Department of Biology, New York University, 100 Washington Square East, New York, NY 10003, USA
* Author for correspondence (e-mail: chris.rushlow{at}nyu.edu)
Accepted 31 January 2005
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SUMMARY |
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Key words: BMP gradient, Zen, Race, Feed-forward
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Introduction |
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In Drosophila, patterning of the embryonic dorsoventral axis
depends on the combined action of two morphogens: Dorsal (Dl) and
Decapentaplegic (Dpp). Dl is a maternally loaded transcription factor that is
responsible for setting up the overall dorsoventral axis of the embryo
(reviewed by Stathopoulos and Levine,
2002). A gradient of Dl protein is formed during early
embryogenesis with peak levels in the ventral nuclei. The gradient acts to
subdivide the axis into three main regions ventral (mesoderm), lateral
(neuroectoderm) and dorsal ectoderm by eliciting several threshold
responses from batteries of zygotic patterning genes. For example,
transcriptional activation of twist (twi) and snail
(sna) require high levels of Dl, while short gastrulation
(sog), brinker (brk) and rhomboid
(rho) can be activated by lower levels of Dl. Genes such as
zerknüllt (zen), decapentaplegic
(dpp) and tolloid (tld) are repressed by Dl and
thus come to be expressed only in the dorsal region. Differential target gene
responses are mediated largely by the affinity of Dl-binding sites in the
target enhancers (Jiang and Levine,
1993
).
Dpp acts to further subdivide the dorsal domain into amnioserosa (the
dorsal most region) and dorsal ectoderm, while also inhibiting neuroectoderm
formation (Ferguson and Anderson,
1992a; Wharton et. al.,
1993
; Biehs et al.,
1996
). Dpp is a member of the TGFß superfamily of ligands,
which are most closely related to the BMPs (bone morphogenetic proteins), and
signals through a pathway comprising the type I and type II serine-threonine
kinase transmembrane receptors and the intracellular Smad proteins, Mother
against Dpp (Mad) and Medea (Med) (reviewed by
Raftery and Sutherland, 2003
).
Upon ligand binding and receptor activation, Mad is phosphorylated (PMad),
thereby allowing translocation into the nucleus along with the co-Smad Medea.
In the nucleus, Smads function as DNA-binding transcription factors (reviewed
by Shi and Massagué,
2003
).
Although dpp RNAs are evenly distributed across the dorsal region
of the precellular embryo, the Dpp activity gradient takes shape during stage
5, as the embryo is undergoing cellularization (reviewed by
Raftery and Sutherland, 2003).
The gradient, which also includes a second BMP ligand Screw (Scw)
(Arora et al., 1994
), is formed
through a dynamic process involving the secreted protein Sog, which emanates
from the adjacent ventral region
(Srinivasan et al., 2002
). As
Sog diffuses dorsally, it sequesters BMPs with the help of another secreted
protein Twisted Gastrulation (Tsg) (Mason
et al., 1994
; Ross et al.,
2001
). This tripartite complex is thought to serve two purposes
that have opposite effects on BMP signaling. First, it antagonizes BMP
signaling by preventing BMP ligands from interacting with their receptors; and
second, it promotes signaling by allowing the redistribution of BMPs to more
dorsal regions (Holley et al.,
1995
; Shimmi and O'Connor,
2003
). Tolloid (Tld), a metalloprotease localized in the dorsal
region (Shimell et al., 1991
),
cleaves Sog, thereby releasing BMPs as active ligands. The net effect is a BMP
gradient that can be visualized by detecting the output of the BMP pathway,
the nuclear Smad proteins (reviewed by
Raftery and Sutherland, 2003
).
Initially PMad is in a broad gradient containing relatively low levels of
protein. This develops into a steep step-wise gradient with increasingly high
levels in a five- to six-cell-wide stripe along the dorsal midline, the
presumptive amnioserosa, and lower levels in the three or four cells adjacent
to either side of the stripe. In more lateral regions of the dorsal ectoderm,
PMad protein is not detectable by antibody staining.
