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First published online 14 November 2007
doi: 10.1242/dev.008409
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Centre de Biologie du Développement, UMR 5547 CNRS/UPS, IFR 109, Institut d'Exploration Fonctionnelle des Génomes, 118 route de Narbonne, 31062 Toulouse cedex 9, France.
* Author for correspondence (e-mail: vincent{at}cict.fr)
Accepted 14 September 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cis-regulatory modules, collier (knot), nautilus (MyoD), Drosophila, Muscle identity
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Liberg et al., 2002;
Pang et al., 2004).
col was initially characterised for its expression and function in a
specific region of the embryonic head that corresponds to both a mitotic
domain (MD2) and a gnathal parasegment (PS0)
(Crozatier et al., 1996).
col is also expressed in, and required for the formation of, a single
somatic muscle, the embryonic Dorsal/Acute 3 (DA3) muscle
(Crozatier and Vincent, 1999),
thereby providing a unique entry site for studying the transcriptional control
of muscle identity.
The embryonic musculature of Drosophila melanogaster is highly
stereotyped, with a standard arrangement of around 30 somatic muscles in each
trunk hemisegment. Each muscle fibre is an individual syncitium that can be
distinguished by its position, shape, epidermal attachment sites and
innervation (Bate, 1993;
Baylies et al., 1998). Muscle
fibres are seeded by founder cells (FCs), which are themselves generated from
progenitor cells singled out from promuscular clusters by Notch-mediated
lateral inhibition (Carmena et al.,
1995; Ruiz Gomez and Bate,
1997). FCs undergo multiple rounds of fusion with fusion competent
myoblasts (FCMs) to form a myofibre. The current view is that `muscle
identity' transcription factors (TFs) endow FCs with the capacity to execute
the fusion and differentiation programme specific to each muscle fibre
(Baylies and Michelson, 2001;
Frasch and Leptin, 2000). The
`identity TF code', at least in part, reflects the initial position of the
promuscular cluster and derived progenitor cell. Pioneering work on the
control of expression of the homeodomain transcription factor Even-Skipped
(Eve) in dorsal muscle progenitors showed that it involved the combinatorial
activity of TFs functioning downstream of Wingless (Wg), Decapentaplegic (Dpp)
and receptor tyrosine kinase (RTK) signalling, [dTCF (Pan - FlyBase), Mothers
against Dpp (Mad) and Pointed (Pnt), respectively]. Integration of this
positional information and tissue-specific (mesodermal) information at the
level of the eve promoter was responsible for activating
Eve-expression in promuscular clusters (equivalence groups) from which Eve
progenitors were selected by Notch (N) signalling
(Carmena et al., 2002;
Carmena et al., 1998;
Halfon et al., 2000;
Halfon et al., 2002).
Large-scale analyses of gene expression in conditions of perturbation of
components of Eve regulation suggested that related transcriptional codes
could be responsible for different patterns of progenitor gene expression
(Estrada et al., 2006;
Philippakis et al., 2006;
Sandmann et al., 2006). The
eve enhancer reproducing Eve expression in muscle progenitors was not
active, however, in recruited FCM nuclei
(Halfon et al., 2000),
indicating that different cis-regulatory elements (and TFs) could be required
for specifying promuscular clusters and maintaining a TF identity code.
Here we used Col expression as both a determinant and read-out of DA3
muscle identity to ask how positional information that defines promuscular
clusters is relayed into the FC identity and extended to fused FCM nuclei. We
first identified the cis-regulatory regions controlling col
transcription at several steps during formation of the DA3 muscle and defined
a DA3-muscle-specific cis-regulatory module (CRM). Detailed analysis of this
CRM revealed the existence of three separate steps: Col activation in
promuscular clusters, upregulation in the selected progenitor and DA3 FC and
activation in the nuclei of FCM incorporated in the growing DA3 myofibre
during the muscle fusion process. Comparison of the DA3 muscle CRM between
several Drosophila species identified a set of conserved sequence
motifs with functional significance supported by the expression patterns of
reporter genes containing the D. virilis (D. vir) DNA.
