Mechanisms of Development 110 (2002) 39–50
www.elsevier.com/locate/modo
Drosophila MEF2 is a direct regulator of Actin57B transcription in
cardiac, skeletal, and visceral muscle lineages
Kathleen K. Kelly, Stryder M. Meadows 1, Richard M. Cripps*
Department of Biology, University of New Mexico, Albuquerque, NM 87131-1091, USA
Received 17 August 2001; received in revised form 2 October 2001; accepted 3 October 2001
Abstract
To identify regulatory events occurring during myogenesis, we characterized the transcriptional regulation of a Drosophila melanogaster
actin gene, Actin 57B. Act57B transcription is first detected in visceral muscle precursors and is detectable in all embryonic muscles by the
end of embryogenesis. Through deletion analysis we identified a 595 bp promoter element that was sufficient for high levels of expression in
all three muscle lineages. This fragment contained a MEF2 binding site conserved between D. melanogaster and Drosophila virilis which
bound MEF2 protein in embryo nuclear extracts. Mutation of the MEF2 site severely reduced promoter activity in embryos, and in Mef2
mutants Act57B expression was severely decreased, demonstrating MEF2 is an essential regulator of Act57B. We also showed that MEF2
likely acts synergistically with factors bound to additional sequences within the 595 bp element. These findings underline the importance of
MEF2 in controlling differentiation in all muscle lineages. Our experiments reveal a novel regulatory mechanism for a structural gene where
high levels of expression in all embryonic muscles is regulated through a single transcription factor binding site. q 2002 Elsevier Science
Ireland Ltd. All rights reserved.
Keywords: Actin; Drosophila; Muscle development; Myocyte enhancer factor-2; Transcriptional regulation
1. Introduction
Studies of muscle development have placed an emphasis
on describing the transcription factors that are required for
myogenesis. Prominent among these are the members of the
myocyte enhancer factor 2 (MEF2) family of transcriptional
regulators. The four murine mef2 genes and the single
Drosophila Mef2 gene are each expressed in all skeletal,
visceral and cardiac lineages during development (Edmondson et al., 1994; Lilly et al., 1994; Nguyen et al., 1994;
Taylor et al., 1995; Subramanian and Nadal-Ginard,
1996), suggesting that they play important roles in the
development of the musculature.
In vertebrates the existence of multiple mef2 genes has
hindered the characterization of null phenotypes due to
overlapping expression patterns and potential functional
redundancy of gene products. However, inactivation of the
murine mef2c gene resulted in an embryonic lethal phenotype with defects in heart morphogenesis and myogenesis
(Lin et al., 1997a), and abnormal development of smooth
* Corresponding author. Tel.: 11-505-277-2822; fax: 11-505-277-0304.
E-mail address: [email protected] (R.M. Cripps).
1
Present address: Department of Molecular and Cellular Biology,
University of Arizona, Tucson, AZ 85721, USA.
muscle cells (Lin et al., 1998). Drosophila mutants in which
all MEF2 function is absent have a failure of muscle differentiation in all muscle lineages, although skeletal, visceral
and cardiac myoblasts are still specified (Bour et al., 1995;
Lilly et al., 1995). Together, these results have revealed an
evolutionarily conserved central role for MEF2 in the
execution of the myogenic program.
Although MEF2 function is required in all muscle
lineages, individual muscles can have startlingly different
phenotypes. It is therefore thought that MEF2 interacts with
tissue specific cofactors to control gene expression in
phenotypically different muscles. In support of this is the
inability of MEF2 alone to convert fibroblast to myoblasts
(Molkentin et al., 1995). Furthermore, MEF2 has been
shown to function synergistically with myogenic bHLH
proteins to activate the myogenic program in vertebrate
skeletal muscles (Kaushal et al., 1994; Molkentin et al.,
1995; Black et al., 1998). MEF2 also functions in collaboration with PAR domain protein-1 in Drosophila skeletal
muscles (Lin et al., 1997b). However, partners for MEF2
to control the differentiation of cardiac and smooth muscle
cells have yet to be identified.
Analysis of the cis-regulatory regions of muscle-specific
genes has been useful in identifying factors and cofactors
required for tissue-specific gene expression. In many cases,
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PII: S 0925-477 3(01)00586-X
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K.K. Kelly et al. / Mechanisms of Development 110 (2002) 39–50
genes with broad expression patterns within the musculature
have separable enhancer regions which control subsets of the
expression pattern of that gene, presumably due to the different arrays of transcription factors present in each muscle
type. An extreme example of this is the Mef2 gene in Drosophila in which transcription is controlled by a very large array
of upstream enhancers, each of which contribute a subset of
the expression of Mef2 during embryogenesis (Lilly et al.,
1995; Schulz et al., 1996; Gajewski et al., 1997; 1998; Cripps
et al., 1998; Cripps et al., 1999; Nguyen and Xu, 1998).
In this paper we present an example of a gene where high
levels of expression in all three embryonic muscle lineages
is controlled by MEF2 through a single enhancer binding
site. Of the two embryonic muscle actin isoforms, Drosophila Actin 57B is the major myofibrillar actin expressed in
skeletal, visceral and cardiac muscle during embryogenesis
(Tobin et al., 1990; Keller et al., 1997). Transcripts of
Act57B are first detected at stage 10 in the visceral muscle
precursors, with expression in skeletal muscle at stage 11
and by stage 16 this transcript is also found in the dorsal
vessel. Using deletion analysis of the upstream promoter
sequences, we have identified a minimal 595 bp element
that is sufficient for activation in all three muscle lineages.
Within the element, a single MEF2 binding site is required
for high levels of expression of this promoter in all muscle
tissues, suggesting that for Act57B, MEF2 is essential for
expression in all embryonic muscles.
potential regulatory elements we sequenced the entire 5.4 kb
EcoRI genomic fragment containing the gene, and mapped
onto that the exon structure described by Fyrberg et al.
(1981) (Fig. 2A). To determine the location of the transcription start site, we performed a primer extension assay upon
total RNA either from control embryos aged 0–2 h (before
Act57B expression), or from embryos aged 15–17 h (after
the onset of Act57B expression). The primer was selected
such that it would hybridize to Act57B mRNA downstream
of the translation start site (Fig. 2A). We also performed
DNA sequencing reactions upon the 5.4 kb plasmid using
2. Results
2.1. Act57B expression pattern
To understand the regulatory events that control Act57B
transcription we initially characterized in detail the expression pattern of Act57B during embryogenesis. We used in
situ hybridization with a digoxigenin labeled 3 0 UTR antisense probe to visualize the endogenous gene transcripts
during embryogenesis. Expression commenced at the
germ band extended stage (stage 10) (Fig. 1A); at this
stage transcripts were detected in the visceral muscle
precursors only. By stage 11, Act57B expression was also
detected in skeletal myoblasts (Fig. 1B). As embryogenesis
proceeded, Act57B transcripts continued to be expressed in
the visceral and somatic muscle lineages (Fig. 1C,D) and by
stage 16 were seen in the developing heart (Fig. 1E). These
findings are consistent with those of Tobin et al. (1990) who
showed Act57B expression in skeletal and visceral embryonic muscles and Keller et al. (1997) who showed Act57B
expression in heart. Our experiments significantly extend
previous work by describing in detail the temporal appearance of Act57B transcripts in all muscle lineages at specific
stages of embryogenesis.