There are several candidate BMP target genes whose expression domains
correlate with the stepwise PMad gradient. For example, hindsight
(hnt; peb FlyBase)
(Yip et al., 1997) and
Race (related to angiotensin-converting enzyme; Ance
FlyBase) (Tatei et al., 1995
)
are expressed specifically in the peak level PMad domain. u-shaped
(ush) (Cubadda et al.,
1997
; Jazwinska et al.,
1999b
), tail-up (tup)
(Thor and Thomas, 1997
;
Ashe et al., 2000
) and
rhomboid (rho) (Bier et
al., 1990
; Ross et al.,
2001
) are expressed more broadly in 12-14 cells encompassing the
adjacent lower level PMad domain. pannier (pnr)
(Ramain et al., 1993
;
Winick et al., 1993
) is
expressed in a broad domain covering about 36 cells (or 25% of the
dorsal-ventral circumference), the border of which does not correlate with a
clear domain of PMad activity. The mechanism underlying threshold responses to
the BMP/Smad gradient is not fully understood.
A possible mechanism emerged from studies on Brk, a transcriptional
repressor (Campbell and Tomlinson,
1999; Jazwinska et al.,
1999a
; Jazwinska et al.,
1999b
; Minami et al.,
1999
). As Dpp signaling represses brk expression,
brk domains are largely complementary to dpp domains, which
allows target genes to be transcribed in the dpp domains. In areas
where dpp and brk overlap slightly, Smads and Brk may
compete for DNA binding on target enhancers as Brk sites often overlap Smad
sites (Kirkpatrick et al.,
2001
; Rushlow et al.,
2001
; Saller and Bienz,
2001
). It has therefore been suggested that a Brk gradient,
inverse to the Smad gradient, acts to spatially restrict target gene
activation and consequently sets borders of expression
(Jazwinska et al., 1999a
;
Ashe et al., 2000
;
Müller et al., 2003
).
However, this mechanism does not explain the threshold responses of those
genes that are not Brk targets, such as Race. Alternatively, a
mechanism involving differential binding site affinity for Smads may play a
role in establishing their borders of expression. Indeed Wharton et al.
(Wharton et al., 2004
) used
modified Race enhancers to demonstrate that it is possible to broaden
expression domains by increasing the affinity of Smad-binding sites.
We demonstrate that Race activation requires the combined input of
Smads and Zen. Zen-binding sites lie adjacent to Smad sites in the
Race enhancer, and we show that Smads facilitate the DNA binding of
Zen and that this requires protein interaction between Smads and Zen. As
zen is regulated by peak levels of the BMP gradient and thus becomes
expressed only in the dorsalmost region
(Rushlow et al., 2001), the
regulation of Race resembles a feed-forward loop whereby one
regulator, BMP/Smad, controls a second regulator, Zen, and then both bind and
activate a common target gene, Race. In addition, we tested the
respective roles of Zen and Smads in setting the expression borders of
Race. When zen was expressed ectopically, Race
expression broadened to encompass both the high and low level regions of the
BMP gradient. Thus, as long as Zen is present, low levels of BMP activity are
sufficient to activate Race, indicating that the purpose of the peak
of the BMP gradient is to set the Zen domain.
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Materials and methods |
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Ectopic expression of zen and zen-Del
The zen cDNA (+10 to +1234 from the transcription starting site)
(Rushlow et al., 1987a) was
cloned into pUAST (Brand and Perrimon,
1993
) via EcoRI and XbaI sites on the 5'
and 3' ends, respectively. The zen-Del cDNA was made by PCR
mutagenesis (Expand High Fidelity PCR system, Roche Applied Science) using
oligos spanning the deletion region (amino acids 152-198) and cloned into
pUAST via the EcoRI and XbaI sites. Transgenic flies were
generated by the standard transformation protocol
(Spradling and Rubin, 1982
).
