Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093-0349, USA
Author for correspondence (e-mail:
jposakony{at}ucsd.edu)
Accepted 25 May 2005
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SUMMARY |
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Key words: Cell-cell signaling, Default repression, Co-repressors, Neural precursor specification, Cell fate, Cis-regulatory logic, Drosophila
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Introduction |
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The capacity of N signaling to specify diverse cell fates in diverse
developmental contexts depends on the ability of the pathway to activate in
each setting the appropriate subset of its target genes (reviewed by
Barolo and Posakony, 2002).
This specificity is founded in turn on the cis-regulatory apparatus of the
targets and on the use of regionally expressed `local activators', which
function cooperatively with Su(H) to effect target gene activation. A cell
that responds to the N-mediated signal and expresses one or more local
activators will activate only those target genes that include binding sites
for both the local activator(s) and Su(H).
Lateral inhibition is the developmental setting classically associated with
N pathway function (Lehmann et al.,
1983; Poulson,
1967
). Although it is also used in the Drosophila
mesoderm for specification of muscle progenitor cells
(Carmena et al., 1995
;
Corbin et al., 1991
), lateral
inhibition has been most comprehensively studied in the context of
neurogenesis in the ectoderm. Here, local expression of proneural genes, which
encode basic helix-loop-helix (bHLH) transcriptional activators, confers on
small groups of cells called proneural clusters (PNCs) the potential to adopt
a neural precursor cell fate (Cubas et
al., 1991
; Skeath and Carroll,
1991
). A single cell in the PNC, which displays the highest level
of proneural protein accumulation, stably adopts the neural fate and laterally
inhibits the remaining cells via N-mediated signaling, remanding them to an
epidermal fate (Cabrera, 1990
;
Doe and Goodman, 1985
;
Hartenstein and Posakony,
1990
; Simpson,
1990
).
Lateral inhibitory signaling in Drosophila PNCs directly elicits
the expression of multiple genes located in the Enhancer of split
Complex [E(spl)-C] (Bailey and Posakony,
1995; Lecourtois and
Schweisguth, 1995
), which are collectively required for inhibition
of the neural precursor cell fate
(Delidakis et al., 1991
;
Nagel et al., 2000
;
Schrons et al., 1992
). These
genes encode proteins belonging to one of two families: bHLH transcriptional
repressors and Bearded (Brd) family proteins
(Lai et al., 2000b
). Each
E(spl)-C gene is associated with a discrete enhancer module that includes
high-affinity binding sites for the proneural proteins Achaete (Ac) and Scute
(Sc), and for Su(H) (Nellesen et al.,
1999
). These sites constitute a cis-regulatory `code' for
expression specifically in PNCs; the proneural proteins serve as the local
activators that cooperate with N-activated Su(H) to trigger robust
transcription of E(spl)-C genes.
Consistent with their function in antagonizing the neural precursor cell
fate, expression of both bHLH repressor and Brd family genes of the E(spl)-C
is generally excluded from the committed precursor
(Jennings et al., 1994;
Jennings et al., 1995
;
Kramatschek and Campos-Ortega,
1994
; Lai et al.,
2000b
; Nolo et al.,
2000
; Zaffran and Frasch,
2000
). Although logical, this asymmetry is also somewhat
paradoxical, given the elevated levels of the proneural proteins in the neural
precursor cell. Earlier work with reporter genes revealed that the exclusion
is transcriptionally based (Kramatschek
and Campos-Ortega, 1994
; Lai
et al., 2000b
; Nolo et al.,
2000
), but the underlying mechanism was not established. On the
basis of genetic experiments, Koelzer and Klein
(Koelzer and Klein, 2003
)
proposed that a repressive activity of Su(H) might be responsible. We
demonstrate explicitly that direct transcriptional repression by Su(H) lies at
the heart of the exclusion phenomenon.