2.2. Structure of the Act57B gene
To localize the transcribed region of Act57B relative to
Fig. 1. Expression of Act57B and an Act57B-lacZ reporter construct. (A–E)
In situ hybridization using an antisense Act57B 3 0 UTR probe. (F) In situ
hybridization of a stage 14 embryo using a 3 0 UTR sense probe, giving no
signal, demonstrating the specificity of the antisense probe. (G–K) Anti-bgalactosidase staining of the 22479/12 Act57B-lacZ reporter. (L) Stage 14
embryo with a lacZ reporter fused to the Act57B intron sequence shows no
staining with b-galactosidase antibody, demonstrating the absence of independent regulatory sequences within the intron. (A,G) Stage 10 embryos
showing initial Act57B activity in visceral mesodermal cells (vm). (B,H)
Stage 11 embryos with gene activity continuing in visceral muscle precursors and also present in skeletal myoblasts (sm). By stages 15–16 (C,D,I,J),
Act57B and reporter gene expression is seen in all skeletal muscles (sk) and
the visceral muscles (vi). At stage 16 Act57B expression is detected in the
heart (ht) (E,K). All embryos (except E and K) are lateral views oriented
with anterior to left and dorsal up. (E) and (K) are dorsal views to show the
heart tube with anterior to left. Scale bars represent 100 mm.
K.K. Kelly et al. / Mechanisms of Development 110 (2002) 39–50
41
the sequence of both the TATA box and start site are
strongly conserved in D. virilis (see below).
2.3. Regulatory sequences required for Act57B expression
Fig. 2. Structure of Act57B. (A) Gene structure with the TATA box, transcription start site (arrow) and translation start (ATG) and stop (TAA)
noted. (B) Primer extension assay to determine the transcription start site.
Total RNA was isolated from embryos aged from 0–2 h (before Act57B
expression) and 15–17 h. The product of reverse transcription was 101 bp
long. The corresponding sequencing reactions were used to determine the
transcription start site and are shown in the left four lanes with basepairs
flanking the transcription start site (arrow) listed. Primer location and orientation is noted in panel (A).
the same primer and electrophoresed these reactions alongside the primer extension products. A primer extension
product was generated only in the 15–17 h collection lane,
and migrated with the thymine at position 101 of the
sequencing reaction. These results indicate that transcription starts with an A ribonucleotide 90 bp upstream of the
AUG initiation codon. Although it is formally possible that
the transcription start site is contained within a more 5 0
exon, this is unlikely since there is a consensus TATA
box located 33 bp upstream of the identified start site and
To identify cis-acting DNA sequences controlling transcription of Act57B, we isolated DNA corresponding to
upstream sequence proximal to the transcription start site
(22479/12) as well as DNA corresponding to the intron
(1100/1831). We tested these sequences for enhancer
activity in vivo in transgenic animals using a lacZ reporter
construct (Fig. 3). The promoter fragment 22479/12 directed lacZ expression in a pattern that recapitulated the endogenous gene expression with activity seen first in the
visceral myoblasts at stage 10 (Fig. 1G), followed by activity at stage 11 in the skeletal muscle precursors (Fig. 1H)
and stage 16 in the heart (Fig. 1K), although staining in the
small heart tube was occasionally irregular. The intron
sequence contained no regulatory regions which were sufficient for muscle specific expression since there was no bgalactosidase accumulation at any embryonic stage (Figs.
1L and Fig. 3); intron sequences may contain regulatory
elements that work in concert with upstream elements.
These findings demonstrate that all the regulatory sequences
for Act57B expression during embryogenesis are contained
within the 22479/12 fragment.
In order to localize regulatory elements within the 22479/
12 promoter element, we tested additional lacZ reporter
constructs with serial deletions of the 5 0 end (Fig. 3). Deletion
of sequences between 22479 bp to 2593 bp had no effect on
either the timing or levels of lacZ expression, and antibody
staining was identical to that shown in Fig. 1 for the 22479/
12 fragment (Fig. 3). Importantly, lacZ expression was still
detected in all three muscle lineages.
Fig. 3. Identification of a minimal promoter sequence that directs muscle specific lacZ expression. Top: gene structure of Actin 57B showing the transcription
start site (arrow), exons (shaded boxes denote the translated sequence) and the intron. Bottom: serial 5 0 end deletions of the promoter sequence were fused to a
lacZ reporter and assayed for muscle specific activity using b-galactosidase antibody in whole mount embryos. Staining was scored in skeletal, visceral and
heart muscles such that (111) denotes strong staining in all muscle lineages and (2) denotes no antibody staining in all lineages. All 5 0 constructs maintained
the same strong level of reporter activity indicating that sequences required for Act57B expression at high levels are contained within the 2593/12 bp
fragment.
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K.K. Kelly et al. / Mechanisms of Development 110 (2002) 39–50
2.4. Identification of conserved sequence between D. melanogaster and D. virilis actin genes
Clearly, all of the sequences controlling stage and tissue
specific expression of Act57B are contained within this short
595 bp promoter element. To aid us in identifying important
regulatory sequences within the 2593/12 region we
sequenced the upstream region of ActC2, the D. virilis
homologue of Act57B (Loukas and Kafatos, 1986; Lovato
et al., 2001). Fig. 4 shows the sequence alignment for the
promoter regions of Act57B and ActC2. Close to the coding
sequence of each gene, there are two regions of strong similarity. One of these is the location of the transcription start
site identified above and the other is the TATA box region.
The strong conservation suggests that they are important to
the expression of Act57B.
Distal to the TATA box, there are additional sequence
similarities at: 289/280, 2106/2101, 2131/2120, 2150/
2144/, 2169/2161, 2277/2197, 2348/2309, 2424/
2397, and 2463/2454. In Fig. 4B, the two largest blocks
of sequence similarity are highlighted. The first of these large
blocks, Region a, at 2277/2197 is 31 bp in length and differs
between the two species at only a single nucleotide. A more
distal sequence, designated Region b, at 2424/2397 is 27 bp
in length and also differs between the two species by a single
nucleotide. That these elements have been conserved over 60
million years suggests strongly that they perform important
regulatory roles in Act57B transcription.
Fig. 4. Identification of evolutionarily conserved sequences in the upstream promoter regions of Act57B and its homolog (ActC2) in D. virilis. (A) The locations
of two large blocks of sequence homology (a and b) identified within upstream sequences of Act57B and ActC2. (B) Sequence alignment of the homologous
regions between D. melanogaster and D. virilis. Large conserved upstream sequences are shaded in salmon, however note also additional smaller regions.
Within Region a there is a consensus MEF2 binding site (white area); there are no know myogenic transcription factors which bind to Region b (salmon box).
Locations of the TATA box (blue box), the translation start site (MET), and the boundary of exon 1 (open box) are indicated.
K.K. Kelly et al. / Mechanisms of Development 110 (2002) 39–50
We performed a database search with the conserved
sequences to identify potential regulatory factor binding
sites (http://genomatix.gsf.de/cgi-bin/matinspector/matinspector.pl). Within Region a there is a consensus MEF2
binding site (Fig. 4B). The next closest sequence to a
consensus MEF2 binding site contains two mismatch
nucleotides and was not conserved in D. virilis. Region b,
as well as other conserved sequence, contained no known
muscle-specific transcription factor binding sites.