Conserved binding sites for the mesodermal TFs Twist (Twi), Nautilus (Nau, the
Drosophila orthologue of MyoD) and Mef2
(Andres et al., 1995;
Huang et al., 1996;
Ip et al., 1992;
Kophengnavong et al., 2000)
and a putative Col-binding site necessary for positive autoregulation were
present in different subdomains of the DA3 muscle CRM, correlating with the
separate phases of col regulation. We show that col
auto-regulation is crucial for a reiterative, two-step activation of
col transcription in each `naïve' FCM incorporated into the DA3
muscle. Nau, which was previously reported to be required for DA3 muscle
formation (Keller et al.,
1998), is also required for col transcription in the DA3
muscle, beyond the FC stage. Pan-FC expression of either Col, Nau or both
proteins resulted in ectopic col transcription in different sets of
muscles. Together, our results show that separate sets of cis-regulatory
elements ensure col activation in the DA3/D05 promuscular cluster,
progenitor and DA3 myofibre. Nau and Col act together in ensuring that all
nuclei within the DA3 myofibre activate Col and express the same
differentiation programme, thereby directly supporting the concept of
combinatorial control of muscle identity.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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);
rp298-Gal4 (Menon and Chia,
2001); col1
(Crozatier et al., 1999) and
nau188 (Balagopalan et
al., 2001) EMS-induced loss-of-function alleles;
vg83b27-R, a 
-ray induced amorphic allele;
UAS-col (Vervoort et al.,
1999); hs-col
(Crozatier and Vincent, 1999);
UAS-nau (Keller et al.,
1997); UAS-lacZ (Bloomington Stock Center, Indiana, USA).
UAS-mcd8::GFP (Grueber et al.,
2003).
Plasmid constructions and transgenic lines
The P5cl construct was described in Crozatier and Vincent
(Crozatier and Vincent, 1999).
Other Pcl constructs were generated by cloning different fragments of
col upstream DNA (for the restriction sites used, see Fig. S2 in the
supplementary material) into pCaSpeRβ-gal or pPTGal4
(Sharma et al., 2002).
Mutagenesis of the putative Nau- and Col-binding sites in P2.6cl was
done by PCR. The D. vir constructs were generated by restriction
digestion of genomic DNA isolated from a 
phage library (J. Tamkun,
University of California, Santa Cruz, CA).
Immunohistochemical staining and in situ hybridisation
Embryos were fixed and processed for antibody staining and/or in situ
hybridisation as described (Crozatier et
al., 1996). The nau intronic probe covers all three
nau introns and the two corresponding exons. The following primary
antibodies were used: rabbit anti-Col (1/400); mouse anti-Col (1/100); rabbit
anti-MHC (1/500; D. Kiehart, Duke University, Durham, NC); mouse
anti-β-gal (1/1000, Promega); rabbit anti-GFP (1/1000 Torrey Pines
Biolabs); Secondary antibodies were Alexa Fluor 488 and Alexa Fluor 647
conjugated goat anti-rabbit, Alexa Fluor 647 conjugated goat anti-mouse
(Molecular Probes 1/300); Rhodamin RedX conjugated donkey anti-mouse (Jackson
Laboratory 1/300); biotinylated goat anti-mouse (Vector Laboratory, 1/1000).
For double fluorescent in situ hybridisation/immunostaining, we used
biotinylated col and digoxygenin-labelled nau intronic
probes and the ABC kit from Vector Laboratory, followed by fluorescent
tyramide staining (Alexa fluor 555 or 488 conjugated tyramide from Molecular
Probes) and Fast Red. Primary antibodies against Col, GFP or MHC were used at
five times the usual concentration. Monoclonal Col antibodies were generated
in collaboration with Jeannine Boyes and Georges Delsol, U 563 INSERM,
Toulouse Purpan.
Sequence alignments and transcription factor binding sites
Pairwise sequence alignments of col upstream sequences from
various Drosophila species
(http://flybase.bio.indiana.edu/static_pages/news/articles/2007_03/genomes_papers3.html)
were done using NCBI-BLAST (bl2seq), Genome Browser (UC Santa Cruz) and
Evoprinter (NINDS, NIH, Bethesda) and manually edited following
eye-inspection. Search for individual binding sites for transcription factors
made use of Genomatix Matinspector, Possum
(http://zlab.bu.edu/~mfrith/possum/),
cis-analyst
(http://rana.lbl.gov/cis-analyst/cgi/viewer.php)
and FlyEnhancer
(http://genomeenhancer.org/fly;
M. Markstein) and manual inspection based on the literature. Access to the
Mef2 and Twi in vivo binding sites
(Sandmann et al., 2007;
Sandmann et al., 2006) was via
the E. Furlong's lab site
(http://furlonglab.embl.de/data/).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Nelson et al.,
2004; Philippakis et al.,
2006). During embryogenesis, col is expressed in the
MD2/PS0 head region, the somatic DA3 muscle, precursor cells of the lymph
gland, a small set of multidendritic (md) neurons of the peripheral nervous
system and specific neurons of the central nervous system (CNS)
(Baumgardt et al., 2007;
Crozatier et al., 2004;
Crozatier et al., 1999;
Crozatier and Vincent, 1999;
Orgogozo and Schweisguth,
2004). We previously generated a lacZ reporter transgene
(P{5col::lacZ}, abbreviated P5cl,
Fig. 1A) containing 5 kb of
col upstream DNA, which faithfully reproduced col
transcription both in the MD2/PS0 and the DA3 muscle, starting at the
progenitor stage and not in promuscular cluster(s)
(Crozatier and Vincent, 1999).
