in D. pseudoobscura and D. melanogaster. Although
the strong conservation of sequence and location suggests that the putative
D. pseudoobscura Ser enhancer may be a functional homolog of the
D. melanogaster Ser minimal wing enhancer, it remains to be tested
whether this enhancer drives reporter gene expression at the identical time
and location in the D. melanogaster wing discs.
![]() |
ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Present address: Biology Department, Trinity College, 238 Life Sciences
Center, Hartford, CT 06106, USA
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