Brookdale Department of Molecular, Cell and Developmental Biology, Box 1026, Mount Sinai School of Medicine, New York, NY 10029, USA
Author for correspondence (e-mail:
manfred.frasch{at}mssm.edu)
Accepted 6 January 2005
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SUMMARY |
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Key words: Drosophila, Dpp, Wg
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Introduction |
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Tissue development in the Drosophila mesoderm has been a favorable
system in which to study these events. Upon the spreading of the mesodermal
cell layer underneath the embryonic ectoderm, the progenitor cells of
different organs, such as the dorsal vessel, somatic and visceral muscles, are
generated at stereotyped locations within the mesoderm. Specifically, in the
dorsal region of the mesoderm (which has been studied in most detail), the
progenitors of cardioblasts, pericardial cells, specific dorsal somatic
muscles and circular midgut muscles are generated
(Campos-Ortega and Hartenstein,
1997; Frasch and Nguyen,
1999
). Signals from the dorsal ectoderm mediated by Dpp are
required, but not sufficient, for the induction of all of these progenitor
cells in the dorsal mesoderm
(Staehling-Hampton et al.,
1994
; Frasch,
1995
). Importantly, the Dpp signals need to act in concert with
mesoderm-intrinsic regulators, which make the mesodermal cells competent to
respond. One of the key regulators intrinsic to the mesoderm is the NK
homeobox gene tinman, which, like dpp, is required for the
induction of all dorsal mesodermal cell types
(Azpiazu and Frasch, 1993
;
Bodmer, 1993
;
Yin and Frasch, 1998
).
tinman itself is initially activated in the early mesoderm by
twist and, just prior to cell specification events, its expression is
prolonged by Dpp signals specifically in the dorsal mesoderm
(Bodmer et al., 1990
;
Frasch, 1995
;
Yin et al., 1997
).
In addition to these dorsal cues, differentially active cues modulate the
specific responses in the mesoderm along the anteroposterior (AP) axis.
Notably Wg, which is expressed in transversely striped domains within the A
compartments of the ectoderm, is required in combination with Dpp for the
specification of the progenitors of cardioblasts, pericardial cells and dorsal
somatic muscles (Baylies et al.,
1995; Wu et al.,
1995
). Conversely, the precursors of the midgut visceral mesoderm
are induced by Dpp but suppressed by Wg
(Frasch, 1995
;
Azpiazu et al., 1996
) (see
http://www.eurekah.com/abstract.php?chapid=2028&bookid=162&catid=20).
Hence, visceral mesoderm precursors arise in domains that are alternating with
those of cardiac and somatic muscle progenitors along the AP axis in the
dorsal mesoderm. Additional cues, which include signals through various
receptor tyrosine kinases (RTKs) and the FGF receptor Heartless, then generate
further subdivisions within the visceral mesoderm as well as diverse
identities among the progenitors of cardiac and somatic muscle tissues
(Carmena et al., 1998
;
Michelson et al., 1998
;
Englund et al., 2003
;
Lee et al., 2003
). Mutual
repression among induced regulatory genes also plays a role
(Han et al., 2002
;
Jagla et al., 2002
). The
combined actions of these regulators results in the spatially restricted
transcriptional activation of target genes, which drive genetic programs
controlling the specification and/or differentiation of individual cells.
Recently, significant progress has been made in resolving the issue of how
combinatorial inputs are integrated at the level of enhancers of target genes
in this system. A relatively simple situation exists for the Dpp-responsive
enhancer of tin, which does not receive any differential inputs along
the AP axis. This enhancer has been shown to contain several copies of binding
sites for Smads, which function as nuclear Dpp signaling effectors, as well as
binding sites for Tin protein. Each of the two types of binding sites are
essential for enhancer activity (Xu et
al., 1998). Thus, it appears that combinatorial binding of
Dpp-activated Smads and mesoderm-intrinsic Tin, together with protein
interactions between Smads and Tin
(Zaffran et al., 2002
),
provides the synergism required for the active state of the enhancer. A more
complex situation, when compared with tin, is found for
even-skipped (eve), a homeobox gene that is induced in
specific segmentally repeated progenitors of pericardial cells and dorsal
somatic muscles within the dorsal mesoderm
(Frasch et al., 1987
;
Su et al., 1999
). This pattern
of eve expression requires not only Dpp but also Wg signals and RTK
signals that are active in smaller areas within the fields where Dpp and Wg
intersect (Frasch, 1995
;
Wu et al., 1995
;
Azpiazu et al., 1996
;
Carmena et al., 1998
). As for
the induction of tin, these external signals require Tin as a
mesoderm-intrinsic activity for the induction of eve
(Azpiazu and Frasch, 1993
;
Bodmer, 1993
). Consistent with
these identified inputs, the corresponding enhancer region of eve has
been found to contain functionally important binding sites for Tin, Smads, the
Wg effector dTCF/Lef-1 and Ets-domain protein-binding sites that are presumed
targets of RTK signals (Halfon et al.,
2000
; Knirr and Frasch,
2001
; Han et al.,
2002
). A comparison between the situation in the eve
versus the tin enhancer raises the question: why are the Smad and Tin
sites in the tin enhancer sufficient for its induction when the
eve enhancer requires additional inputs from Wg via the dTCF/Lef-1
sites? Additional functional studies have provided answers to this question. A
model was proposed, in which bound dTCF/Lef-1 acts as a repressor that
abrogates the activity of the bound Tin and Smad proteins in the absence of Wg
signals, whereas in the presence of Wg signals the repressive activity of
dTCF/Lef-1 is abolished (Knirr and Frasch,
2001
). Consequently, Wg signals allow Tin/Smads (together with RTK
signal effectors) to induce eve in segmentally repeated clusters of
cells within the dorsal mesoderm.