To identify the missing cis-regulatory information, we tested a longer
construct containing the entire 9 kb region separating col from
CG10200, the next predicted upstream gene
(http://flybase.bio.indiana.edu/;
P9cl, Fig. 1A). In
addition to the head and DA3 muscle, P9cl expression reproduced
col expression in md neurons and a subset of neurons in the CNS. A
DNA fragment located further upstream, between CG10200 and the next predicted
gene CG10202, was independently shown to drive col expression in the
anteroposterior organiser of the wing imaginal disc
(Hersh and Carroll, 2005).
However, neither this construct nor P9cl reproduced Col expression in
promuscular clusters (Fig. 1D).
The col transcription unit is immediately flanked at its 3' end
by another gene, BEAF32 (Fig.
1A), making rather unlikely the presence of cis-regulatory
elements within this region. However, it contains ten different introns, of
total length around 30 kb, the cis-regulatory content of which remains to be
assessed (see Discussion). To delineate more precisely the CRM driving col expression in the DA3 muscle, we tested a series of constructs containing 2.6, 2.3, 1.6 and 0.9 kb of DNA upstream of the col transcription start site, respectively (Fig. 1A). P2.6cl retained the information necessary for col expression in MD2/PS0 and the DA3 progenitor and muscle (Fig. 1C), although we noted that P2.6cl expression in muscle progenitors was less robust than P9cl. P2.3cl was also activated in MD2/PS0 at stage 6 and the DA3 muscle. However, unlike P9cl or P2.6cl, P2.3cl was not activated in the DA3/DO5 progenitor but only at the FC stage (Fig. 1C; ectopic lacZ expression was observed in clusters of neuroectodermal cells at embryonic stage 11). This difference indicated that cis-regulatory elements required for col expression in the DA3/DO5 progenitor reside between positions -2.6 and -2.3 and act separately from those required for expression in the DA3 FC and muscle. P1.6cl was only active in MD2/PS0, whereas no expression at all could be detected with P0.9cl (data not shown). Together, expression data from this series of reporter constructs allowed the mapping of the CRM required for col-specific expression in the DA3/DO5 muscle progenitor and DA3 FC/myofibre to a DNA fragment between positions -2.6 and -1.6 upstream of the col transcription start (Fig. 1E).
|
;
Yuh et al., 2002). Among these
species, D. virilis (D. vir) is the most distant from D.
melanogaster (D. mel)
(Tamura et al., 2004). We
first verified that Col expression in D. vir was similar to that in
D. mel embryos (Fig.
2A and see Fig. S1 in the supplementary material) and could infer
from this that the regulatory information controlling col
transcription in the DA3 muscle lineage has been conserved. Sequence
comparison of 9 kb of the col upstream region between D. mel, D.
vir and four other Drosophila species, D. yakuba, D.
ananassae, D. pseudoobscura and D. mojavensis revealed numerous
stretches of high sequence conservation, of sizes up to 100 bp (see Fig. S1 in
the supplementary material). Ten conserved motifs of size >20 bp, numbered
1 to 10 from 5' to 3', were found in the same order and at the
same relative position between position -2.6 and the start of transcription in
all six Drosophila species (Fig.
2B and see Fig. S2 in the supplementary material). To test the
relevance of this conservation, we generated lacZ reporter constructs
containing either D. vir or D. mel DNA
(Fig. 2B).