The setting for these studies is the PNCs of the wing imaginal disc, from which sensory organ precursor (SOP) cells of the adult peripheral nervous system (PNS) arise. Using enhancer-reporter transgenes that are active specifically in the inhibited (non-SOP) cells of PNCs, we have found by mutational analysis that Su(H) binding sites are crucial not only for direct activation of E(spl)-C genes in non-SOP cells but also for their direct repression in SOPs. Loss of this repression results in ectopic expression of the reporter genes in SOPs, which is dependent on the integrity of proneural protein binding sites. The developmental importance of direct Su(H)-mediated repression of E(spl)-C genes in the SOP is demonstrated by our observation that de-repression of a single bHLH repressor gene [owing to mutations in its Su(H) binding sites] can result in loss of the SOP cell fate. Both loss- and gain-of-function experiments establish essential roles for the adaptor protein Hairless (H) and the transcriptional co-repressor proteins Groucho (Gro) and C-terminal binding protein (CtBP) in the repression activity of Su(H) in the SOP. Our results constitute the first evidence for direct transcriptional repression by Su(H) in the most classic setting for N signaling, and reveal how this repression serves to protect the fate of neural precursor cells during lateral inhibition.
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Materials and methods |
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Gene diagrams
Gene diagrams in Figs 1,
2,
3 were created using the
GenePalette software tool (Rebeiz and
Posakony, 2004)
(www.genepalette.org).
Transgene construction
E(spl)m-RFP and
E(spl)m
-GFP reporter constructs were prepared by
cloning a 1.0 kb BamHI-XhoI genomic DNA fragment
(1083 to 71) from the E(spl)m
gene into the
multiple cloning site (MCS) of the pRed H-Stinger and pGreen H-Stinger
insulated P element transformation vectors
(Barolo et al., 2000a
;
Barolo et al., 2004
),
respectively. An orthologous fragment from D. virilis E(spl)m
was cloned into pGreen H-Stinger as a BamHI-XhoI restriction
fragment, to make Dv E(spl)m
-GFP. E(spl)m8-GFP was
constructed by cloning a 1.1 kb genomic EcoRI-XhoI DNA
fragment (1174 to 72) into the MCS of pGreen H-Stinger.
An E(spl)m8 transgene was constructed by replacing the
lacZ reporter gene in the insulated pPelican vector
(Barolo et al., 2000a) with a
2.4 kb genomic SpeI-KpnI DNA fragment (1174 to
+1243).
An E(spl)m-Gal4 driver was constructed by cloning
the 1.0 kb genomic DNA sequence described above into the MCS of the H-GAL4
vector as an EcoRI-BamHI fragment. H-GAL4 is based on the
CaSpeR P-element transformation vector
(Pirrotta, 1988
) and consists
of the GAL4-coding sequence downstream of the Hsp70 minimal
(43) promoter and upstream of a fragment containing the Hsp70
polyadenylation signal and site; a region of the Pelican/Stinger MCS
(Barolo et al., 2000a
) is
immediately upstream of the promoter. Proper expression of
E(spl)m
-Gal4 in non-SOPs was verified by crossing to
flies carrying UAS-Stinger (Barolo
et al., 2000a
).
Wild-type and mutant UAS-H misexpression constructs were cloned
into the pUAST vector (Brand and Perrimon,
1993). UAS-H and UAS-H
C were
described previously; a stop codon placed after codon 1070 in
UAS-H
C eliminates the seven C-terminal amino acids
(PLNLSKH), which constitute the CtBP-binding motif
(Barolo et al., 2002
).
UAS-H[Gm] encodes a H protein in which the Gro-binding motif YSIHSLLG
is changed to AAAHSAAG; this abolishes the in vitro
interaction between the two proteins (see
Barolo et al., 2002
).
UAS-H[Gm]
C expresses a H protein that lacks both
motifs.