2.5. Requirement of conserved sequences
To determine the functional importance of conserved
regulatory sequences within the 2593/12 fragment we
generated an additional 5 0 deletion construct (2278/12)
deleting Region b, and a second construct containing
Region b, but not Region a (2593/2245). These constructs
were then assayed for their ability to direct lacZ reporter
activity in transgenic flies. As shown in Fig. 5, lacZ expression in the 2277/12 transgenic lines was decreased in visceral (Fig. 5B) and skeletal (Fig. 5F) lineages, and cardiac
expression was also detected although strongly reduced
(Fig. 5J) compared to the 2593/12 construct. Reporter
constructs consisting of the upstream fragment containing
Region b were unable to confer muscle-specific lacZ expression in any of the muscle lineages (Fig. 5C,G,K). These
results suggest that Region b may regulate Act57B expression levels while Region a specifies expression in muscle
43
tissue, possibly via MEF2 activity. To further address this
we fused the upstream region, containing Region b, back
with the downstream sequence containing a mutated MEF2
site in Region a. While mutating the MEF2 site reduced
reporter activity, we were able to recover the muscle specific reporter expression patterning in skeletal and visceral
muscle although cardiac muscle failed to stain (Fig.
5D,H,L). These results suggested a regulatory role in muscle
development for both MEF2 and additional transcription
factors binding in Region b and Region a. Furthermore,
although expression in cardiac tissue was generally more
sensitive to deletion or mutation of these promoter
sequences, our results in general show similar sequence
requirements for all three lineages.
To confirm that the 2278/12 or 2593/12 mutant MEF2
constructs show significantly less activity than the 2593/12
minimal construct, we performed quantitative b-galactosidase assays of embryos homozygous for the lacZ reporter
constructs 2593/12, 2278/12, and 2593/12 mutant
MEF2. We assayed three independent lines for each of the
constructs using 10 mg of total embryo protein; the mean bgalactosidase activity for each construct is shown in Table 1.
Using Student’s t-test (Gardiner, 1997) we found that bgalactosidase activity of lines carrying the 2278/12 or
the mMEF2 constructs were significantly (P , 0:05)
reduced compared to the full-length 2593/12 element.
Furthermore, the level for the minimal construct 2593/12
was greater than the sum of b-galactosidase levels for
Fig. 5. LacZ reporter constructs used to define the functions of conserved upstream regions. The conserved regions a and b within the D. melanogaster Act57B
promoter are shaded. Reporter constructs were assayed for lacZ expression at embryonic stages 11 (A–D), and 16 (E–L). (A–H), lateral view, dorsal to top and
anterior to left. (I–L) dorsal view to show the heart tube, anterior to left. The 2593/12 construct shows strong expression at all stages (A,E,I). A 5 0 deletion of
the promoter construct to 2278/12 strongly diminished lacZ reporter activity indicating the presence of regulatory sequences within the 2593 bp to 2278 bp
region (B,F,J). Promoter-lacZ reporter constructs consisting of this upstream 2593 to 2278 sequence were unable to confer any muscle-specific reporter
expression (C,G,K). Fusing the 2593 to 2278 sequence with the downstream region where the MEF2 site is mutated (mMEF2) recovered muscle-specific lacZ
expression, however reporter activity was diminished demonstrating the importance of this single MEF2 site in Act57B expression (D,H,L). In (L), the irregular
reporter expression in cells surrounding the heart was not consistently observed in other lines bearing this construct and likely represent a mild enhancer-trap
phenotype. Scale bars represent 100mm.
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K.K. Kelly et al. / Mechanisms of Development 110 (2002) 39–50
Table 1
Mean b-galactosidase activity for lacZ reporter constructs a
Construct
Mean †
SEM
n
2 593/12
2 278/12
2 593/12 mutant MEF2
0.593 ng
0.144 ng*
0.079 ng*
0.247
0.034
0.182
3
3
3
a
SEM, standard error of mean. n, number of independent transgenic lines
tested. † Refers to ng of b-galactosidase per 10 mg total protein. * Differ
significantly from 2593/12 (P , 0:05) using Student’s t-test.
constructs 2278/12 and 2593/12 mutant MEF2. This
suggests that factors bound at the MEF2 site act synergistically with factors bound at the upstream region to activate
Act57B transcription.
2.6. Requirement of MEF2 for Act57B expression
The results described above demonstrate that a consensus
MEF2 binding site is required for normal activity of the
2593/12 fragment. To determine if MEF2 plays a direct
regulatory role in Act57B expression we first sought to
determine if MEF2 binds to the consensus sequence of the
promoter (Fig. 6A). In electrophoretic mobility shift assay
(EMSA), in vitro translated MEF2 protein bound to a double
stranded oligonucleotide containing the putative MEF2
binding site within the 2593/12 element (Fig. 6A, lane
2). MEF2 binding was specific since an unlabeled wild
type oligonuculeotide competed strongly for MEF2 binding
(Fig. 6A, lanes 3–4) and a mutant oligonucleotide was
unable to compete (Fig. 6A, lanes 5–6). Note that the mutant
sequence within this experiment contained the same mutation tested in transgenic flies (Fig. 5).
To determine if MEF2 was present in nuclei of embryos
and capable of binding to the Act57B promoter MEF2 binding site we performed an EMSA using embryonic nuclear
extract (Fig. 6B). The MEF2 consensus sequence was
bound specifically by nuclear protein (Fig. 6B, lanes 2–6).
To demonstrate the protein bound was in fact MEF2, an antiMEF2 antibody (Lilly et al., 1995) blocked formation of the
oligonucleotide/protein complex (Fig. 6B, lanes 7–8). These
results indicate that MEF2 was present in the embryonic
nuclear extract and was able to bind to the consensus
sequence in the Act57B promoter. In addition to the MEF2/
probe interaction, we saw binding activity with a faster electrophoretic mobility. This interaction was non-specific as
shown by equal competition with wild type (lanes 3–4) and
mutant (lanes 5–6) unlabeled oligonucleotides.
Knowing that MEF2 bound the consensus sequence of
Act57B promoter we sought to address the regulatory role
of MEF2 in vivo using in situ hybridization on Mef2 mutant
(Mef2 P544/Mef2 P544) embryos (Fig. 7). Comparing the
Act57B expression patterns in wild type (Fig. 7A–C) and
Mef2 mutants (Fig. 7D–F) emphasized the importance of
MEF2 regulation in embryogenesis. There was a reduction
in Act57B expression at stage 10 in the mutants (compare
Fig. 7A and Fig. 7D) with a small amount of discontinuous
staining in the visceral myoblasts. By stage 13, Act57B
expression was severely reduced in the skeletal myoblasts
of Mef2 mutants (compare Fig. 7B and Fig. 7E) and at stage
16 there was no staining in cardiac muscle of in situ hybridized Mef2 P544/Mef2 P544 mutant embryos (compare Fig. 7C
and Fig. 7F). Ranganayakulu et al. (1995) and Bour et al.
(1995) both documented cell death in the somatic muscle
lineage at later stages of development in Mef2 mutants,
stage 16 and stage 15 respectively. However, Lilly et al.