P.3.4clvir corresponds to D. mel P2.6cl, whereas
P3.4-1.3clvir and P2.6-0.9cl are truncated
versions covering motifs 1 to 10. All four reporter genes showed expression in
the DA3 muscle, starting at the progenitor stage, confirming the evolutionary
conservation of a DA3-muscle-specific CRM
(Fig. 2A). A Gal4 driver line
containing only the -2.6 to -1.6 region (P2.6-1.6cG), harbouring only
motifs 1 to 7 (Fig. 2B), was
also specifically expressed in the DA3 muscle
(Fig. 2A). This confirmed that
the DA3 muscle CRM is located between positions -2.6 and - 1.6. We noticed,
however, that expression of P2.6-1.6cG was weaker and more sporadic
than P2.6-0.9cl, suggesting the existence of cis-regulatory
element(s) between positions -1.6 and -0.9 contributing to robust DA3 muscle
expression. We then searched within the conserved motifs 1 to 10 for consensus
binding sites of known TFs that could account for col activation in
the DA3 muscle. This identified a binding site for the mesodermal basic
helix-loop-helix (bHLH) protein Twi (Ip et
al., 1992; Kophengnavong et
al., 2000) (within motif 2 Fig.
2B), correlating well with the position of the muscle progenitor
cis-element (Fig. 1E) and a
potential EBF/Col-binding site (Travis et
al., 1993) within motif 7. Further visual inspection of the
sequence alignments identified other conserved TF-binding sites, including one
Mef2-binding site (Andres et al.,
1995) within the -1.6 to -0.9 fragment and one consensus binding
site for Nau (Huang et al.,
1996; Kophengnavong et al.,
2000). On the one hand, the position of the Mef2 site correlated
well with the requirement of the -1.6 to -0.9 fragment for robust DA3 muscle
expression (Fig. 2A). On the
other hand, the presence of a Nau-binding site was particularly intriguing as
Nau is required for DA3 muscle formation
(Keller et al., 1998).
Potential binding sites for other TFs
(Bergman et al., 2005;
Vlieghe et al., 2006) could be
found in the DA3 CRM, but we limited here our annotation to the conserved
sites (see Fig. S2 in the supplementary material). The relative paucity of
known TF-binding sites in the conserved sequence motifs found in the DA3
muscle CRM leaves largely open the question of the roles of these motifs in
col regulation.
|
).
By using different col-lacZ reporter genes, we mapped the
cis-regulatory element(s) responsible for this muscle-specific activation to
the DA3 muscle CRM (Fig. 2B-D
and data not shown). As it is restricted to the DA3 and VL1 (and possibly DA2)
muscles, we reasoned that col auto-activation was dependent upon a
specific combination of TF expressed in these muscles. Of the known TFs
expressed in somatic muscles, only Vestigial (Vg) and Nau are expressed in DA3
and VL1 (Bate et al., 1993;
Dohrmann et al., 1990;
Keller et al., 1998;
Paterson et al., 1991).
nau mutant embryos lack a subset of muscle fibres, with DA3 being the
most severely affected (Balagopalan et al.,
2001; Keller et al.,
1998). By contrast, no muscle phenotype has yet been described for
vg loss-of-function mutations. vg mutants show reduced wings
and severe flight muscle defects but are viable and fertile, allowing the
study of their maternal plus zygotic phenotype. We did not observe abnormal
Col expression or abnormal DA3 muscle formation in vg mutant embryos,
indicating that Vg is not required for formation of this muscle (data not
shown).
col activation in nuclei of fused myoblasts: a reiterative process endowing all nuclei of the DA3 myofibre with the same transcriptional programme
In situ hybridisation with a col intronic probe that labels
nascent transcripts revealed that col transcription is activated in
the nuclei of those FCMs that are recruited to form the DA3 muscle
(Crozatier and Vincent, 1999).
To further investigate the mechanisms behind this observation, we compared the
patterns of Col accumulation and col transcription during the process
of DA3 muscle formation (Fig.
3A-C). We found that, throughout the FC/FCM fusion phase (stage
12-15), each DA3 muscle syncitium contains on average one or two nuclei, which
stain positive for Col but do not transcribe col (see also
Fig. 4). Close-up analysis of
fusion events in stage 13 embryos further revealed that only nuclei containing
high levels of Col protein activated col transcription
(Fig. 3A). This strongly
suggested that Col accumulation is a prerequisite for auto-activation in newly
fused FCM nuclei. In support of this interpretation, all the DA3 muscle nuclei
transcribe col after completion of the muscle fusion process
(Fig. 3B), although this
uniform expression phase is only transient, as col transcription
declines abruptly during stage 16 to become undetectable
(Fig. 3C). From these
observations, we conclude that activation of col transcription occurs
through a reiterative two-step mechanism, ensuring the same transcriptional
programme to all nuclei of the DA3 myofibre. In a first step, nuclei from FCMs
newly incorporated into the growing syncitium import some of the Col protein
present in the muscle precursor (inset in
Fig. 3A). In a second step,
col transcription is turned on in these nuclei.