Herein, we define the distinct molecular inputs into a third enhancer,
namely that of the NK-homeobox gene bagpipe (bap), which is
induced by Dpp in the early dorsal mesoderm during the same period when
tin and eve are being induced
(Staehling-Hampton et al.,
1994; Frasch,
1995
). bap is a crucial regulator of the development of
the trunk visceral mesoderm and, hence, of midgut muscle development
(Azpiazu and Frasch, 1993
;
Zaffran et al., 2001
) (see
http://www.eurekah.com/abstract.php?chapid=2028&bookid=162&catid=20).
bap is induced in metameric clusters of cells within the dorsal
mesoderm that alternate with those expressing eve and other early
cardiac and dorsal muscle markers along the AP axis (see
Fig. 1). This pattern is
explained by the finding that the activity of Dpp to induce bap is
abrogated by Wg signals (Azpiazu et al.,
1996
), which contrasts with the situation for eve, where
Wg synergizes with Dpp. Recent studies have shown that Wg signals act
indirectly during this process and function by inducing the forkhead domain
genes sloppy paired 1 and sloppy paired 2 (slp1 and
slp2) in striped domains within the mesoderm
(Lee and Frasch, 2000
). Slp
proteins, in turn, act as segmental repressors of bap induction
(Riechmann et al., 1997
;
Lee and Frasch, 2000
) (see
Fig. 1). In common with
eve (and tin), the induction of bap expression by
Dpp signals also require synergism with mesodermal tin
(Azpiazu and Frasch, 1993
) (see
Fig. 1). Our functional
dissection of the corresponding bap enhancer reveals interesting
similarities and differences to the mesodermal tin and eve
enhancers. Specifically, we show that a 267 bp element, bap3.2, which
recapitulates the endogenous bap pattern in the dorsal trunk
mesoderm, includes combinatorial binding sites for Tin and Smad proteins that
are essential for its induction. By contrast, binding sites for Slp are
required for the segmental repression of bap enhancer activity. We
also show that the Slp-binding sites exert an additional, positive function
during bap induction, in part through binding of Biniou (Bin), a
forkhead domain protein that is activated downstream of bap but
provides positive feedback on bap
(Zaffran et al., 2001
).
Finally, we show that this bap enhancer includes elements that
prevent the induction of bap by Dpp in the dorsal ectoderm and,
thereby, contribute to the observed germ layer specific response. Similar
elements were previously found in the Dpp-responsive enhancer of tin
(Xu et al., 1998
), and we
provide data to indicate that the same repressing mechanism prevents induction
of both tin and bap in the dorsal ectoderm. Altogether, the
data presented illustrate how Wg signals can either antagonize or cooperate
with Dpp signals at the molecular level. More generally, they extend our
knowledge of how the molecular integration of combinatorial signals at the
level of target enhancers generates mesoderm-specific outputs with precisely
defined spatial patterns, which prefigure specific tissue primordia.
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Materials and methods |
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For site-directed mutagenesis and deletion within the bap3.2.1 DNA fragment, PCR reactions were performed with bap3.2.1 as a template and with different primer sets, which were designed to introduce new restriction sites to mutate or delete specific sites. Detailed information on the sequences of primers used for PCR can be obtained upon request. All constructs were sequenced to confirm that only the intended mutations were introduced, and were then cloned into the transformation vector. The mutated DNA sequences of each construct are shown in Figs 5, 7.
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DNase I footprinting assays
Footprinting assays were performed essentially as described by Yin et al.