(1995) using nautilus, apterous, and S59 probes, and Bour
et al. (1995) using a b3-tubulin probe, demonstrated that
skeletal myoblasts are present in normal numbers as late
as stage 14. Therefore, the observed reduction in Act57B
transcription levels in skeletal myoblasts at stage 13 (Fig.
7B,E) is not artefactually reduced due to death of myoblasts,
but a result from an absence of MEF2 transcription factor to
maintain high levels of Act57B expression.
By generating a line of flies mutant for Mef2 (Mef2 P544/
Mef2 P544) and carrying the 21742/12-lacZ construct we
were also able to demonstrate a requirement for MEF2 in
controlling the expression of the Act57B promoter-lacZ
construct. b-galactosidase expression in the Mef2 mutants
Fig. 6. Electrophoretic mobility shift assays showing MEF2 protein bound
specifically to the consensus sequence within the Actin 57B promoter. (A)
In vitro translated MEF2 protein bound to the MEF2 site (complex b), is
competed with a wild type MEF2 oligonucleotide sequence (wtMEF2), and
is competed only weakly by a mutant MEF2 oligonucleotide sequence
(mMEF2), demonstrating specificity for MEF2 binding within the context
of the Act57B promoter. (B) Gel mobility shift using nuclear extracts
collected from embryos aged 12-20 h. In the absence of competitor, a
complex with three closely spaced bands of different MEF2 isoforms is
observed (b), wild type MEF2 oligonucleotide (wtMEF2) competes and
mutant MEF2 oligonucleotide (mMEF2) does not compete. Addition of
MEF2 antibody (aMEF2) diminished the intensity of the multi-band
bound complex and much of the signal was restricted to the wells (not
shown). Inhibition of complex formation by a MEF2 antibody demonstrates
the presence of MEF2 protein in the nuclear extract that specifically bound
to the consensus sequence. The additional band seen with high antibody
concentration is likely the result of a non-specific interaction (b, bound
probe complex consisting of three bands and f, free probe). Competitors
were at 50 £ and 300 £ of the labeled oligonucleotide.
K.K. Kelly et al. / Mechanisms of Development 110 (2002) 39–50
45
Fig. 7. MEF2 regulates Act57B expression in Drosophila embryogenesis. (A–F) In situ hybridization using a 3 0 UTR Act57B antisense probe to address the
regulatory role of MEF2 in Act57B muscle specific expression. (A–C) Wild type embryos, (D–F) Mef2 mutants (Mef2 P544/Mef2 P544). (G–L) Expression of
21742/12 promoter-lacZ construct in SM6b, eve-lacZ/Mef2 P544 embryos (G–I) and Mef2 P544/Mef2 P544 mutants (J–L), stained with anti-b-galactosidase
antibody. Note the stripes of b-galactosidase from the eve-lacZ reporter used to distinguish mutant and non-mutant embryos (arrowheads in G and H).
(A,D,G, J) Stage 10; in the MEF2 mutants, Act57B and lacZ reporter expression is reduced compared to wild type in the visceral muscle precursors (vm). At
stage 13 (B,E,H,K) there is little or no detectable Act57B expression in the skeletal myoblasts (sk) and the b-galactosidase antibody staining seen in (K) is
reduced compared to wild-type siblings (H). At stage 16 (C,F,I,L) expression in the heart (ht) is absent in MEF2 mutants for both Act57B and b-galactosidase.
Embryos are oriented with anterior to left and dorsal up. Scale bar represents 100mm.
was strongly reduced compared to 21742/12-lacZ in a
heterozygous background at all stages and in all muscle
lineages (compare Fig. 7G–I and Fig. 7J–L). Taken
together, these results provide strong evidence that MEF2
is essential for regulation of Actin 57B expression in all
muscle lineages during embryogenesis.
It is worth noting that the effects of mutating the MEF2
binding site within the promoter fragment (Fig. 5D) reduced
reporter gene expression, however the expression of a wildtype promoter construct in a Mef2 mutant background (Fig.
7J) was much more seriously reduced. Since there are no
additional MEF2 binding sites in the minimal promoter
fragment, the residual expression in Fig. 5D may be attributed to recruitment of MEF2 to the promoter by an additional cofactor in the absence of direct MEF2 binding.
Furthermore, if additional cofactors are themselves regulated by MEF2, in the Mef2 mutant background expression
of these cofactors would be reduced, in turn further reducing
Act57B reporter activity. This conclusion is supported by
our findings that sequences in addition to the MEF2 site
are important to Act57B transcription.
3. Discussion
Muscle differentiation occurs through the regulated
synthesis of structural proteins that carry out diverse
processes such as myofibril assembly and function,
myoblast fusion, muscle patterning, and interaction with
non-muscle components such as nerve and tendon cells. In
order to fully comprehend the events that occur during this
time, a detailed knowledge of the transcriptional regulatory
molecules that control the expression of these structural
genes is needed.
In this manuscript we have begun to dissect the regulatory
mechanisms controlling expression of a myofibrillar actin
gene, Act57B. Actin is one of the earliest structural genes
expressed in the developing musculature (Fernandes et al.,
1991), therefore regulators of actin expression also probably
regulate some of the key morphogenetic processes that
occur early in myogenesis. Act57B is the predominant
actin gene expressed in all muscle lineages in the embryo,
and is also expressed at larval and adult stages (Fyrberg et
al., 1983; Tobin et al., 1990). Through deletion analysis, we
have identified a single 595 bp element that completely
recapitulates the embryonic expression pattern. The transcriptional regulator MEF2 is expressed in a manner that
precedes and parallels that of Act57B (Bour et al., 1995;
Lilly et al., 1995), suggesting a role for MEF2 in regulation
of this actin gene. Contained within the 595 bp regulatory
element is a consensus MEF2 binding site, which we have
shown specifically binds MEF2 using gel shift assays. Mutation of this site in lacZ-reporter constructs severely
diminishes reporter activity. In addition, we observed a
severe reduction in the endogenous Act57B expression in
the Mef2 mutant background.
Taken together these findings demonstrate that MEF2 is a
direct and essential transcriptional regulator of Act57B
expression during embryogenesis. MEF2 functions over a
broad developmental time starting at stage 10 in the visceral
muscle precursors and in all muscles by the end of embryogenesis. Taylor (2000) described a putative nuclear
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K.K. Kelly et al. / Mechanisms of Development 110 (2002) 39–50
protein, Dmeso18E, whose expression in visceral and
somatic myoblasts is dependent upon MEF2 function at
stage 11–12. Along with a reduction in Dmeso18E expression, disruption of Act57B expression represents the earliest
phenotype observed to date in Mef2 mutants.
A number of MEF2 targets have been previously characterized, both in Drosophila and vertebrate systems (reviewed
in Black and Olson, 1998). These include the Drosophila
tropomyosin (TmI) gene, where expression in skeletal and
visceral muscle cells, but not the heart, is dependent upon
MEF2 (Lin et al., 1996). Paramyosin gene expression in
skeletal and visceral muscle depends critically upon a
conserved MEF2 site (Arredondo et al., 2001). Also, MEF2
regulates Drosophila b3 tubulin (bTub60D) and aPS2 integrin
expression through enhancers specific to early somatic and
visceral muscle respectively (Ranganayakulu et al., 1995;
Damm et al., 1998). Similar modular regulation is observed
in vertebrate muscle protein genes.