Col and Nau are required for col transcription during DA3 muscle fusion
First evidence for col autoregulation during DA3 muscle formation
came from the observation that col transcription is not maintained in
the DA3 FC in col mutant embryos
(Crozatier and Vincent, 1999).
In order to investigate this phenotype in more detail, we constructed a
P9col-Gal4 driver (P9cG), allowing us to express a
membrane-bound form of GFP in the DA3 muscle progenitor and to specifically
follow the fate of this progenitor in col mutant embryos
(UAS-mcd8GFP/P9cg; Fig.
3D). In wt embryos, mCD8GFP remains expressed and is detected both
intracellularly and at the plasma membrane of the DA3 myofibre. In
col mutant embryos, mCD8GFP expression is lost early but stability of
the protein at the plasma membrane allows the detection of the mutant DA3
fibres. This experimental set-up confirmed that fusion of FCMs with the DA3 FC
is drastically impaired in col mutant embryos and that col
transcription is neither maintained in the DA3 FC nor activated in the nuclei
of FCM, which sometimes fuse to form an abortive DA3 muscle precursor
(Fig. 3E). These data establish
that col auto-regulation and the muscle DA3 identity programme are
intimately connected.
|
). The presence of a consensus Nau-binding site in the
col DA3 muscle CRM raised the possibility that one Nau function could
be to regulate col transcription. To address this possibility, we
first compared nau and col transcription in wt DA3 muscles,
using in situ hybridisation to primary transcripts and Col immunostaining.
This revealed that col and nau are transcribed together in
the DA3 progenitor, FC and muscle precursor up to early stage 13
(Fig. 4A-C). Subsequently, only
col transcription persists in the DA3 muscle
(Fig. 4D). We then looked at
col transcription in embryos homozygous for the null allele
nau188 (Balagopalan et
al., 2001; Wei et al.,
2007). Based on Col and Myosin heavy chain (MHC) antibody staining
(Fig. 4E,F and data not shown)
the DA3 muscle was completely absent in around 5% of segments, abnormal in
orientation in 45% and rather normal-looking in about 50% of segments,
consistent with previous reports (Keller
et al., 1998). Low amounts of Col protein were observed in nuclei
of the `normal-looking' DA3 muscles (Fig.
4E,F), allowing us to look at col transcription in these
nuclei. In wt embryos at stage 15, each DA3 muscle syncitium contains on
average nine nuclei, which are all strongly stained with Col antibodies, and
seven to eight are positive for col transcription
(Fig. 4E; see also
Fig. 3B). The DA3 fibres
present in nau188 embryos contained only seven nuclei on
average, with most showing a low level of Col protein. However, at most two or
three of those transcribed col
(Fig. 4F). This result
indicated that Nau activity is required, in addition to Col, for activation of
col transcription in the FCM nuclei that are recruited by the DA3 FC.
The Col protein that is detected in nau mutant embryos probably
derives from earlier, Nau-independent col transcription. Supporting
this conclusion, one nucleus, probably the FC nucleus, shows high levels of
col transcripts in nau mutant embryos at late stage 12, when
DA3 muscle precursors contain two or three nuclei
(Fig. 4G,H). In summary,
nau and col are expressed in the DA3 FC and both Nau and Col
are required for col activation in the nuclei of newly recruited FCM,
thereby ensuring that all nuclei within the DA3 myofibre acquire the same
identity.