(Yin et al., 1997) with
single-end-labeled bap3.2.1 probes. Different amounts of GST-Tin, GST-Mad,
GST-Medea (Xu et al., 1998
),
GST-Bin (Zaffran et al.,
2001
), GST-Bap and GST-Slp were added to the reaction. For
producing GST-Bap, a SspI/EcoRI DNA fragment from the
bap cDNA (filled in by Klenow reaction), containing the full coding
sequences, was cloned into the SmaI site of pGEX3X. For GST-Slp, a
DNA fragment (EcoRV/NotI) from a slp1 cDNA (a gift
from L. Pick) containing the full protein-coding sequence was cloned into the
SmaI site of pGEX3X.
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Results |
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Repressing sequences prevent ectopic induction of bap enhancer in the dorsal ectoderm
A shorter regulatory DNA fragment, bap3.2.1, was derived from the bap3.2
regulatory element by removing the first 57 bp from the 5'-end and the
last 30 bp from the 3'-end of bap3.2 (see
Fig. 3A, Fig. 5). bap3.2.1 drives
expression in the same segmented pattern as bap3.2; interestingly, however,
this expression occurs not only in the dorsal mesoderm but also in the dorsal
ectoderm (Fig. 2K, compare with
2I). The lacZ
expression patterns of bap3.2.1-lacZ in dorsal ectoderm and mesoderm
can largely be superimposed onto one another. The only major difference
between the two germ layers is observed in parasegments 13 and 14, where
bap3.2.1-lacZ produces two additional expression clusters in the
ectoderm that is neither seen with any of the reporter constructs nor with
endogenous bap (Fig.
2K, see also 2A).
Likewise, a 165 bp genomic DNA fragment, bapV2 from D. virilis, which
corresponds to the bap3.2.1 element of D. melanogaster (see
Fig. 3B and
Fig. 5), also displays enhancer
activity in both dorsal mesoderm and dorsal ectoderm with this pattern
(Fig. 2L). The presence of
ectopic lacZ expression in the ectoderm with bap3.2 derivatives was
further confirmed in embryo cross-sections. Whereas bap3-lacZ embryos
show no detectable lacZ expression in the ectoderm
(Fig. 2M), there are traces of
ectodermal lacZ expression in bap3.2-lacZ embryos
(Fig. 2N) and strong dorsal
ectodermal lacZ expression in bap3.2.1-lacZ embryos
(Fig. 2O).
Altogether, these observations imply that the first 57 bp and the last 30
bp of the bap3.2 regulatory element have a key role in the repression of
bap enhancer induction in the dorsal ectoderm and show that the
mechanism of repression of ectodermal bap induction is evolutionarily
conserved. The similar patterns of enhancer activity in both germ layers upon
deletion of these repressor sequences support the notion, based on our genetic
data, that the major spatial inputs regulating bap expression in the
mesoderm are also active in the ectoderm
(Azpiazu et al., 1996;
Lee and Frasch, 2000
). Indeed,
one of these candidate inputs from the ectoderm, namely Dpp, leads to the
activation of Mad in a dorsal domain in the mesoderm (and ectoderm) the
ventral border of which coincides with the ventral borders of bap
induction (Fig. 2P).
Early TVM enhancer of bap contains combinatorial binding sites for key signaling effectors and mesodermal regulators
To investigate whether the bap regulators identified genetically,
including tin, dpp, slp (downstream of wg) and bin,
can act directly on the early TVM regulatory element of bap, DNaseI
protection experiments with recombinant Tin, Bap, Smad (Mad and Medea), Slp
and Bin proteins was performed on the 180 bp bap3.2.1 DNA sequence from D.