Transcription factor binding site mutations were made using the Transformer
(Clontech) or Chameleon (Stratagene) mutagenesis kits. The E-box proneural
protein binding site GCAGGTG was changed to
GAAGCTT
(Van Doren et al., 1992);
Su(H) sites of the form YGTGRGAA were changed to YGTGRCAA
(Bailey and Posakony, 1995
);
these mutations abolish binding of the respective proteins in vitro.
Cloning and sequencing of E(spl)m orthologs
A 40-mer primer containing D. melanogaster sequence that included
both the S2 Su(H) site and the E box of E(spl)m was
used to recover orthologous upstream sequence from libraries of D.
virilis or D. hydei genomic restriction fragments that had been
ligated to pBlueScript IIKS (Stratagene) using the RAGE (Rapid Amplification
of Genomic DNA Ends) method (Mizobuchi and
Frohman, 1993
). Additional rounds of RAGE were used to obtain
complete E(spl)m
PNC cis-regulatory modules from D.
hydei and D. virilis. D. pseudoobscura sequence was obtained
from the Human Genome Sequencing Center at Baylor College of Medicine
(www.hgsc.bcm.tmc.edu/projects/drosophila/).
Bristle scoring
To measure the phenotypic effect of relieving E(spl)m8 from direct
repression by Su(H), 50 flies (25 females and 25 males) of the following
genotypes were scored at early adulthood for missing macrochaete bristles at
20 positions each: w1118; w1118
homozygous for the wild-type E(spl)m8 transgene (six lines); or
w1118 homozygous for the mutant E(spl)m8 Sm
transgene (10 lines).
To measure the SOP fate-promoting activity of wild-type and mutant forms of
H, both left and right orbital regions of 25 adult females of each genotype
were scored for supernumerary bristles. All genotypes carried one copy of
E(spl)m-Gal4, driving expression of one copy of
UAS-H, UAS-H
C, UAS-H[Gm] or
UAS-H[Gm]
C. Two different insertions were tested for
each UAS construct.
Antibody staining
Wing imaginal discs were dissected from late third-instar larvae, fixed in
4% paraformaldehyde in PBS for 30 minutes, washed, and incubated with anti-Hnt
(monoclonal, Developmental Studies Hybridoma Bank), anti-Sens (polyclonal;
gift of H. Bellen), or anti-ß-galactosidase (monoclonal, Roche; gift of
W. McGinnis) primary antibody followed by Cy3 (Jackson Labs; for
ß-galactosidase) or Alexa 647 (Molecular Probes; for Hnt and Sens)
secondary antibody. Pupal notum in Fig.
1 was dissected and stained at 14 hours APF.
Confocal microscopy
A Leica TCS SP2 microscope (equipped with Leica Confocal Software v2.5;
Leica Microsystems) was used for confocal imaging. Images in Figs
1,
2,
3,
4,
5 are average projections of
stacks taken along the apicobasal axis at 1 µm increments. Z-axis
range was delimited to collect the full signal from all fluorophores.
Fluorophores were excited separately at 488 nm (GFP), 543 nm (RFP, Cy3) or 633
nm (Alexa 647); emissions were collected at 490-530 nm (GFP), 630-710 nm (RFP,
Cy3) or 640-740 nm (Alexa 647). All GFP signals were collected at the same
gain.
De-repression of E(spl)m-GFP expression in SOPs
Wing discs were dissected from late third-instar larvae and stained with
anti-ß-galactosidase antibody to detect A101-positive SOPs.
Using constant excitation and collection parameters, expression of one copy of
E(spl)m-GFP was scored by confocal microscopy in 16
discs at each of seven SOP positions: ANWP, ANP, PNP, APA, PSA, PDC and PSC
(see figure legends for abbreviations). Four genotypes were analyzed: (1)
w1118; E(spl)m
-GFP/+; A101/+ +; (2)
w1118; E(spl)m
-GFP/+; A101/HE31
+; (3) w1118; E(spl)m
-GFP/+; A101/+
groE48; and (4) w1118;
E(spl)m
-GFP/+; A101/HE31 groE48.