The combinatorial activity of Nau and Col controls col expression
To further test the hypothesis of a combinatorial role of Nau and Col in
conferring the DA3 muscle its identity, we examined the activation pattern of
P2.6cl at stage 15 after either Nau alone, Col alone or Nau+Col were
ectopically expressed in all muscle FCs (rp298Gal4 driver)
(Menon and Chia, 2001).
rp298Gal4-driven Col expression resulted in ectopic P2.6cl
expression in several muscles other than DA3, including DA2, DT1 and VL2,
although this expression was most robust in VL1, as seen in hs-col
experiments (Fig. 5A,B),
without major phenotypic effects, at least at the level of muscle fibre
morphology (data not shown). By contrast, rp298Gal4-driven Nau
expression, while altering the pattern of muscle fibres, as previously
documented with a heat-shock construct
(Keller et al., 1997),
provoked ectopic expression of P2.6cl only in a single muscle, the
DA2 muscle (Fig. 5C). These
data confirmed that, despite a more general role than Col in somatic
myogenesis (Keller et al.,
1997; Wei et al.,
2007), Nau is generally unable by itself to ectopically activate
col transcription. When Col and Nau were expressed together,
P2.6cl was activated in the same muscles as with Col alone, but much
more strongly (compare Fig. 5B with
D), confirming that Nau potentiates the ability of Col to activate
its own transcription. Interestingly, P2.6cl was activated by Nau+Col
in a few muscles, including the SBM muscles, which did not respond to the
presence of Col alone, indicating that Nau and Col may act synergistically.
Still, many muscles remained refractory to this combination and did not
express P2.6cl, suggesting that other competence factors are lacking
or that negative regulation exerted by Notch and/or other factors may be
dominant in these muscles.
|
|
),
consistent with sequence conservation of the COE DNA-binding domain
(Dubois and Vincent, 2001)
(V.D., unpublished). The closest match to the consensus EBF recognition site
found within the DA3 CRM is the sequence ATGTCTGGGGAT, which is part of the
conserved motif 7 (Fig. 6F and
see Fig. S2 in the supplementary material). Gel-shift assays and
immunoprecipitation of DNA-protein complexes formed by co-incubation of Col
with synthetic oligonucleotides overlapping this predicted EBF-binding site
failed to reveal strong binding in vitro (V.D., unpublished). Nevertheless,
DA3-specific expression of P2.6clcol in vivo was almost
undetectable when this site was mutated
(Fig. 6B,F), suggesting that it
mediates col auto-regulation. To provide a different test of this in
vivo function, we looked at P2.6clcol activation in
conditions of ectopic Col expression. Unlike P2.6cl
(Fig. 6D),
P2.6clcol expression was activated very weakly, if at all,
in the VL1 (and DA2) muscles (Fig.
6E). These results reinforce the conclusion that the predicted
EBF/Col-binding site present within the conserved motif 7 is required for
positive col autoregulation.
|
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Rushton et al., 1995). The
current view is that the properties specific to each muscle result from the
selective expression, in each FC, of distinct combinations of `muscle
identity' TFs. However, experimental evidence for such a combinatorial code
has remained sparse. We addressed here this question, using as a paradigm Col
expression as both a determinant and read-out of DA3 muscle identity.
Three separate steps in the transcriptional control of muscle identity
Functional dissection of the DA3 muscle CRM present in the col
upstream region showed that col expression in the DA3 FC can be
separated from its expression in the DA3/D05 progenitor and the promuscular
cluster. It thus revealed the existence of three steps in the transcriptional
control of muscle identity (Fig.
7). That col expression in the DA3/D05 progenitor could
be uncoupled from that in promuscular clusters was in apparent contradiction
with the previous conclusion from pioneering studies on Eve expression in
dorsal muscle progenitors that this expression issued from Eve activation in
promuscular clusters. Restriction of Eve expression to progenitors was
considered a secondary step, mediated by N-signalling during progenitor
selection by lateral inhibition (Carmena
et al., 1998; Halfon et al.,
2000). To reconcile our data and this model, we propose that the
muscle DA3 CRM is only active in the DA3/D05 progenitor because it lacks some
positively acting cis-elements necessary to counteract N-mediated repression
of col transcription (Fig.
7A). We have indeed previously shown that col
transcription is repressed by N during the progenitor selection process
(Crozatier and Vincent, 1999).
We also noted that a Twi-binding site is present in the `progenitor' subdomain
of the DA3 CRM (Fig. 2B and
Fig. 7A). The functional
importance of this site is supported by its in vivo occupancy in 4- to
6-hour-old embryos when selection of the DA3/DO5 progenitor takes place
(Sandmann et al., 2007).
Together, Twi in vivo binding and the col/P2.6cl/P2.3cl
expression data suggest that Twi activity contributes to col
expression in the DA3/DO5 progenitor but may not be sufficient to override N
repression of col transcription before progenitor selection.