An underlying feature of these genes is that the enhancers
that have been characterized control a subset of the whole
expression pattern of the gene in question. Our characterization of Act57B regulation contrasts with these results by
demonstrating that MEF2 is required for high levels of
expression of Act57B in all muscle lineages, and furthermore that MEF2 controls this expression in all lineages via a
single site. To our knowledge, this is the only example of
MEF2 directing expression of a structural gene in all muscle
types through a single binding site. Furthermore, our deletion analyses did not identify any sequence elements in this
gene which contribute to expression in specific lineages
since the isolated a 595 bp element contains all of the regulatory sequences required for Act57B expression in skeletal,
visceral, and cardiac muscle lineages. Mutation or deletion
of this 595 bp element results in general effects upon expression in all lineages.
The results presented in this manuscript also indicate that
MEF2 function alone is not sufficient to induce Act57B
expression at high levels in muscle cells. LacZ reporter
constructs with the MEF2 site and flanking sequences, but
lacking region b (construct 2278/12), had reduced reporter
activity. In addition, ectopic expression of Mef2 under
control of a heat shock promoter did not induce expression
of Act57B (unpublished observations). This is consistent
with the findings of Lin et al. (1996) where the TmI MEF2
site alone was unable to direct expression of a lacZ reporter.
We also show that low levels of Act57B expression occur
in the absence of MEF2 function, either when the MEF2 site
is mutated in the minimal reporter construct (construct
2593/12 mMEF2) or in a Mef2 mutant background. The
simplest explanation for these results is that MEF2 functions
with additional factor(s) to regulate muscle gene expression
and that the presumptive cofactor is also restricted in its
activity to the musculature.
However, the identity of this putative cofactor is far from
clear. PDP1 has been shown to be a MEF2 cofactor in Drosophila, regulating TmI gene expression in the somatic meso-
derm (Lin et al., 1997b), however there are no PDP1 binding
sites contained within the minimal Act57B enhancer fragment. In vertebrate systems the myogenic bHLHs (MyoD,
myogenin, Myf5, MRF4) have been shown to regulate gene
expression with MEF2 through E-boxes (reviewed in Molkentin and Olson, 1996). Again, there are no E-boxes
contained within the minimal Act57B enhancer. Since the
upstream region required for high promoter activity contains
areas of strong sequence conservation between D. melanogaster and D. virilis, it is likely that these might represent
binding sites for the putative cofactors. It is unclear at this
time exactly what factors may bind to this region.
All of the data presented in this paper indicate that the
Act57B locus is under the positive control of MEF2 and
potential cofactors. Moreover, none of our deletion
constructs show inappropriate expression, suggesting the
absence of cis-acting negative regulatory sequence within
the enhancer. However, there is a time during early embryogenesis when MEF2 is present, but Act57B is not
expressed. This could simply be due to the absence of positively acting cofactors at this stage in development. Alternatively, this could be the result of negative regulators
interacting directly with factors bound to the Act57B promoter. Histone deacetylases have been shown to interact
directly with MEF2 (Miska et al., 1999; Lemercier et al.,
2000; Lu et al., 2000a) and this interaction has a potentially
important role in regulating vertebrate myogenesis (Lu et
al., 2000b; McKinsey et al., 2000). Although the Drosophila
genome sequence (Adams et al., 2000) has revealed several
potential open reading frames for histone deacetylases, it is
not known if these are expressed or functional in the developing Drosophila mesoderm. Future analysis of muscle
specific structural genes will help illuminate the network
of positive and negative regulatory factors which contribute
to the development of multiple physiologically distinct
muscle lineages.
4. Experimental procedures
4.1. Drosophila strains and culture
Drosophila stocks were raised at 258C on Drosophila Jazz
Food Mix (Applied Scientific Inc., South San Francisco,
CA) in plastic vials or half pint bottles.
To study Act57B expression patterning in Mef2 mutants,
we used in situ hybridization of Mef2 P544 embryos (Lilly et
al., 1995). The Mef2 P544 allele was maintained over an SM6b
balancer chromosome which contains an eve-lacZ reporter.
Homozygous Mef2 mutants were identified based on the
absence of lacZ staining using a lacZ antisense probe (see
in situ hybridization below), or by the abnormal gut phenotype of Mef2 null individuals at stage 16 (Bour et al., 1995;
Lilly et al., 1995).
To study Act57B-lacZ reporter activity in a Mef2 mutant
background, flies from the 21742/12 promoter-lacZ
K.K. Kelly et al. / Mechanisms of Development 110 (2002) 39–50
construct linked to the third chromosome were crossed into
an SM6b/Mef2 P544 background using standard genetic
crosses. This generated yw; SM6b,eve-lacZ/Mef2 P544; P[w 1
Act57B-lacZ]C6-25 which was stained with anti-b-galactosidase antibody both to distinguish the mutant and nonmutant offspring as well as to assay the activity of the
Act57B-lacZ reporter.
4.2. In situ hybridization
A pBR322 plasmid containing Act57B was obtained from
Dr Sarah Tobin (Stanford University, CA) and the entire
Act57B gene is contained with a single 5.4 kb EcoRI fragment of this plasmid. This fragment was subcloned into
pBluescript KS II (Stratagene, La Jolla, CA) and clones in
each orientation were isolated: E119–5 has the 3 0 end of the
gene closest to the M13 reverse sequencing primer, and
E119–22 has the 5 0 end of the gene closest to the M13 reverse
sequencing primer. To generate a piece of the 3 0 UTR
sequence for use as an in situ hybridization probe, a PCR
was performed upon E119-5 using a 1 strand primer immediately 5 0 to the translation stop site (Act57B 3 0 1 : 5 0 ATCGTCCACCGCAAGTGC-3 0 ), and M13 reverse sequencing primer. This generated a 1.4 kb product which was
ligated into pGEM-T Easy (Promega Corp., Madison, WI)
and clones with inserts in each orientation were isolated
(E125-1 and E125-2). For antisense probe generation,
E125-1 was linearized with AccI and probe was generated
with T7 RNA polymerase (Roche Molecular Biologicals,
Indianapolis, IN). For sense probe generation, E125-2 was
linearized with HindIII and probe was generated with T7
RNA polymerase. Digoxigenin-labeled probes were applied
to embryos aged 0–16 h according to O’Neill and Bier
(1994). Following hybridization, signal was detected using
Alkaline Phospatase BCIP/NBT substrate (Vector Laboratories, Burlingame, CA). Stained embryos were cleared and
mounted in 80% (v/v) glycerol for photography.
In order to assay the expression of Act57B in the Mef2
mutants (Mef2 P544/Mef2 P544) we hybridized the embryos
with the Act57B and a lacZ antisense probes. The lacZ
probe was generated by linearizing the E72-1 plasmid
(pBluescriptSK plasmid containing lacZ) with NdeI and
probe synthesis with T7 RNA polymerase was as described
above. MEF2 mutants were identified either by the lack of
the eve-lacZ expression from the SM6b balancer, or by the
gut morphology of stage 16 embryos.