Flies carrying UAS-zen and UAS-zen-Del were crossed
to stripe-2 eve-Gal4 drivers (gift from S. Small) and the expression
of ectopic zen or zen-Del proteins was confirmed by staining
with anti-Zen antibodies. Guinea pig or rabbit anti-Zen antibodies were
generated (Covance) as described by Rushlow et al.
(Rushlow et al., 1987b
). To
obtain uniform early embryonic expression of the UAS-zen and
UAS-zen-Del transgenes, a maternal Gal4 driver was used in
which the GAL4-VP16 fusion protein is expressed maternally under the
control of the
tubulin 67C promoter. These were further
crossed into a zenw36/TM3, hb-lacZ background for
the zen mutant rescue experiments.
In vitro mutagenesis and transgenic analysis
The Race 533 bp enhancer DNA was kindly provided by M. Levine
(Rusch and Levine, 1997).
Deletions and point mutations were created by PCR mutagenesis. An internal
deletion of 66 bp (nucleotides 432-497 of the Race enhancer) deletes
most of the Mad-binding region. The two proximal Zen-binding sites were
mutated as follows: ATATTAAT was changed to ATCTAGAT, and ATTAAAAATAAATAAT was
changed to TAGAAAAATAACTGCA. Race-lacZ constructs were prepared by
subcloning the wild-type and mutated versions of the Race enhancer
into a modified Casper transformation vector that contains the minimal
promoter sequence from the even-skipped gene fused to the
lacZ reporter gene (eve-lacZ Casper)
(Small et al., 1992
). At least
three transformant lines for each construct were tested.
In situ hybridization and antibody staining
Wild-type, mutant and transgenic embryos were fixed, hybridized with
zen, Race, hnt or lacZ antisense RNA probes, and stained
(Roche Molecular Biochemicals), dehydrated and mounted in araldite
(Polysciences) as described by Rushlow et al.
(Rushlow et al., 2001).
Anti-PMad polyclonal antibodies were kindly provided by P. ten Dijke
(Persson et. al., 1998
) and
used at a final dilution of 1:1000 in PBS. Secondary anti-rabbit antibody
staining was performed using the Vectastain ABC kit (Vector Labs). Embryo
preparations were photographed using DIC optics on a Nikon FX-A
microscope.
Bacterial expression of Zen, Zen-Del, Mad and Medea
The GST-Zen and GST-Zen-Del fusion constructs were cloned by introduction
of EcoRI sites at the initial ATG and at the 15th nucleotide
downstream of the stop TAA codon in the zen cDNA by PCR mutagenesis,
followed by excision of the EcoRI fragment from the PCR product and
ligation into the EcoRI site of the pGEX-4T-2 vector (Pharmacia).
Expression plasmids encoding GST-MadN and GST-Medea fusion protein
containing the N-terminal MH1 domains were obtained from A. Laughon
(Kim et al., 1997) and M.
Frasch (Xu et al., 1998
),
respectively. The expression and the purification of the recombinant proteins
were carried out as described before
(Kirov et al., 1993
). The
concentration of the isolated proteins was determined by SDS-PAGE after
staining with Coomassie R-250, together with defined amounts of bovine serum
albumin.
In vitro DNA binding assays
DNAse I footprint analyses were carried out as previously described
(Kirov et al., 1993). The 533
bp Race enhancer was originally cloned into the pBluescript KS+
vector (Stratagene) NotI site
(Rusch and Levine, 1997
).
Three fragments were generated for footprint analysis using the BssHI
and XhoI sites on each side of the vector polylinker:
BssHI-ApoI (50 to 163),
HinfII-TthIII (107 to 349) and TthIII-XhoI
(349-603). The electrophoretic mobility shift assays were performed as
described before (Kirov et al.,
1993
) except that the electrophoresis was run at room temperature.
The sequence of the 42 bp oligonucleotide (Race sequences 486-527)
spanning the wild-type Smad-binding sites and the proximal Zen-binding site is
(consensus core sites are underlined):
5'-TCAGACGCGACTAAGCCGATCTCGCATTAAAAATAAATAATG-3'.