Only six out of 16 discs from larvae of the last genotype included PSA SOPs
that were present to score.
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Results |
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Activation of N-regulated genes of the E(spl)-C in imaginal disc PNCs makes
use of a combination of Su(H)-binding sites and binding sites for the
proneural proteins Ac and Sc (Bailey and
Posakony, 1995; Nellesen et
al., 1999
; Singson et al.,
1994
). Consistent with this `Su(H) plus proneural' cis-regulatory
code, the E(spl)m
module includes five high-affinity Su(H)
sites and a single high-affinity proneural site
(Fig. 1A, construct 1)
(Lai et al., 2000a
;
Nellesen et al., 1999
). We
examined the effects on reporter gene activity of mutating only the proneural
site (Em, Fig. 1A,
construct 2), only the five Su(H) sites (Sm,
Fig. 1A, construct 3) or all
six sites (EmSm, Fig.
1A, construct 4). We first observed that the integrity of the `E
box' proneural protein binding site is strictly required for detectable
reporter expression in nearly all wing disc PNCs; residual expression is
observed along the entire wing margin and in a very small subset of PNCs
(Fig. 1E). This result
demonstrates that, as for other N pathway target genes, the proneural proteins
make an essential input as direct transcriptional activators of
E(spl)m
in PNCs.
Mutation of the five Su(H)-binding sites in the E(spl)m PNC
module (Sm, Fig. 1A,
construct 3) yields a dramatic alteration in the spatial pattern of its
activity (Fig. 1F-H). Reporter
gene expression in non-SOP cells is drastically reduced or eliminated, and
strong ectopic expression is now observed in SOPs
(Fig. 1F-G). Direct comparison
of the wild-type (RFP) and Sm (GFP) reporter transgenes in the same
disc emphasizes the stark contrast in their specificities
(Fig. 1H). This finding
indicates, first, that Su(H) has an essential role as a direct transcriptional
activator of E(spl)m
in the N-responsive non-SOPs, and,
second, that it acts as a direct transcriptional repressor of the gene in
SOPs.
Finally, we observed that mutation of the proneural protein binding site in addition to the Su(H)-binding sites (EmSm, Fig. 1A, construct 4) abolishes detectable PNC expression of the reporter gene (Fig. 1I). Most importantly, this result shows that both the residual non-SOP and the ectopic SOP expression of the Sm mutant (Fig. 1F) is strictly dependent on direct proneural input. It also indicates that the residual activity of the Em proneural site mutant along the wing margin and in a few PNCs (Fig. 1E) requires direct input from Su(H).
The bHLH repressor gene E(spl)m8 is also subject to direct transcriptional repression by Su(H) in SOPs
The bHLH repressor-encoding genes of the E(spl)-C, exemplified by
E(spl) itself [referred to hereafter as E(spl)m8 to
distinguish it readily from E(spl)m], likewise make use of a
`Su(H) plus proneural' cis-regulatory code for their activation in PNCs during
lateral inhibition (Fig. 2A,
construct 1) (Bailey and Posakony,
1995
; Nellesen et al.,
1999
; Singson et al.,
1994
). We sought to determine whether direct repression by Su(H)
in SOPs applies as well to this class of N pathway target genes. We found that
a 1.1 kb genomic DNA fragment from immediately upstream of E(spl)m8
(Fig. 2A, construct 2) confers
PNC-specific expression on a heterologous promoter-reporter construct in late
third-instar wing discs (Fig.
2B). Expression is also observed along the wing margin (see also
Bailey and Posakony, 1995
). As
with E(spl)m
, double labeling (using anti-Hnt to mark SOPs)
reveals that the PNC activity of this fragment is predominantly in non-SOPs
and excluded from SOPs (Fig.
2B-D). Mutation of the three Su(H) binding sites (Sm,
Fig. 2A, construct 3) abolishes
most non-SOP expression (Fig.