4.3. Primer extension assay
Total RNA was extracted from wild type embryos aged
0–2 h (control) and 15–17 h at 258C using TRI reagent
according to the manufacturer’s instructions (Molecular
Research Center Inc., Cincinnati, OH). A primer extension
oligonucleotide (5 0 -ATCGTCACACATTTTGTTTTAG-3 0 )
was synthesized that was complementary to the Act57B
coding sequence. Total RNA (10–60 mg) and primer (100
pg) were coprecipitated and resuspended in TE (8 ml). To
47
the samples 5 £ PE buffer (2 ml) (Carey and Smale, 2000)
was added. The primer was annealed for 3 min in a 758C
water bath which was then allowed to cool slowly to 358C.
The dNTPs (1ml each of 5 mM dGTP, dATP, dTTP, and 2.5
ml 50 mM 32P dCTP), PowerScript reverse transcriptase (0.5
ml) (Clontech, PaloAlto, CA) and buffer (34 ml) (Carey and
Smale, 2000) were added to the reaction. The reactions were
then incubated for 1 h at 378C. After precipitation, the
products of the primer extension were resuspended in
Sequenase stop solution (4 ml) (USB Corp., Cleveland,
OH). DNA sequencing reactions were performed using the
same oligonucleotide primer and the T7 Sequenase version
2.0 DNA sequencing kit (USB Corp., Cleveland, OH).
Primer extension and sequencing reaction products were
separated on a denaturing polyacrylamide gel (Sambrook
et al., 1989). After electrophoresis at 2000 V/40 mA the
gel was fixed, dried for 1 h, and exposed to film overnight.
4.4. Generation of promoter-lacZ constructs
Genomic fragments for fusion to lacZ reporter constructs
were generate by PCR. The 1100/1831 intron construct
was generated using 57B Ex1 1 (5 0 -ATGAAGTTGCTGCTCTGG-3 0 ) and 57B E £ 2- (5 0 -ACCGGCCTTGCACATGCCGGAGC-3 0 ). The largest 22479/12 construct
was generated using M13 reverse primer and 57B Ex1PCR (5 0 -CTAAAGTATCGCCGCGTTGGT-3 0 ) using
E119-22 as a template. Each of these products were ligated
into pGEM-T Easy, and transferred from there to the Pelement vector CaSpeR-hs-AUG-bGal (CHAB; Thummel
and Pirrotta, 1992) using appropriate flanking restriction
sites. The 5 0 deletion constructs were generated using the
following primers. 21742/12: 57B 5 0 1 II (5 0 GGGAATTCTACTTAAATGTATTTTCCGG-3 0 ) and 57B
Ex1- PCR II (same as Ex1- PCR but incorporating an
XhoI site at the 5 0 end of the primer; 21484/12: 57B 5 0
1III
(5 0 -GGGAATTCCTCTGGCAACTCTGGAGG-3 0 )
and 57B Ex1- PCR II; 2978/12: 57B 5 0 1IV (5 0 GGGAATTCAAAGCCGGCATGTAACGG-3 0 ) and 57B
Ex1- PCR II; 2583/12: 57B 5 0 1V (5 0 -GGGAATTCCGTAACGAACCGACC-3 0 ) and 57B Ex1- PCR II; 2278/12:
57B 5 0 1VI (5 0 -GGGAATTCTGCCATCCCATATTGTGC3 0 ) and 57B Ex1- PCR II. The sequence in italics contains an
EcoRI site which was used for the subsequent cloning step.
These products were ligated into pGEM-T Easy and subsequently directionally cloned into CHAB using the engineered EcoRI and XhoI sites. Note that all of the
constructs tested were cloned into CHAB although all
upstream constructs contained the endogenous Act57B transcription start site we supplied each with an heterologous
hsp70 promoter in case the small amount of 5 0 leader from
Act57B was poorly functional or non-functional.
Mutation of the MEF2 site was carried out by PCR-site
directed mutagenesis (Horton, 1993). Oligonucleotides with
the MEF2 site mutated to a NotI site were used to generate
primary products from a genomic DNA template: 57B 5 0
48
K.K. Kelly et al. / Mechanisms of Development 110 (2002) 39–50
1V and 57B mm- (5 0 -CCGATCCGCCGCGGCCCCGATCCTTCAGCATAGG-3 0 ); 57B Ex1- PCR II and 57B
mm 1 (5 0 -CTGAAGGATCGCGGCCGCGGCGGATCGGCCGGTGG-3 0 ) (italicized sequence represents the engineered NotI site). Primary PCR products were gel purified
and used as templates in a secondary PCR to generate the
final Act57B enhancer with the MEF2 site mutated. This was
ligated into pGEM-T Easy and subcloned into CHAB as
described above. The presence of the mutation was
confirmed both by NotI digestion and sequencing.
CHAB constructs were injected into D. melanogaster
embryos following Bernstein and Cripps (2000); three to
five independent homozygous transgenic lines were generated for each construct.
collected from homozygous transgenic lines for 2 h on agar/
grape juice plates and aged for 14 h at 258C. Soluble
proteins were extracted from approximately 100 embryos
by homogenization in assay buffer (Ashburner, 1989), and
the suspension was spun briefly to clarify it. To control for
variation in protein extraction, protein yield was quantified
using Bradford reagent (5 ml extract, 70 ml water, 925 ml
Bradford reagent) (Bradford, 1976). We used b-galactosidase standards at 0.1 ng/ml, 0.5 ng/ml, 1 ng/ml, and 5 ng/ml,
to determine absolute soluble protein concentrations. Identical amounts of total embryo protein for each line (10 mg)
homogenization buffer (for a final volume of 1 ml) and b-dgalactopyranoside (10 ml of a 100 mM stock) were mixed
and incubated at 378C. Optical density readings were taken
at 0.5, 1, and 2 h at 574 nm.
4.5. Immunohistochemistry
Embryos were collected on agar/grape juice plates for 16
h at 258C. Immunohistochemistry of whole-mount embryos
was performed as described in Patel et al. (1987) for three to
five independent lines for each construct. The primary antibody was an anti-b-galactosidase monoclonal antibody
(1:400, Promega Life Science, Madison, WI) and localization was detected using Vectastain Elite Staining Kit
(Vector Laboratories, Burlingame, CA). Embryos depicting
average b-galactosidase expression for each of the enhancer-lacZ constructs were cleared either in 80% (v/v)
glycerol or in methyl salicylate (Patel, 1994) and mounted
for photography.
To depict accurately the differences in lacZ reporter
construct expression in Fig. 5, equivalent numbers of
embryos homozygous for each lacZ reporter construct
were processed in parallel with identical antibody incubation and staining reaction times. Photo documentation of
these embryos was performed using the same film exposure
times and any electronic brightness/contrast manipulations
were done on these embryo photos en masse.
4.6. Sequence analysis of D. virilis ActC2 upstream
sequence
The isolation of a pBluescript plasmid containing ActC2
was described elsewhere (Lovato et al., 2001). To determine
the sequence upstream of the translation start site, this plasmid was first sequenced using 5 0 -TCCGTTTCTCTATCCG3 0 which annealed in exon 1 at 1213 bp. Subsequent primers
were generated from the obtained sequence. This process was
repeated until we had upstream sequence to 2950 bp from
the transcription start site. To identify sequences similar
between D. melanogaster and D. virilis, sequence comparisons were performed using the lalign program (http://
www.ch.embnet.org/software/LALIGN_form.html).