melanogaster. The DNA footprinting results demonstrate that both Tin and
Bap proteins can bind to the predicted Tin-binding site, which includes a
perfect match to the canonical Tin-binding motif TCAAGTG
(Fig. 4;
Fig. 5)
(Chen and Schwartz, 1995;
Gajewski et al., 1997
). In
addition to the Tin-binding site, a site with a TAAG core motif can strongly
bind Bap but not Tin (CTTA in opposite strand;
Fig. 4 and
Fig. 5; note that the same core
motif is found in binding sites of a Bap ortholog, Nkx3.2)
(Kim et al., 2003
). With
regard to Dpp signaling mediators, there are five Mad-protected regions, three
of which are also protected by recombinant Medea (Mad/Medea-1 to -3;
Fig. 4 and
Fig. 5). Site 1 includes an
AGAC motif that was initially identified as a Smad binding motif in
vertebrates (Zawel et al.,
1998
; Shi et al.,
1998
) whereas sites 3-5 contain GC-rich sequences with CGGC motifs
that were first shown to bind Smad proteins in Drosophila
(Kim et al., 1997
;
Shi, 2001
). Site 2 may be a
combination of the two types (TGAC motif and CG-rich sequences). We do not
observe a clear correlation of either type of site with the binding of Mad
versus Medea. Finally, recombinant Slp proteins protect a wide stretch that
includes an inverted repeat of core binding motifs for forkhead transcription
factors (TAAACA) (Pierrou et al.,
1994
; Kaufmann et al.,
1995
), but extends further downstream
(Fig. 5 and
Fig. 4). During the course of
our work, it was reported that Slp can bind to tandem repeats of CAAA
sequences, which are present in three copies in the 3' region of the
protected region (Andrioli et al.,
2002
). Gel mobility shift and competition assays with Slp using
wild-type oligonucleotides and a version in which the TAAACA motifs were
mutated indicated that Slp can bind to both the TAAACA and the CAAA motifs
with roughly equal affinity (data not shown). In addition, the FoxF family
protein Bin binds to the TAAACA inverted repeat region, but less well to the
CAAA repeat region when compared with Slp
(Fig. 4). Taken altogether,
these binding data are consistent with the hypothesis that the known
mesodermal regulators of bap, namely Tin and Bin (and possibly
autoregulatory Bap), as well as the signaling inputs from Dpp and Wg (through
Smads and Slp, respectively) are integrated via direct binding to the early
TVM enhancer of bap.
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The construct bap3.2.1-tin-m with a mutated Tin/Bap-binding site completely fails to activate lacZ expression in the dorsal mesoderm (Fig. 6A,D). By contrast, and as predicted because there is no Tin, the ectodermal lacZ expression remains unaffected (Fig. 6D; the ectodermal expression is also unperturbed in bap3.2.1-bap-m, Fig. 6C). twist-driven ectopic expression of tin in the whole mesoderm does not cause any ectopic expression of bap or bap3.2.1-lacZ ventrally (data not shown), suggesting that Tin binding to this bap enhancer element is essential, but also not sufficient to activate bap in the mesoderm. Most likely, Tin needs to cooperate with other localized activators that bind to the same regulatory element to activate bap expression in the mesoderm. However, bap3.2.1 enhancer activity in the dorsal ectoderm, which lacks Tin, does not require an intact Tin-binding site. These results point to an intricate molecular mechanism that makes bap3.2.1 enhancer activity differentially sensitive to regulators in the mesoderm versus ectoderm.
To study the in vivo function of Smad-binding sites in the early bap regulatory element, a series of mutation or deletion constructs were generated. A reporter with bap3.2.1-Smad1-m, with mutations in the AGAC core sequence of the 5' most Mad/Medea-binding site (Mad/Medea-1), does not display any lacZ expression in either mesoderm or ectoderm (Fig. 6A,E). Hence, this Smad-binding site (Mad/Medea-1) is essential for bap3.2.1 enhancer activity in both mesoderm and ectoderm. In addition, mutations in the second Mad/Medea-binding site (Mad/Medea-2; derivative bap3.2.1-Smad2-m) result in a loss of lacZ expression in the ectoderm and a near-loss of expression in the mesoderm (Fig. 6A,F). The above results and the highly conserved sequences of Mad/Medea-1 and -2 among different Drosophila species (Fig. 5) suggest that both Smad-binding sites have essential and non-redundant functions in regulating bap expression during embryogenesis. Deletion of both the third and fourth Smad-binding sites (Mad/Medea-3 and Mad-4; derivative bap3.2.1-Smad3,4-d) causes weak lacZ expression in both mesoderm and ectoderm (Fig. 6A,G). Thus, these two Smad-binding sites are not quite as crucial as sites 1 and 2, but have additive or synergistic effects in inducing high levels of enhancer activity. Consistent with this notion, these two Smad-binding sites are absent from the homologous bap regulatory element of D. virilis (Fig. 5). The most 3' Smad-binding site (Mad-5) does not closely match the GC-rich Mad/Medea-binding motif and is not well conserved among the five Drosophila species (Fig. 5). Deletion of this Smad-binding site (derivative bap3.2.1-Smad5-d) does not cause any change of lacZ expression in either mesoderm or ectoderm (data not shown), implying that it does not have an essential function in vivo even though Mad can bind to it in vitro.
Mutations in the C1 region from D. melanogaster (bap3.2.1-C1-m) and, likewise, within the bapV2 element from D. virilis were also examined for their effects in embryos. In both cases, lacZ expression is absent in both mesoderm and ectoderm (Fig. 6H,I), suggesting that the highly conserved C1 sequence plays an essential role in mediating the function of bap activators that function in conjunction with Dpp in both germ layers. By contrast, mutation of the C2 region does not affect enhancer activity (data not shown).