2E) and yields strong ectopic expression in SOPs
(Fig. 2E-G). We conclude that
Su(H) normally acts as a direct repressor of both the Brd family genes and the
bHLH repressor genes of the E(spl)-C (see
Lai et al., 2000b
) in SOPs of
the adult PNS.
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We compared the bristle patterns of w1118 adults
carrying two copies of either a wild-type E(spl)m8 transgene
(Fig. 2A, construct 1) or the
same transgene with its Su(H)-binding sites mutated [E(spl)m8 Sm]. We
consider this a very stringent assay of the requirement for Su(H)-mediated
repression in the SOP for two reasons. First, we are testing the effects of
derepressing a single N pathway target gene, although there are several other
such genes (both bHLH repressor and Brd family) residing in the E(spl)-C
alone. Second, the level of ectopic E(spl)m8 expression generated by
a de-repressed genomic DNA transgene is expected to be much lower than that
achieved by a UAS-E(spl)m8 construct activated by strong GAL4
drivers, as in the prior studies (Nakao
and Campos-Ortega, 1996; Tata
and Hartley, 1995
). We found that whereas flies carrying the
wild-type E(spl)m8 transgene display only very mild bristle loss
(Fig. 4A,E), flies carrying
E(spl)m8 Sm exhibit a significantly more severe bristle-loss
phenotype (P=0.002; Fig.
4B,E). Staining of late third-instar wing discs with
anti-Senseless (Sens) antibody to visualize SOPs showed that this bristle loss
was due to a failure of SOP specification
(Fig. 4D,D'; compare with
Fig. 4C,C'). We conclude
that loss of direct Su(H)-mediated repression of a single N pathway target
gene can be sufficient to extinguish the SOP fate, thus altering the adult
bristle pattern.
|
Earlier work can be interpreted to suggest that a similar protein complex
might mediate repression by Su(H) in the SOP. At several macrochaete and many
microchaete positions on the adult fly, simultaneous reduction of the doses of
H and gro in an otherwise wild-type background leads to
significant bristle loss (A.G. Bang, PhD thesis, University of California San
Diego, 1993) (Barolo et al.,
2002; Price et al.,
1997
); we show now that this is due to a failure of commitment to
the SOP cell fate (Bang et al.,
1991
) (see Fig. S1 in the supplementary material). In light of the
results described here, a plausible interpretation of these findings is that H
and Gro are normally part of a repressive Su(H)-containing complex in the SOP,
and that reduction of their doses sufficiently compromises the repressive
activity as to partially de-repress N pathway target genes like
E(spl)m8, leading to failure of SOP specification. As a test of this
model, we thought it might be possible to detect such de-repression of a
suitable reporter gene; Fig. 5
shows that this expectation is borne out. Late third-instar wing discs from
wild-type larvae (Fig.
5A,C-E,I) or larvae heterozygous for null alleles of either
H or gro (not shown) only rarely exhibit detectable activity
of an E(spl)m
-GFP reporter transgene in SOPs. By
contrast, we found that wing discs from larvae doubly heterozygous for null
alleles of both H and gro (HE31
groE48/+ +) show substantial frequencies of ectopic GFP
expression in SOPs (Fig.
5B,F-I). Moreover, the SOP expression observed in the double
heterozygotes is considerably stronger than that detected rarely in a
wild-type background (Fig. 5I).
These results demonstrate that normal levels of H and gro
activity are required for the Su(H)-dependent repression of N pathway target
genes in SOPs, and are consistent with the participation of a
Su(H)-H-Gro-containing protein complex in this repression
(Barolo et al., 2002
).
|
A key prediction of the model is that the ability of H to bind Gro (via the
motif YSIHSLLG) (Barolo et al.,
2002) and CtBP (via the motif PLNLSKH)
(Barolo et al., 2002
;
Morel et al., 2001
) should be
required for the SOP fate-promoting activity of H. We tested this prediction
by using an E(spl)m
GAL4 driver to express different forms of
H specifically in the non-SOP cells of the PNCs. The orbital region of the
adult fly head is a particularly favorable territory in which to assay the
production of supernumerary bristles by H overexpression
(Fig. 6A,B). Expression of a
wild-type UAS-H transgene results in the appearance of an average of
approximately four ectopic bristles in the orbital region
(Fig. 6B,C). This activity is
significantly impaired by mutating either the Gro recruitment motif
(UAS-H[Gm]) or the CtBP-binding motif
(UAS-H
C) (Fig.