4.7. b -Galactosidase assay
To directly compare b-galactosidase levels in transgenic
lines carrying our promoter-lacZ constructs, embryos were
4.8. Nuclear extract preparation and electrophoretic
mobility shift assay
For nuclear extracts, embryos were collected for 8 h and
aged 12 h on agar/grape juice plates at 258C. 500 mg of
embryos of ages 12–20 h were recovered and subsequently
subject to the nuclear extraction protocol of Lichter and
Storti (1991). This yielded 0.1 mg of extract.
For electrophoretic mobility shifts, the probe and unlabeled wild type competitor were 5 0 -AAGGATCTATTTTTAGGCGGAT-3 0 , with GG overhangs on the 5 0 ends. The
wild type probe was labeled by filling in the G overhangs
with 32P dCTP using Klenow enzyme (Roche Molecular
Biochemicals, Indianapolis, IN) and diluted to 50,000
cpm/ml. The mutant competitor was 5 0 -AAGGATCGCGGCCGCGGCGGAT-3 0 , again with 5 0 GG overhangs.
Wild type and mutant competitors were diluted to 50 £
and 300 £ concentrations of the 50,000 cpm probe. MEF2
lysate was generated using the plasmid pCDNA-DMEF2
provided by Dr Brian Black (University of California, San
Francisco, CA) and Promega TNT kit (Promega, Corp.,
Madison, WI) following the manufacturer’s protocol. Binding reactions consisted of polydI.dC (1 mg for reactions
containing the in vitro MEF2 translated protein and 2 mg
for reactions containing the nuclear extract), 5 £ buffer (2
ml), MEF2 lysate (3 ml) or nuclear extract (1 ml of 2 mg/ml
stock), and water to bring the final volume to 10 ml. Nonspecific binding was allowed to proceed for 15 min on ice.
Labeled probe and competitors were then added and incubated 15 min at room temperature. Entire reactions were
loaded onto a 3% non-denaturing polyacrylamide gel
which was run at 20–25 mA at 48C. The gel was then
dried for 1 h and exposed to film overnight.
Acknowledgements
We are grateful to Brian Black and Sarah Tobin who
supplied essential reagents for this work. We thank Diane
Marshall for the use of her microscope and Tyanna Lovato
and Nicolas Brainard for excellent technical assistance. This
K.K. Kelly et al. / Mechanisms of Development 110 (2002) 39–50
work was supported by a Beginning Grant-in-Aid from the
American Heart Association, Desert/Mountain Affiliate to
R.M.C., and a Predoctoral Research Fellowship from the
American Heart Association, Desert/Mountain Affiliate to
K.K.K.
References
Adams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., Gocayne, J.D.,
Amanatides, P.G., Schere, S.E., Li, P.W., Hoskins, R.A., Galle, R.F.,
et al., 2000. The genome sequence of Drosophila melanogaster.
Science 287, 2185–2195.
Arredondo, J.J., Ferrers, R.M., Maroto, M., Cripps, R.M., Maroc, R., Bernstein, S.I., Cervera, M., 2001. Control of Drosophila paramyosin/miniparamyosin gene expression. J. Biol. Chem. 276, 1–8.
Ashburner, M., 1989. Drosophila: A Laboratory Handbook and Manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Bernstein, S.I., Cripps, R.M., 2000. Generation of transgenic Drosophila
melaogaster by P-element-mediated germline transformation. In:
Norton, P.A., Steel, L.F. (Eds.). Gene Transfer Methods. Eaton Publishing, Natick, MA, pp. 93–125.
Black, B.L., Olson, E.N., 1998. Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Ann. Rev. Cell
Dev. Biol. 14, 197–230.
Black, B.L., Molkentin, J.D., Olson, E.N., 1998. Multiple roles for the
MyoD basic region in transmission of transcriptional activation signals
and interactions with MEF2. Mol. Cell Biol. 18, 69–77.
Bour, B.A., O’Brien, M.A., Lockwood, W.L., Goldstein, E.S., Bodmer, R.,
Taghert, P.H., Abmayr, S.M., Nguyen, H.T., 1995. Drosophila MEF2, a
transcription factor that is essential for myogenesis. Genes Dev. 118,
401–415.
Bradford, M., 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal. Biochem. 72, 248–254.
Carey, M., Smale, S.T., 2000. Transcriptional Regulation in Eukaryotes:
Concepts, Strategies, and Techniques. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY.
Cripps, R.M., Black, B.L., Zhao, B., Lien, C.-L., Olson, E.N., 1998. The
myogenic regulatory gene Mef2 is a direct target for transcriptional
activation by Twist during Drosophila myogenesis. Genes Dev. 12,
422–434.
Cripps, R.M., Zhao, B., Olson, E.N., 1999. Transcription of the myogenic
regulatory gene Mef2 in cardiac, somatic and visceral muscle cell
lineages is regulated by a Tinman-dependent core enhancer. Dev.
Biol. 214, 420–430.
Damm, D., Wolk, A., Buttgereit, D., Löher, K., Wagner, E., Lilly, B.,
Olson, E.N., Hasenpusch-Theil, K., Renkawitz-Pohl, R., 1998. Independent regulatory elements in the upstream region of the Drosophila b3
tubulin gene (bTub60D) guide expression in the dorsal vessel and the
somatic muscles. Dev. Biol. 199, 138–149.
Edmondson, D.G., Lyons, G.E., Martin, J.F., Olson, E.N., 1994. Mef2 gene
expression marks the cardiac and skeletal muscle lineages during mouse
embryogenesis. Development 120, 1251–1263.
Fernandes, J., Bate, M., Vijayraghavan, K., 1991. Development of the
indirect flight muscles of Drosophila. Development 113, 67–77.
Fyrberg, E.A., Bond, B.J., Hershey, N.D., Mixter, K.S., Davidson, N., 1981.
The actin genes of Drosophila: protein coding regions are highly
conserved but intron positions are not. Cell 24, 107–116.
Fyrberg, E.A., Mahaffey, J.W., Bond, B.J., Davidson, N., 1983. Transcripts
of the six Drosphila actin genes accumulate in a stage – and tissuespecific manner. Cell 33, 115–123.
Gajewski, K., Kim, Y., Lee, Y.M., Olson, E.N., Schulz, R.A., 1997. D-Mef2
is a target for tinman activation during Drosophila heart development.
EMBO J. 16, 515–522.
Gajewski, K., Kim, Y., Choi, C.Y., Schulz, R.A., 1998. Combinatorial
49
control of Drosophila Mef2 gene expression in cardiac and somatic
muscle cell lineages. Dev. Genes Evol. 208, 382–392.
Gardiner, W.P., 1997. Statistics for the Biosciences. Prentice Hall Europe,
Hemel Hempstead, Hertfordshire, UK.
Horton, R.M., 1993. In vitro recombination and mutagenesis of DNA. In:
White, B.A. (Ed.). PCR Protocols: Current Methods and Applications,
vol 15. Humana Press, Totowa, NJ.
Kaushal, S., Schneider, J.W., Nadal-Ginard, B., Mahdavi, V., 1994. Activation of the myogenic lineage by MEF2A, a factor that induces and cooperates with MyoD. Science 266, 1236–1240.