The Smad-binding site mutations are (mutated core sites are underlined):
5'-TTAGATGCGAGTAAGATGATCTCGCATTAAAAATAAATAATG-3'.
The Zen-binding site mutations are (mutated core sites are underlined):
5'-TCAGACGCGACTAAGCCGATCTCTCTAGAAAAATAACTCGAG-3'.
The sequence of the 128 bp DNA fragment is (consensus core sites are
underlined; vector sequences are in lower case):
5'gaattcgcccttAACGTCGGCTTATCTTCGCGCCTACCTGGCCGAGAACCCCAGACGGATTGGAAACATCAGACGCGACTAAGCCGATCTCGCATTAAAAATAAATAATGCTCGAGaagggcgaattc-3'.
The end-labeled oligonucleotides or fragment, which was isolated from a
subclone of the Race enhancer by EcoRI digestion, were added
to the reactions containing Zen, Mad, Medea or combinations of different
proteins and incubated on ice for 30 minutes before loading on the gel.
Protein interaction assays
GST pull-down assays were carried out as previously described
(Kirov et al., 1996).
Wild-type (and mutant forms) of zen were cloned into the pAR vector
(NdeI and EcoRI sites)
(Rosenberg et al., 1987
) by
introduction of an NdeI site at the initial (or internal sites to
make truncated proteins) and an EcoRI site four nucleotides
downstream of the stop codon (or at internal sites to make truncated
proteins). These constructs were expressed in vitro with the TNT Coupled
Reticulocyte lysate system (Promega) using the T7 promoter in the pAR vector.
Expression of these proteins was confirmed by electrophoresis on a 12% SDS
polyacrylamide gel.
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Results |
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dpphr4/+ heterozygous embryos have normal Race expression (Fig. 1D). Similarly, zenw36/+ heterozygotes also have normal Race expression (data not shown). However, in double heterozygous dpphr4/+; zenw36/+ embryos, Race expression is absent in the middle body region (Fig. 1E). zen expression appears normal in these embryos (Fig. 1F) as does PMad staining (data not shown). Thus, although the level of Dpp activity in these embryos is sufficient to drive refined zen expression, it is not able to compensate for the reduction in zen dose in order to properly activate Race. Likewise, there is not enough Zen to compensate for the reduction in Dpp activity. Again, we conclude that Race responds to high levels of combined Zen and PMad activities.
Racecan be activated by low levels of PMad if Zen is present
To further investigate the combinatorial requirement for dpp and
zen, we performed additional dosage studies. We first examined
Race in embryos with four copies of dpp (4X dpp).
The expression domains of peak-level PMad, zen, and Race are
broader in these embryos covering about 12-14 cells
(Fig. 2D-F). This demonstrates
a clear correlation between peak levels of PMad/Zen and high-level Dpp target
gene expression. Next, we examined Race expression in embryos with 4X
dpp that lack zen activity. Race transcripts are
present, but only in the dorsalmost five or six cells
(Fig. 2I), comparable with that
in 2X dpp embryos (Fig.
2C), even though peak levels of PMad and refined zen
cover 12-14 cells in these embryos (Fig.
2G,H). Therefore, Dpp can activate Race in a
zen-independent manner but only if expressed above wild-type levels,
as was also concluded by Rusch and Levine
(Rusch and Levine, 1997).
However, Race is not activated across the entire domain of PMad, but
only in the dorsal most region, suggesting that Dpp signaling is higher along
the dorsal midline in a 4X dpp embryo than in a wild type 2X
dpp embryo, though not obvious in our antibody staining
experiment.
|
These results reveal a dual role for Dpp in Race activation. Peak levels of BMP/Smads define the domain of zen in the dorsalmost cells. Next, BMP/Smads, together with Zen, activate downstream targets such as Race; however, this function does not require peak-level Smads as Race was activated in regions of low-level Smads when Zen was ectopically expressed (Fig. 2L). Thus, we propose that the role of the peak of the BMP gradient is to set the zen domain. Hence, Zen defines the expression borders of the downstream high level targets (see Discussion). We sought to investigate the molecular mechanism underlying the combinatorial requirement of Zen and Smads for the activation of Race transcription.