From the above data we conclude that the activation of bap in the mesoderm normally requires combinatorial binding of mesodermal Tin and Dpp-activated Smad proteins. Binding of Bap (and Bin, see below) increases enhancer activity via a feedback regulatory loop. In addition, yet unidentified activating binding factors are required, potentially as general DNA-binding Smad co-activators.
The Slp-binding site mediates segmental repression of bap enhancer induction and overlaps with an essential mesodermal activation site
Based on the observation that Slp represses bap expression within
the slp-expressing domains of the mesoderm
(Riechmann et al., 1997;
Lee and Frasch, 2000
), we
predicted that mutations made at the Slp-binding site would result in uniform
lacZ expression along the AP axis, similar to the endogenous
bap expression in slp mutant embryos. However, several
different mutations, particularly within the forkhead domain consensus sites,
caused a complete loss (bap3.2.1-slp-m1,
Fig. 7A-C) or severe reduction
(bap 3.2.1-slp-m2, Fig. 7A,D;
bap 3.2.1-slp-m3, Fig. 7A,E) of
lacZ expression in the mesoderm. The levels of ectodermal enhancer
activity are not affected by these mutations. These observations show that
there are one or several activators that require this site and whose function
is mesoderm specific. These activators are likely to include Bin, which is
needed for prolonged expression of bap at stage 11 and binds to this
site (Fig. 4)
(Zaffran et al., 2001
).
However, there must be at least one additional, yet unidentified, binding
factor that is required for initiation of enhancer activity through this
site.
In spite of this complication, the observed ectopic ectodermal expression
of these enhancer derivatives and the known presence of Slp in the same
pattern in both mesoderm and ectoderm enabled us to study the potential
repressive activity of the Slp-binding sequences further. With
bap3.2.1-slp-m1, in which both of the canonical forkhead domain-binding sites
are mutated, reporter gene expression in the ectoderm is expanded only
slightly along the AP axis compared with the strictly segmented
bap3.2.1-lacZ expression (Fig.
7B,C). As Slp is able to bind CAAA sequence repeats
(Andrioli et al., 2002), which
are present in three copies within the 3' half of the protected sequence
stretch, it was possible that Slp can still bind to bap3.2.1-slp-m1 and
repress lacZ expression in the ectoderm. Indeed, electrophoresis
mobility shift assays (EMSA) with recombinant Slp proteins and bap3.2.1-slp-m1
DNA oligo probes showed that Slp was still able to bind, presumably through
the CAAA sequence motifs (data not shown). No segmental de-repression is
observed when only one of the two canonical forkhead domain sites is mutated
(bap3.2.1-slp-m2, Fig. 7D;
bap3.2.1-slp-m3, Fig. 7E),
presumably because of the unaffected binding of Slp to the intact site and/or
the CAAA sequence motifs. Similarly, mutations in the CAAA repeats still allow
segmental repression in both ectoderm and mesoderm
(Fig. 7F and data not shown).
Presumably, Slp is able to repress enhancer activity through binding to the
canonical forkhead domain sites in this situation, which can also bind the
unknown mesodermal activator. By contrast, the introduction of mutations in
both types of Slp-binding sequences (bap3.2.1-slp-m5,
Fig. 7A) or the deletion of the
entire sequence protected by Slp (bap3.2.1-slp-d1,
Fig. 7A), results in almost
complete segmental de-repression of reporter gene expression in the ectoderm
(Fig. 7G-I). The inability of
Slp to repress these enhancer derivatives is further confirmed by the observed
co-expression of enhancer-driven lacZ with Slp in the dorsal ectoderm
(Fig. 7I, compare with the
normal mutually exclusive expression,
7H). Taken together, the above
results suggest that in the normal context, the Slp-protected DNA fragment
mediates segmental repression by Slp proteins in the mesoderm both through the
canonical forkhead domain sites and the Slp-specific CAAA motifs. Conversely,
the activation of the enhancer in the mesoderm requires binding of Bin and a
yet unidentified activator to the canonical forkhead domain sites or sequences
overlapping with them.