6C), suggesting that both co-repressors make a functional
contribution. Loss of both motifs (UAS-H[Gm]
C)
essentially abolishes the capacity of H to promote ectopic bristle development
in this assay (Fig. 6C). Our
results are strongly consistent with the interpretation that the SOP cell's
requirement for H activity (Bang
et al., 1995
; Bang et al.,
1991
) is based on the recruitment by H of Gro and CtBP to confer
repressive activity on Su(H), thus preventing inappropriate expression of
inhibitory N pathway target genes.
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Discussion |
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|
Consistent with the model proposed here, prior studies have shown that loss
of Su(H) function in imaginal disc tissue has complex effects on gene
expression in PNC cells. Lateral inhibition fails
(Schweisguth, 1995;
Schweisguth and Posakony,
1992
), as Su(H) is not available to transduce the N signal by
activating E(spl)-C genes (Bailey and
Posakony, 1995
; Lecourtois and
Schweisguth, 1995
). Hence, the cells of the mutant PNC exhibit
many of the characteristics of SOPs, such as high levels of Ac and
A2-6 (scabrous) expression
(Schweisguth and Posakony,
1994
), and expression of the early SOP markers A101
(neuralized) and A37 (neuromusculin)
(Schweisguth and Posakony,
1992
). They also display very high levels of Dl
(Schweisguth and Posakony,
1994
). However, Su(H) mutant PNC cells also express
multiple E(spl)-C genes (Bailey and
Posakony, 1995
; Koelzer and
Klein, 2003
; Lai et al.,
2000b
; Nellesen et al.,
1999
), a characteristic of non-SOPs; this is due to activation by
the high proneural levels prevailing in these cells, in the absence of default
repression by Su(H) (Bailey and Posakony,
1995
) (this paper).
|
The above considerations lead us to conclude that Su(H) mutant PNC
cells are an unsatisfactory surrogate for SOPs. In the present study, we have
investigated instead the behavior of authentic SOPs, and have shown that this
fate is indeed at risk under conditions in which direct `default repression'
of E(spl)-C genes by Su(H) is compromised. We find that several conditions
that partially or completely relieve this repression [including the
Sm mutation of an E(spl)m8 transgene (this paper), a H
gro/+ + double-heterozygote background (this paper)
(Barolo et al., 2002) and a
H loss-of-function genotype (Bang
et al., 1995
; Bang et al.,
1991
)] cause, to various extents, the loss of expression of even
the earliest SOP markers and irreversible failure to execute the SOP fate.
Cis-regulatory logic: the `Su(H) plus proneural' code in PNC expression of E(spl)-C genes
In earlier work, we have demonstrated that a `Su(H) plus proneural'
cis-regulatory code directs specific expression of E(spl)-C genes (both bHLH
repressor and Brd family) in imaginal disc PNCs
(Bailey and Posakony, 1995;
Nellesen et al., 1999
;
Singson et al., 1994
). The
results reported here enlarge our understanding of the regulatory logic
embodied in this code. Mutational analysis of the Su(H) and proneural
protein-binding sites in the E(spl)m
PNC module demonstrates
that each activator makes an essential contribution to the activity of the
module in non-SOP cells of the PNC; indeed, we found that neither class of
binding site is sufficient to activate detectable reporter gene expression in
most non-SOPs. This is a classic example of `cooperative activation'
(Barolo and Posakony, 2002
) by
the combination of a signal-regulated factor [Su(H)] and a regionally
expressed `local activator' (Ac/Sc). The `Su(H) plus proneural' cis-regulatory
code is thus very effective in eliciting robust and specific expression of
E(spl)m
in non-SOPs (Fig.