Mad facilitates the binding of Zen to the Race enhancer
We assayed for Mad and Medea binding to the Race enhancer, a 533
bp DNA fragment that lies 1.5 kb upstream of the Race transcription
start site (Rusch and Levine,
1997). When fused with a lacZ reporter gene, this DNA
fragment drives a similar expression pattern to that of wild-type
Race mRNA (Fig. 3B)
(Rusch and Levine, 1997
). We
performed DNA-binding assays with recombinant GST fused Mad protein and GST
fused Medea protein, both of which contain the DNA-binding MH1 domain and the
linker region (Kim et al.,
1997
; Xu et al.,
1998
).
|
A 66 bp deletion of this region (nucleotides 432-497) abolishes the in
vitro footprint binding of Mad (data not shown), and also greatly reduces in
vivo expression of the Race-enhancer reporter gene,
Mdel-lacZ (Fig. 3C),
suggesting that Mad can directly activate Race via binding to this
region. The observed residual activity of this reporter might be due to the
remaining Smad-binding site (box g). We also detected four Zen-binding sites
in the Race enhancer, three of which are shown in the footprint in
Fig. 3A (lanes 6-9; nucleotides
in red). Point mutations in all four of the ATTA core sites abolished
lacZ reporter expression (Rusch
and Levine, 1997), as did mutations in the three most proximal
sites (Fig. 3D;
ZBS-lacZ).
The above data favor the existence of a relatively short regulatory module
containing a cluster of Smad-binding sites bordered by Zen-binding sites that
regulates the essential aspects of early Race expression. Similarly,
short sequences containing multiple Smad sites and other transcription factor
binding sites have been found to regulate the Dpp targets tin
(Xu et al., 1998) and
Ubx (Saller and Bienz,
2001
) in Drosophila.
The closely apposed Smad- and Zen-binding sites in the Race regulatory module hinted at the possibility that their combined activity, which is essential for Race activation, might partially depend on their direct interaction. The most immediate result from such an interaction could be to facilitate the binding of one or the other protein to DNA. To test this, we tried to detect cooperative binding of Mad and Zen proteins to DNA. In preliminary experiments, we found the range of concentrations of Zen and Smad proteins that produce complexes with a 42 bp oligonucleotide spanning a cluster of three Smad-binding sites and the two most proximal Zen sites. Then by incubating one component with suboptimal amounts of the other, which by itself is not sufficient to produce complexes, we expected that the DNA binding of the protein of suboptimal concentration would increase if there is cooperative DNA binding, and/or possibly form supershift complexes containing both proteins.
Mad and Medea form similar complexes with the 42 bp oligonucleotide
(Fig. 4B, lanes 2-3), and do
not bind oligonucleotides in which the three Smad sites (CAGAC, GACT, GCCG)
were mutated (TAGAT, GAGT, GATG respectively; lanes 6-7). In experiments with
the larger 122 bp fragment that contains seven Smad sites
(Fig. 4A), mutation of the two
CAGAC sites (boxes d and e in Fig.
3A and Fig. 4A)
prevented Medea binding (data not shown), indicating that Medea binds the
CAGAC site, as was recently shown by Pyrowolakis et al.
(Pyrowolakis et al., 2004).
Zen forms two complexes with the 42 bp oligonucleotide that have different
mobilities from the Smad complexes (Fig.
4B, lane 4), and does not bind to oligonucleotides in which the
core sites (ATTA, TAAT) were mutated (TAGA, TCGA, respectively;
Fig. 4B, lane 12). When a
concentration of Mad that does not produce detectable Mad complexes was
incubated with varying Zen protein concentrations and the122 bp DNA fragment,
the intensity of the Zen complexes increased compared with when Mad was not
added to the reactions (Fig.