Characterization of sequence elements preventing the induction of bap and tin in the dorsal ectoderm
Information from several different enhancer derivatives has shown that the
germ layer-specific induction of bap, as well as of tin,
relies in part on repressive sequences that prevent ectopic induction of both
genes in the dorsal ectoderm. For example, the bap enhancer
derivative bap3.2.1, which differs from bap3.2 by the absence of 57 bp from
the 5'-end and 30 bp from the 3'-end, is induced ectopically in
the dorsal ectoderm with the same pattern as its normal expression in the
dorsal mesoderm (Fig. 2K,
Fig. 6B,
Fig. 8D). Similarly, a
shortened version of the bap enhancer from D. virilis,
bapV2, drives segmental lacZ expression ectopically in the dorsal
ectoderm, in contrast to a longer version, bapV1, which is largely mesoderm
specific (Fig. 2J,L). An
analogous situation was previously described for tin. In this case,
it was observed that the deletion of two identical sequence motifs, tinD1a and
tinD1b, within the Dpp-responsive enhancer of tin causes ectopic
enhancer induction by Dpp in the dorsal ectoderm
(Xu et al., 1998). Together,
these observations suggest that the mechanisms for the repression of ectopic
induction of mesodermal genes by Dpp in the ectoderm have been conserved in
different Drosophila species, and that different Dpp targets in the
mesoderm use closely related mechanisms to ensure their germ layer-specific
induction.
DNA sequence comparison among the 5'-57 bp and the 3'-30 bp
sequences of bap3.2, as well as tinD1a and tinD1b from the tin
enhancer, identified three regions, termed R1, R2 and R3, in bap3.2 that
showed sequence similarities with one another and with tinD1a and tinD1b
(Fig. 8B). Four to five
additional copies of this type of motifs are found at conserved positions
within 200 bp of sequences upstream of bap3.2 in different
Drosophila species (data not shown). These observations raise the
possibility that these motifs might represent binding sites for a yet unknown
repressor preventing mesodermal gene induction in the ectoderm. To further
characterize the DNA elements mediating the ectodermal repression, various
derivatives of bap3.2 were generated that were either truncated or contained
mutated or swapped sequences in the identified repressing regions
(Fig. 8A; Materials and
methods). All three putative binding sites for the ectodermal repressor were
found to contribute to the repression, albeit with slightly different degrees
of inhibitory activities. Comparisons of the ectodermal enhancer activities of
bap3.2
R3, bap3.2
R1-2 and bap3.2
R1 suggest that the three
sites have partially redundant activities, with R1 having the strongest effect
in ectodermal repression (Fig.
8E,F; data not shown). When mutations are introduced all three
sites, R1, R2 and R3, within bap3.2 (bap3.2R1-3mut), the resulting
de-repression in the ectoderm is almost as complete as with deletions of these
sequences (Fig. 8G, compare
with 8D). Hence, the identified
sequence motifs appear to be largely responsible for the repression of
induction in the ectoderm. The sequence similarities of tinD1a and tinD1b, and
their analogous biological activities within the tin enhancer would
suggest that these sequences are able to replace the R1, R2 and R3 sequences
functionally within the bap enhancer. To test this possibility, both
tinD1a and tinD1b were added to either the 5'- or the 3'-end of
bap3.2.1 (bap3.2.1-tinD1.5' and bap3.2.1-tinD1.3', respectively).
As predicted, tinD1a and tinD1b strongly repress bap3.2.1 enhancer activity in
the ectoderm, without affecting it in the mesoderm
(Fig. 8H and data not shown).
These results suggest that similar mechanisms, probably via binding of
identical repressor factor(s), are involved in preventing the ectopic
induction of bap and tin in the ectoderm. The ectopic
ectodermal expression of the enhancers of bap and tin is
directly controlled by Dpp signals, as shown with mutations of Smad-binding
sites, which prevent the induction in the dorsal ectoderm of the enhancers
that lack the repressing sequences (Fig.
6E-G) (Xu et al.,
1998
). Consequently, the putative repressors must normally
interfere with Dpp signaling outputs at the level of the target enhancers.
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Discussion |
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The induction of mesodermal tissues in Drosophila is a process during which Wg signals can modulate the responses to Dpp signals by either synergizing with them or antagonizing them. Whereas previous studies have described the functional architecture of mesodermal enhancers that are targeted either by Dpp alone or by synergistic Dpp and Wg signals, our present study describes an example of an enhancer whose response to Dpp is suppressed by Wg signals. A comparison of the functional organization of these enhancers provides new insight into molecular strategies of nuclear signal integration to produce differential developmental responses.
Nuclear Dpp signaling outputs and their suppression by Wg-induced Slp
Our data show that bap is a direct target of Dpp signals. Thus, we
can rule out an indirect pathway of bap being activated solely by
tin, whose mRNA expression is known to depend on Dpp inputs during
the time of bap activation
(Azpiazu and Frasch, 1993).