7A).
However, our work reveals that the use of this code puts the SOP at risk of
inappropriately activating N pathway target genes, such as
E(spl)m and E(spl)m8, for a specific reason: the
elevated level of proneural protein accumulation in SOPs
(Cubas et al., 1991
;
Skeath and Carroll, 1991
). We
found that, in the absence of direct repression by Su(H), the proneural
proteins can act through the single E box binding site in the
E(spl)m
PNC module to drive strong ectopic reporter gene
expression in the SOP in contrast to their insufficiency in most
non-SOPs. Thus, even in the absence of any activating contribution from Su(H),
which is required in non-SOPs, E(spl)-C genes would respond to the high
proneural levels in the SOP were it not for direct repression by Su(H)
(Fig. 7B). The elegant logic of
the `Su(H) plus proneural' code insures instead that only non-SOPs activate
effective levels of the genes by which the N pathway inhibits the SOP cell
fate.
Recently, Cave et al. (Cave et al.,
2005) have reported that a specific configuration of Su(H)-binding
sites known as the Suppressor of Hairless Paired Site (SPS)
(Bailey and Posakony, 1995
) is
essential for transcriptional synergy between proneural proteins and Su(H) in
driving specific expression in PNCs. The results reported here on
transcriptional regulation of E(spl)m
contradict this
conclusion. We have clearly demonstrated that the strong expression of
E(spl)m
in the non-SOP cells of the PNC depends crucially on
cooperation between proneural activators and Su(H), yet none of the Su(H)
sites of this gene are in the SPS configuration. Thus, until the mechanistic
basis for proneural/Su(H) synergy is more fully elucidated, we believe that
the term `Su(H) plus proneural' remains the most accurate and most general
description of the PNC cis-regulatory code.
Default repression and N signaling in Drosophila development
Direct repression of E(spl)-C genes in the SOP during lateral inhibition is
a conspicuous example of what we have termed `default repression', a property
of developmental signaling pathways whereby pathway target genes are repressed
by a signal-regulated transcription factor in the absence of signaling
(Barolo and Posakony, 2002). We
proposed that default repression has evolved in order to prevent inappropriate
(signal-independent) activation of pathway target genes in cells that express
local activators but do not respond to the signal. Indeed, as discussed above,
the SOP is in particular need of default repression because it is
characterized (perhaps unusually) by elevated accumulation of the local
activators for the PNC, the proneural proteins. That Su(H) can keep N pathway
target genes off in SOPs even in the face of exceptionally high local
activator levels (Fig. 7A) is
testament to the efficacy of default repression as a regulatory strategy.
It is now clear that default repression by Su(H) is a crucial feature of
the operation of the N pathway in all three of the developmental situations in
which it is known to function (Table
1) (Bray, 1998):
lateral inhibition (this paper), binary cell fate decisions in lineages
(Barolo et al., 2002
;
Barolo et al., 2000b
), and
formation of tissue boundaries (Morel and
Schweisguth, 2000
). This conclusion is based on an analysis, in
all three cases, of the consequences of mutating Su(H)-binding sites in one or
more N pathway-activated genes (Table
1); we emphasize that attribution of a default repression activity
to a signal-regulated transcription factor can be made only after such
cis-regulatory experiments have been performed. It is likely that default
repression by Su(H) is an integral part of N pathway function during
Drosophila development.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
---|
Our sequences of the E(sp1)m PNC enhancers from D.
hydei and D. virilis have been deposited in GenBank; Accession
Numbers DQ076189 and DQ076190, respectively.
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/15/3333/DC1
* Present address: Department of Cell and Developmental Biology, University
of Michigan Medical School, Ann Arbor, MI 48109-0616, USA
Present address: Department of Molecular and Cell Biology, University of
California Berkeley, Berkeley, CA 94720-3200, USA
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