4C, lanes 10-12 compared to lanes 6-8). Complexes that could be
interpreted as Mad/Zen supershifts were not clearly visible as distinct
complexes. Similar experiments using the DNA fragments with mutated Smad sites
showed little if any enhancement of Zen binding by small amounts of Mad (data
not shown), indicating that the observed cooperative binding depends on Mad
interaction with DNA.
|
Mad-Zen protein interaction is necessary for Race activation
To test for direct physical protein-protein interactions between Zen and
Mad proteins, we performed GST pull-down assays with GST-MadN and
in vitro translated full-length and truncated Zen proteins,
(Fig. 5A,B). Full-length Zen
clearly interacts with Mad (Fig.
5C, lanes 1-2). Our results confirm the recent report of the
Mad-Zen interaction found in a genome-wide yeast two-hybrid screen
(Giot et al., 2003). By
testing a series of truncated Zen proteins
(Fig. 5C, lanes 3-14), we
mapped the domain of the Zen protein involved in the interaction with Mad to
be within the 48 amino acids C-terminal to the homeodomain (amino acids
152-199). Removal of this region by an internal deletion
(Fig. 5A, Zen-Del) also
abolishes the ability of Zen to interact with GST-MadN
(Fig. 5C, lanes 15-16). Though
the recombinant Zen protein with this deletion, Zen-Del, binds DNA similarly
to full-length Zen (Fig. 4D, lanes 6-9), the cooperative binding to the wild-type DNA fragment containing
Mad- and Zen-binding sites is not observed
(Fig. 4D, lanes 10-13).
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Discussion |
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Raceis activated by a combinatorial mechanism
Specific activation or repression of transcription by a combination of
transcription factors is a common theme in the regulation of developmentally
important genes (Howard and Davidson,
2004). The results from our genetic analysis and the molecular
dissection of the Race enhancer clearly show that Race is
activated by the combined action of Smads and Zen. Although Smads can single
handedly activate Race when overexpressed
(Fig. 2I), under normal
circumstances concurrent Zen activity is required. Why are both Smads and Zen
necessary?
Zen may act to restrict target gene expression specifically to the
presumptive amnioserosa. As the Dpp pathway is used repeatedly during
development, other factors must function in combination with Dpp to ensure
tissue specificity (see Affolter and Mann,
2001; Reim et al.,
2003
). The ectopic expression studies support this idea. In normal
embryos, Race is activated only in regions where there are peak
levels of PMad and Zen. In embryos where Zen is ubiquitously expressed,
Race can now be activated in regions where there are lower levels of
PMad (Fig. 2L), indicating that
high level PMad is not the determining factor for amnioserosa tissue
specificity. Rather PMad allows expression, and Zen determines the border of
expression. We interpret the overexpression studies where Dpp can activate
Race alone (Fig. 2I)
(Rusch and Levine, 1997
) to be
situations where there are such high levels of Smads that Race and
hnt become activated promiscuously, and hence differential regulation
is lost. In normal embryos, the combination of Smads and Zen ensures that the
high level target genes are activated only in the presumptive amnioserosa.
On the other hand, why the need for Smads? One role for Smads is suggested
from the observation that Smads facilitate the binding of Zen to the
Race enhancer (Fig.
4). It is well established that Hox proteins often require
co-factors for DNA binding to target enhancers (reviewed by
Mann and Affolter, 1998). For
example, composite sites that also bind the co-factor Extradenticle (Exd)
ensure a greater selectivity for binding over the higher frequency Hox core
site such as TAAT. In other examples, binding sites for signaling pathway
effectors lie close to Hox/Selector-binding sites (see
Guss et al., 2001
;
Affolter and Mann, 2001
). The
closely apposed Zen- and Smad-binding sites in the Race enhancer is
one such scenario, as Zen can be thought of as a Selector gene
(Rushlow and Levine, 1990
).