Rather, tin acts simultaneously and synergistically with Dpp. In
fact, recent data with tin alleles lacking the Dpp-responsive
enhancer show that bap can be induced in the absence of Dpp-induced
tin products, as long as the twist-activated tin
products are present (S. Zaffran and M.F., unpublished). We show that the
molecular basis for this observed synergism of tin and dpp
relies on the combinatorial binding of Tin and Dpp-activated Smad proteins to
the bap enhancer. Several possible molecular mechanisms could
underlie the strict requirement for combinatorial binding of Tin and Smads.
For example, the relatively low binding affinity and specificity of Smads
might be enhanced by bound Tin, which can engage in protein interactions with
Mad and Medea (Zaffran et al.,
2002
). The combined presence of Tin and Smads in close vicinity or
in complexes may also be a prerequisite for the assembly of higher order
complexes with transcriptional co-activators such as CBP/p300
(Liu et al., 1997
;
Feng et al., 1998
;
Janknecht et al., 1998
;
Pouponnot et al., 1998
;
Waltzer and Bienz, 1999
). In
addition, Tin may counteract the function of yet unknown repressors of nuclear
Dpp signaling activity so that they can only repress in the ectoderm.
Unlike Dpp, Wg signals act indirectly upon the early bap enhancer.
Previous genetic and molecular data showed that Wg induces the expression of
the forkhead domain-encoding gene slp via crucial dTCF/Lef-1 binding
sites in both mesoderm and ectoderm (Lee
and Frasch, 2000). slp, in turn, functions as a repressor
of bap (Riechmann et al.,
1997
; Lee and Frasch,
2000
). Our present data show that slp products exert this
function by direct binding to the Dpp-responsive bap enhancer, which
obviously results in a suppression of the synergistic activity of bound Tin
and Smad complexes. Slp proteins contain eh1 motifs that can potentially bind
the Groucho co-repressor and Slp has known repressor activities in other
contexts (Lee and Frasch,
2000
; Andrioli et al.,
2002
; Gebelein et al.,
2004
). In addition, the vertebrate counterpart of Slp, FoxG
(BF-1), is known to interact with Groucho and histone deacetylases
(Yao et al., 2001
). Thus, we
propose that Slp overrides nuclear Dpp signaling activities by dominantly
establishing an inactive state of the chromatin at the bap locus.
It is likely that additional components are involved in the antagonistic
interaction of Slp with Tin/Smad complexes. As we have shown, the Slp-binding
site includes sequences that are also required positively for the mesodermal
response to Dpp, although not for ectopic responses in the ectoderm. In a
genome-wide expression analysis, we did not find any forkhead domain genes
other than bin that are mesoderm specific
(Lee and Frasch, 2004).
However, the function of an essential co-activator in the mesoderm interacting
with this site could be fulfilled by a ubiquitously expressed forkhead domain
protein, and in part by Bin, which is required for the prolongation of the Dpp
response (Zaffran et al.,
2001
). In the yeast one-hybrid screens with this site that yielded
Slp clones we also isolated a clone of fd68A, a uniformly expressed
ortholog of vertebrate FoxK1 (Myocyte Nuclear Factor), but
genetic confirmation of its involvement in bap induction is currently
lacking (Lee and Frasch,
2004
). Regardless of the identity of this factor, Slp could either
compete with this protein and with Bin for DNA binding, or it could disrupt
their productive functional interactions with the Tin/Smad complexes.
Interestingly, the latter type of mechanism has been proposed to operate
during the interference of the slp ortholog BF-1 with
TGFß signaling in the vertebrate cerebral cortex
(Dou et al., 2000
).
Nuclear mechanisms guaranteeing germ layer-specific signaling outputs
Inductive responses that are germ layer- or cell type-specific and exclude
the signal-producing cells are a recurring theme in developmental systems.
Although this type of target specificity can involve different levels of the
signaling cascade, including the tissue-specific expression of receptors or
signaling effectors, we have shown that germ layer-specific induction of
bap is controlled by nuclear events. This is crucial because
activated Smads are present in dorsal nuclei of both germ layers
(Knirr and Frasch, 2001)
(Fig. 2P). We have identified
two mechanisms, which are probably functionally intertwined, that ensure
mesoderm-specificity of the response to Dpp. The first is the requirement for
Tin to synergize with activated Smads, as discussed above. Tin is present
exclusively in the mesoderm and is therefore not available to fulfill such a
function in the ectoderm. Hence, in developmental terms, Tin provides the
mesoderm with the unique competence to respond to Dpp and induce bap.