Additional binding sites for Twi present in the col upstream region,
between positions -8.7 and -8.3, are also bound by Twi in vivo
(Sandmann et al., 2007) and
probably contribute to the robustness of P9cl expression in
progenitor cells, but the question of which cis-regulatory elements mediate
col activation in promuscular clusters remains open. From their Eve
expression studies, Michelson and colleagues developed a computational
framework to identify other FC-specific genes
(Estrada et al., 2006;
Philippakis et al., 2006).
This framework, named Codefinder, integrates transcriptome data and clustering
of combinations of binding sites for five different TFs (Pnt, dTCF, Mad, Twi
and Tin). col/kn was selected by Codefinder owing to the presence of
five clusters of binding sites, four of which are located within introns
(Philippakis et al., 2006). It
remains to be determined which of these could be responsible for col
activation in promuscular clusters, but it is interesting to note that another
in vivo Twi-binding site in 4-6-hour-old embryos correlates with the
3'-most cluster (Sandmann et al.,
2007). In addition to Twi, conserved binding sites for Nau and
Mef2 are found within the DA3 CRM. The Mef2 binding site is located in a
region required for robust DA3-muscle expression of a reporter gene
(Fig. 2B,
Fig. 7B; and see Fig. S2 in the
supplementary material). A direct control of col transcription by
Mef2 during the muscle fusion process is further supported by the recent
finding that Mef2 binds in vivo to the col upstream region between 6
and 8 hours of embryonic development
(Sandmann et al., 2006).
Propagation of transcriptional identity from the founder cell to fusion-competent myoblasts
Detailed analysis of col auto-activation revealed a reiterative,
two-step process: import of pre-existing Col protein in the FCM nuclei that
incorporate into the growing DA3 myofibre precedes activation of col
transcription (Fig. 3). This
process ensures that all incorporated FCM nuclei acquire the same identity.
Nau is required for maintaining col transcription in the DA3 muscle
precursor and this control is probably direct. The presence of a putative
EBF-binding site in the DA3 muscle CRM also correlates with the Col
requirement for maintaining its own transcription beyond the FC stage
(Crozatier and Vincent, 1999).
Thus, despite the failure of our assays to detect strong Col binding to this
site in vitro, it appears to be essential for col auto-regulation in
vivo. This suggests that in vivo binding is potentiated by one or more
specific co-factor(s) present in the DA3 muscle. One co-factor is probably
Nau, as the ability of Col to activate its own transcription in newly
recruited FCM is dependent upon Nau activity
(Fig. 7B). Nau is not
sufficient, however, as many muscles containing both Nau and Col proteins do
not activate col transcription
(Fig. 5). Interestingly, mouse
EBF (also known as Ebf1 and Olf1 - Mouse Genome Informatics) and E2A (Tcfe2a -
Mouse Genome Informatics), a bHLH protein of the same subgroup as MyoD
(Simionato et al., 2007), have
been shown to act on the same target promoter and synergistically upregulate
transcription of B-lymphocyte-specific genes, although no direct physical
interaction between EBF and E2A could be found in vitro. This suggested that
functional interaction of EBF and E2A, similar to Col and Nau, requires yet
another factor (O'Riordan and Grosschedl,
1999). Taking into account the restricted pattern of ectopic
col activation in hs-col conditions, we hypothesised that Vg
could be another component of the DA3 combinatorial identity
(Bate, 1993;
Frasch, 1999). However, we
found that Vg is not required for DA3 muscle specification, leaving open the
question of which factor may bridge Col and Nau functions.
Temporal and combinatorial control of muscle identity
Unlike col or P2.6cl, P2.3cl is expressed in the DA3 FC and muscle
precursor but not the DA3/DO5 progenitor, showing that col
transcription in the progenitor and muscle precursor is under separate
control. These two phases of col regulation are intimately linked,
however, as Col is required for activating its own transcription in the nuclei
of FCM recruited by the DA3 FC. This regulatory cascade may explain how
pre-patterning of the somatic mesoderm and muscle identity are
transcriptionally linked in the Drosophila embryo. As discussed
above, the ability of Col to auto-regulate depends upon the presence of Nau,
another muscle identity TF. Col and Nau act as obligatory co-factors for
maintenance/activation of Col expression in all nuclei of the DA3 muscle, thus
bringing to light a clear case of combinatorial coding of muscle identity.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/24/4347/DC1
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ACKNOWLEDGMENTS |
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