Our studies add to this idea of Smads and Selector cooperativity by
demonstrating enhanced binding of Zen in the presence of Smads. Though the
enhancement we observe in our in vitro assays is not dramatic, it is possible
that in the embryo a moderate enhancement is functionally significant as is
the twofold doubling of the dpp dose.
Another potential role of Smads was suggested from previous overexpression
studies (Rusch and Levine,
1997). Zen was only able to activate Race in the absence
of Dpp if fused to a strong activation domain derived from VP16. This suggests
that Smads provide a transactivation function different from that of Zen. The
Smad MH2 domain has been shown to interact with the transcriptional
co-activators CBP and p300 (Waltzer and
Bienz, 1999
; Shi and
Massagué, 2003
). Zen has not yet been analyzed for
interaction with transcriptional co-activators, however, the activation domain
of Zen lies within the C-terminal 119 amino acids
(Han et al., 1989
), and does
not overlap with the homeodomain or the Mad interaction domain
(Fig. 5). Mechanistically, the
difference in the activation potential between Zen and Smads could be due to
their ability to recruit different co-activators to the transcriptional
machinery.
Mechanisms of threshold responses to the BMP morphogen gradient
Gradients of morphogens provide positional information to the cells by
activating different genes at different threshold concentrations. In early
Drosophila embryos, the transcriptional threshold responses to the
Bicoid (Driever et al., 1989)
and Dl (reviewed by Stathopoulos and
Levine, 2002
) morphogens have been extensively studied. The major
mechanisms by which thresholds are established exploits the DNA-binding
affinities of Bcd and Dl to their operator sites, as well as synergistic
interactions with other transcription factors bound to the
cis-regulatory sequences.
The BMP morphogen gradient also elicits different threshold responses from
its targets, and, as discussed above, a combinatorial mechanism is used to
activate Race, a high level Dpp target. Our genetic results indicate
that Race (Fig. 1),
and also another high level target hnt (M.X., unpublished), are
activated only when a specific threshold of Zen and Smad activities are
reached. In sog mutant embryos, Zen and Smad concentrations are
relatively high, though below peak levels
(Rushlow et al., 2001), and
there is just enough of their combined activity to weakly activate
Race (Fig. 1C) and
hnt (data not shown). By contrast, in the double heterozygous embryos
dpphr4/+; zenw36/+,
Race is not activated (Fig.
1E) because Zen and Smad concentrations are below the threshold
levels required for activation.
A simple way to explain these results is if the Race enhancer has
low affinity to Zen and Smad proteins in vivo. To transcribe Race
effectively would then require relatively high concentrations of the proteins,
which are indeed reached in the dorsalmost cells. It has been known for some
time that the enhancers of the high level Dl targets contain binding sites
with lower affinity for Dl compared with genes responding to lower levels of
Dl (reviewed by Stathopoulos and Levine,
2002). Recently, it has been shown that increasing the affinities
of Smad-binding sites in the Race enhancer broadens the Race
expression domain, which argues that the affinities of the Smad-binding sites
in this high level Dpp target gene enhancer are low
(Wharton et al., 2004
). Our
results suggest that cooperative binding between Smads and Zen, which is
dependent on their physical interaction, should increase their binding to the
Race enhancer (Figs 4,
5,
6). It is possible that
interacting with Smads at the protein level either increases the binding
affinity of Zen or effectively increases the local concentration of Zen when
Smads bind the adjacent sites. This in turn leads to a robust transcriptional
response of Race. The overexpression results are consistent with such
a model. Ectopic Zen can only activate Race if some detectable level
of PMad is present (Fig. 2L),
and in addition Zen must contain the Smad interaction domain
(Fig. 6B).