Perhaps surprisingly then, there is an additional component involved, which
actively prevents induction of the bap enhancer by Dpp in the
ectoderm. As we have shown, the Dpp-responsive core enhancer of bap
is flanked by sequences that appear to function as binding sites for yet
unidentified repressor(s), which keep the enhancer silent in the ectoderm. A
very similar situation was previously described for the Dpp-responsive
enhancer of tin (Xu et al.,
1998
) and, as shown herein by sequence comparisons as well as
functional swapping of the putative ectodermal repressing sequences from the
tin and bap enhancers, they appear to bind the same
repressor(s). Brinker, a known nuclear repressor of Dpp signaling, can be
excluded as a candidate because of its different sequence preference and
absent expression in the dorsal ectoderm
(Jazwinska et al., 1999
;
Sivasankaran et al., 2000
;
Kirkpatrick et al., 2001
;
Rushlow et al., 2001
;
Saller and Bienz, 2001
;
Zhang et al., 2001
). The
situation is reminiscent of an endodermal labial enhancer, in which a
homeotic response element and a repressor element interact to control the
spatially restricted activity of a minimal Dpp response element
(Marty et al., 2001
).
Why would induction of tin and bap in the mesoderm
require Tin as a co-factor of Smads, whereas in the ectoderm, which lacks Tin,
the induction of tin and bap needs to be actively repressed?
In the case of the tin enhancer, the ectodermal repressor elements
are overlapping with the Tin-binding sites. Based upon this situation, we
proposed a model in which the repressor would be present in both germ layers,
but in cells of the mesoderm it is competed away from binding to the enhancer
by Tin (Xu et al., 1998). This
model is compatible with data showing that ectopic expression of Tin in the
ectoderm is able to activate the Dpp-responsive enhancer of tin, even
in the presence of the putative repressor binding elements. However unlike
full-length Tin, an N-terminally truncated version with an intact homeodomain
is not able to allow induction of the tin enhancer in the ectoderm
(Zaffran et al., 2002
).
Furthermore, the putative repressor binding sites in the bap enhancer
are separate from the Tin site. Hence, Tin does not compete for binding but
may rather block or override the repressor factor(s) functionally. Thus, the
positive activity of Tin would dominate over the negative action of this
repressor in the mesoderm. By contrast, the repressing activity of Slp
dominates over the positive action of Tin. Through this intricate balance of
positive and negative switches, Tin could ensure that bap is induced
by Dpp only in the mesoderm, while bound Slp prevents Tin from promoting Dpp
inputs towards bap in striped domains within this germ layer.
However, we can still not fully explain why the absence of both the functional
Tin and ectodermal repressor sites allows enhancer induction in the ectoderm,
while preventing it in the mesoderm. The additional positive and negative
binding factors involved will need to be identified to gain a full
understanding of the germ layer-specific induction of these Dpp-responsive
enhancers.
Mesodermal enhancers of tin, eve and bap - variations on a theme
The bap enhancer described herein represents the third example of
well-characterized Dpp-responsive enhancers from mesodermal control genes. The
other two are from tin, which is induced in the entire dorsal
mesoderm, and eve, which is active in a small number of somatic
muscle founder cells and pericardial progenitors in the dorsal mesoderm. The
activities of the bap and eve enhancers along the
anteroposterior axis are reciprocal, which is due to the fact that the
eve enhancer requires inputs from Wg, whereas bap enhancer
activity is suppressed by Wg. A comparison of the molecular architecture of
these three enhancers reveals that they all share a number of important
features (Fig. 9) (Xu et al., 1998;
Halfon et al., 2000
;
Knirr and Frasch, 2001
;
Han et al., 2002
). Most
notably, all three enhancers feature several Tin- and Smad-binding sites in
close vicinity that are essential for the activation of the enhancer in the
mesoderm. Each enhancer includes both types of known Smad-binding motifs,
which have `AGAC' and `CG'-rich cores, respectively. Hence, the basic
activation mechanisms of each of the three enhancers downstream of Dpp are
likely to be closely related. As discussed above, in the enhancers of both
tin and bap, binding sites for a nuclear repressor of Dpp
signals are key for the germ layer specificity of the inductive response.
Although we do not know whether the same repressive mechanism operates at the
eve enhancer, we note that motifs related to the presumed repressor
binding motifs are present and their function can now be tested in vivo (M.F.,
unpublished). As in the case of bap, the tin enhancer
includes also additional sites that are required for Dpp-inducible enhancer
activity, which may bind essential Smad co-factors. However, based upon the
divergent sequences of these sites (C1 site in the bap and `CAATGT'
motifs in the tin enhancer) (Xu
et al., 1998
), they appear to bind different types of factors in
each case.
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ACKNOWLEDGMENTS |
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Footnotes |
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