Keller, C.A., Erickson, M.S., Abmayr, S.M., 1997. Misexpression of nautilus induces myogenesis in cardioblasts and alters the pattern of somatic
muscle fibers. Dev. Biol. 181, 197–212.
Lemercier, C., Verdel, A., Galloo, B., Curtet, S., Brocard, M.-P., Khochbin,
S., 2000. mHDA1/HDAC5 histone deacetylase interacts with and
represses MEF2a transcriptional activity. J Biol. Chem. 275, 15594–
15599.
Lichter, J.B., Storti, R.V., 1991. In vitro transcription analysis of the Drosophila tropomyosin and other muscle genes. Biochem. Biophys. Acta
1088, 419–424.
Lilly, B., Galewsky, S., Firulli, A.B., Schluz, R.A., Olson, E.N., 1994. DMEF2: A MADS box transcription factor expressed in differentiatng
mesoderm and muscle cell lineages during Drosophila embyrogenesis.
Proc. Natl Acad. Sci. USA 91, 5662–5666.
Lilly, B., Zhao, B., Ranganayakulu, G., Paterson, B.M., Schulz, R.A.,
Olson, E.N., 1995. Requirement of MADS domain transcription factor
D-MEF2 for muscle formation in Drosophila. Science 267, 688–693.
Lin, M-H., Nguyen, H.T., Dybala, D., Stroti, R.V., 1996. Myocyte-specific
enhancer factor 2 acts co-operatively with a muscle activator region to
regulate Drosophila tropomyosin gene muscle expression. Proc. Natl
Acad. Sci. USA 93, 4623–4628.
Lin, Q., Schwarz, J., Bucana, C., Olson, E.N., 1997a. Control of mouse
cardiac morphogenesis and myogenesis by transcription factor MEF2C.
Science 276, 1404–1407.
Lin, Q., Lu, J., Yanagisawa, H., Webb, R., Lyons, G., Richardson, J.A.,
Olson, E.N., 1998. Requirement of the MADS-box transcription factor
MEF2C for vascular development. Development 125, 4565–4574.
Lin, S.-C., Lin, M.-H., Horvath, P., Reddy, K.L., Storti, R.V., 1997b. PDP1,
a novel Drosophila PAR domain bZIP transcription factor expressed in
developing mesoderm, endoderm and ectoderm, is a transcriptional
regulator of somatic muscle genes. Development 124, 4685–4696.
Loukas, M., Kafatos, F.D., 1986. The actin loci in the genus Drosophila:
establishment of chromosomal homologies among distantly related
species by in situ hybridization. Chromosoma 94, 297–308.
Lovato, T.L., Meadows, S.M., Baker, P.W., Sparrow, J.C., Cripps, R.M.,
2001. Characterization of muscle actin genes in Drosophila virilis
reveals significant molecular complexity in skeletal muscle types.
Insect Mol Biol 10, 333–340.
Lu, J., McKinsey, T.A., Nicol, R.L., Olson, E.N., 2000a. Signal-dependent
activation of the MEF2 transcription factor by dissociation from histone
deacetylases. Proc. Natl Acad. Sci. USA 97, 4070–4075.
Lu, J., McKinsey, T.A., Zhang, C.-L., Olson, E.N., 2000b. Regulation of
skeletal myogenesis by association of the MEF2 transcription factor
with class II histone deacetylases. Mol. Cell 6, 233–244.
McKinsey, T.A., Zhang, C.-L., Lu, J., Olson, E.N., 2000. Signal-dependent
nuclear export of a histone deacetylase regulates muscle differentiation.
Nature 408, 106–111.
Miska, E.A., Karlsson, C., Langley, E., Nielsen, S.J., Pines, J., Kouzarides,
T., 1999. HDAC4 deacetylase associates with and represses the MEF2
transcription factor. EMBO J. 18, 5099–5107.
Molkentin, J.D., Olson, E.N., 1996. Defining the regulatory networks for
muscle development. Curr. Opin. Genet. Dev. 6, 445–453.
Molkentin, J.D., Black, B.L., Martin, J.F., Olson, E.N., 1995. Cooperative
activation of muscle gene expression by MEF2 and Myogenic bHLH
proteins. Cell 83, 1125–1136.
Nguyen, H.T., Xu, X., 1998. Drosophila Mef2 expression during mesoderm
50
K.K. Kelly et al. / Mechanisms of Development 110 (2002) 39–50
development is controlled by a complex array of cis-acting regulatory
modules. Dev. Biol. 203, 550–556.
Nguyen, H.T., Bodmer, R., Abmayr, S.M., McDermott, J.C., Spoerel, N.A.,
1994. D-mef2: A Drosophila mesoderm-specific MADS box-containing
gene with a biphasic expression profile during embyrogenesis. Proc.
Natl Acad. Sci. USA 91, 7520–7524.
O’Neill, J.W., Bier, E., 1994. Double-label in situ hybridization using biotin
and digoxigenin-tagged RNA probes. BioTechniques 17, 870–875.
Patel, N., 1994. Imaging neuronal subsets and other cell types in wholemount Drosophila embryos and larvae using antibody probes. In:
Fyrberg, E.A., Goldstein, L.S.B. (Eds.). Methods in Cell Biology, vol
44. Academic Press Inc, Boston, MA.
Patel, N., Snow, P.M., Goodman, C.S., 1987. Characterization and cloning
of fasciclin III: A glycoprotein expressed on a subset of neuron and axon
pathways in Drosophila. Cell 48, 975–988.
Ranganayakulu, G., Zhao, B., Dokidis, A., Molkentin, J.D., Olson, E.N.,
Schulz, R.A., 1995. A series of mutations in the D-MEF2 transcription
factor reveal multiple functions in larval and adult myogenesis in
Drosophila. Dev. Biol. 171, 169–181.
Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
Schulz, R.A., Chromey, C., Lu, M.-F., Zhao, B., Olson, E.N., 1996. Expression of the D-MEF2 transcription factor in the Drosophila brain
suggests a role in neuronal cell differentiation. Oncogene 12, 1827–
1831.
Subramanian, S.V., Nadal-Ginard, B., 1996. Early expression of the different isoforms of the myocyte enhancer factor-2 (MEF2) protein in
myogenic as well as non-myogenic cell lineages during mouse embryogenesis. Mech. Dev. 57, 103–112.
Taylor, M.V., 2000. A novel Drosophila, Mef2-regulated muscle gene
isolated in a subtractive hybridization-based molecular screen using
small amounts of zygotic mutant RNA. Dev. Biol. 220, 37–52.
Taylor, M.V., Beatty, K.E., Hunter, H.K., Baylies, M.K., 1995. Drosophila
MEF2 is regulated by twist and is expressed in both the primordial and
differentiated cells of the embryonic somatic, visceral, and heart musculature. Mech. Dev. 50, 29–41.
Thummel, C.S., Pirrotta, V., 1992. New pCaSpeR P element vectors. Dros.
Inform. Serv. 71, 150.
Tobin, S.L., Cook, P.J., Burn, T.C., 1990. Transcripts of individual Drosophila actin genes are differentially distributed during embryogenesis.
Dev. Genet. 11, 15–26.