How are the lower level target genes activated? ush and
rho are expressed in a broader domain, the border coinciding exactly
with that of low level PMad staining. The Zen domains, however, do not;
refined zen is not broad enough, while early Zen is too broad
encompassing the entire dorsal domain, though Zen could possibly be graded in
this region (Rushlow et al.,
1987b). Thus, it is possible that this class of target genes
relies on a mechanism that uses numerous high-affinity Smad sites, and/or
synergistic action of Smads with other co-factor(s) besides Zen. Such a
mechanism resembles the activation of target genes in the neurogenic ectoderm
of the embryo by Dl (reviewed by
Stathopoulos and Levine,
2002
). It has been shown that the threshold responses from these
genes depend on high-affinity Dl-binding sites, as well as synergistic
interactions of Dl with bHLH transcription factors
(Ip et al., 1992a
;
Jiang and Levine, 1993
).
The pnr expression domain, which is about three times broader than
ush, may represent a third threshold of Dpp activity. However,
pnr is a different type of target gene compared with the prior
classes in that it is repressed by Brk, which is present in a reverse gradient
to Dpp (Jazwinska et al.,
1999a; Ashe et al.,
2000
; Müller et al.,
2003
). In brk mutants, pnr expands into the
ventral region, while Race and ush, for example, are
unchanged. We expect that the pnr gene enhancer contains Brk-binding
sites, whereas we observed in this study that the Race enhancer does
not (M.X., unpublished). Brk binding sites often overlap with GNCN sites
(Sivasankaran et al., 2000
;
Rushlow et al., 2001
;
Zhang et al., 2001
), and it is
possible that in the embryo, as in the wing disc, a concentration-dependent
competition between Smads and Brk establishes the expression domains of the
target genes regulated by both inputs
(Rushlow et al., 2001
).
However, whether direct competition for binding can generate threshold
responses remains to be seen. In summary, it appears that different classes of
Dpp target genes are regulated by different combinations of transcription
factors.
Raceactivation by Dpp and Zen resembles a feed-forward loop
One of the simple regulatory motifs used in transcriptional networks is the
feed-forward (Lee et al.,
2002) or self-enabling (Kang
et al., 2003
) mechanism, whereby one regulator controls a second
regulator and then both bind a common target gene. It has been shown both in
prokaryotes (Shen-Orr et al.,
2002
) and yeasts (Lee et al.,
2002
) that this mode of regulation appears relatively frequently
and is favored over others, e.g. autoregulation motifs, single input motifs in
which one regulator controls several genes, or regulator chain motifs whereby
one gene regulates a second which regulates a third, and so on. Such an
over-representation of the feed-forward motif is probably due to its potential
to provide enhanced sensitivity and temporal control to the transcriptional
response. The feed-forward loop is especially suitable for eliciting precise
threshold responses of morphogen targets as it allows a strong response of the
target gene to small changes in the activity of the regulator that initiates
the loop (Dpp), because of the combined action with the second regulator
(Zen). In fact Bcd and Dl use mechanisms that are reminiscent of the
feed-forward loop to activate their high level targets. Bcd regulates zygotic
hunchback (hb) and together Bcd and Hb activate the
downstream target even-skipped (eve) stripe 2
(Small et al., 1992
), and Dl
activates sna with the help of Twi
(Ip et al., 1992b
). It is
striking that the three morphogen gradients involved in specifying the
Drosophila embryonic axes use the feed-forward strategy to regulate
downstream target genes.
The primary role of the BMP gradient peak is to set up Zen
An unexpected implication from our results concerns the role of the high
end of BMP morphogen gradient. In Drosophila embryos, the refined
zen domain depends on peak levels of BMP activity, and we have shown
that Zen can activate high level targets as long as there is some level of
PMad present to facilitate DNA binding. It can be then concluded that, for the
high level targets, the role of Dpp is twofold: to set the domain of
zen, which we can refer to as a primary target gene; and then to act
in combination with Zen to activate the other, secondary, target genes such as
Race and hnt. In addition, with respect to the BMP gradient
in the Drosophila embryo, we further propose that the sole purpose of
the peak of the gradient is to set up the zen domain.
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ACKNOWLEDGMENTS |
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