CF2 activity and enhancer integration are required for proper muscle gene expression in Drosophila

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CF2 activity and enhancer integration are required for proper muscle gene expression in Drosophila Elena Garcı´a-Zaragoza1, Jose´ Antonio Mas1, Jorge Vivar, Juan J. Arredondo*, Margarita Cervera* Instituto Investigaciones Biome´dicas ‘‘Alberto Sols’’, Departamento de Bioquı´mica, Facultad de Medicina, UAM-CSIC, Arzobispo Morcillo 4, 28029 Madrid, Spain

A R T I C L E I N F O

A B S T R A C T

Article history:

The creation of the contractile apparatus in muscle involves the co-activation of a group of

Received 24 January 2008

genes encoding muscle-specific proteins and the production of high levels of protein in a

Received in revised form

short period of time. We have studied the transcriptional control of six Drosophila muscle

10 March 2008

genes that have similar expression profiles and we have compared these mechanisms with

Accepted 14 March 2008

those employed to control the distinct expression profiles of other Drosophila genes. The

Available online 27 March 2008

regulatory elements controlling the transcription of co-expressed muscle genes share an Upstream Regulatory Element and an Intronic Regulatory Element. Moreover, similar clus-

Keywords:

ters of MEF2 and CF2 binding sites are present in these elements. Here, we demonstrate

CF2

that CF2 depletion alters the relative expression of thin and thick filament components.

Enhancers

We propose that the appropriate rapid gene expression responses during muscle formation

Gene expression

and the maintenance of each muscle type is guaranteed in Drosophila by equivalent dupli-

Muscle genes

cate enhancer-like elements. This mechanism may be exceptional and restricted to muscle

Phylogenetic footprinting

genes, reflecting the specific requirement to mediate rapid muscle responses. However, it may also be a more general mechanism to control the correct levels of gene expression during development in each cell type. Ó 2008 Elsevier Ireland Ltd. All rights reserved.

1.

Introduction

The formation of muscles during development occurs through the highly regulated synthesis of the sarcomeric proteins required for the correct assembly of myofibrils, which is necessary to ensure that adequate forces can be generated by each muscle. Thus, during muscle formation contractile proteins have to reach high levels of expression in a very short period of time. Accordingly, the activation of the majority of the genes encoding muscle-specific proteins must be highly coordinated for the proper assembly of the contractile apparatus. In parallel, the correct stoichiometry of distinct sets of muscle

isoforms must be established, both to maintain myofibril integrity and for each particular fibre to function correctly according to their specific contractile properties (i.e., rate of force generation, relaxation rate and fatigability of the fibres, (Bernstein et al., 1993; Buckingham et al., 1992; Buckingham, 1994). In thin filaments, the major structural components of the highly complex sarcomeres, the functional units of myofibrils, are Actin and Tropomyosin in combination with the members of the troponin complex (Troponin T, Troponin C and Troponin I), while Myosin and Paramyosin form the thick filaments. It is these contractile proteins that regulate muscle contraction and whose expression is co-activated during myogenesis.

* Corresponding authors. Tel.: +34 91 497 5402; fax: +34 91 585 4401. E-mail addresses: [email protected] (J.J. Arredondo), [email protected] (M. Cervera). 1 These authors contributed equally to this work. 0925-4773/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mod.2008.03.003

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In Drosophila, the contractile proteins are encoded by single-copy genes, except for Actin and Troponin C. Furthermore, apart from the paramyosin gene, they all have an initial untranslated exon followed by a relatively large intron. Regulatory elements controlling the spatio-temporal expression of the majority of these genes have already been described (Arredondo et al., 2001a; Hanke and Storti, 1988; Hess et al., 1989, 2007; Karlik and Fyrberg, 1986; Kelly et al., 2002; Marco-Ferreres et al., 2005; Marin et al., 2004; Mas et al., 2004). However, much remains to be learned about the mechanisms that co-ordinate muscle gene expression. Indeed, it remains unclear whether the capacity of muscle genes to achieve high levels of protein expression in very short time periods is due to a common basic regulatory mechanism of activation. Since flies and vertebrates mostly share evolutionary conserved molecular pathways controlling muscle formation, we have taken advantage of the simplicity of the Drosophila musculature to address this issue. Myogenesis is a multi-step process both in Drosophila and in vertebrates. Drosophila develops two sets of muscles during its life cycle, producing separate muscles in the embryonic/ larval stages and in the adult (Baylies et al., 1998; Bernstein et al., 1993). In contrast to mammals, there are very few specialized muscle types and each muscle type is composed of only one type of fibre. In the specification of each muscle type, the regulatory mechanisms that activate gene expression and transcription are critical (Sandmann et al., 2007, 2006). This elaborate process requires multiple enhancers to which specific multi-protein complexes bind and interact with the transcriptional machinery. These complexes dictate when and where a gene is transcribed, but they can also control the rate of transcription in order to ensure that the correct amount of protein is reached in a particular cell type. The properties of muscle-specific enhancers remain unclear because their detailed characterization in transgenic embryos is a laborious process and thus, few enhancers have been analysed in depth (Frith et al., 2001; Hess et al., 2007; Marco-Ferreres et al., 2005; Mas et al., 2004; Pennachhio and Rubin, 2001; Tanaka et al., 2008). However, it is known these enhancers interact with unique combinations of transcription factors in each cell type (Arbeitman et al., 2002; Arnone et al., 2004; Bagni et al., 2002; Black and Olson, 1998; Furlong, 2004; Sandmann et al., 2007, 2006; Tanaka et al., 2008; Taylor, 1998). The studies carried out to date have led to the definition of a robust model to explain how myogenic factors initiate and maintain the muscle phenotype. Nevertheless, the mechanisms that control the levels of expression in different myogenic lineages are still largely unknown. In recent years, a mechanism that regulates Drosophila Troponin T and Troponin I genes has been described (Marin et al., 2004; Mas et al., 2004). This mechanism involves the differential, yet concerted, interaction of two functionally identical modular enhancers, The Upstream Regulatory Element (URE) and the Intronic Regulatory Element (IRE) found in the 5 0 region and in the first intron, respectively, of both the TnT and TnI genes. In the case of TnT, we have demonstrated that these two elements collaborate in a synergistic manner, to ensure the full recapitulation of endogenous TnT gene expression. Moreover, these two elements independently activate TnT transcription in all muscle types and they are therefore

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functionally equivalent. However, the contribution of each regulatory element in achieving the correct TnT expression varies in each particular muscle. In order to fully understand how the correct protein levels are produced in distinct muscle types, we have focused our attention on the transcriptional control of a number of co-regulated Drosophila genes: myosin heavy chain (Mhc), paramyosin (PM), tropomyosin 1 (Tm1) and tropomyosin 2 (Tm2), troponin T (TnT) and troponin I (TnI). Our data show that regulatory elements controlling the transcription of all these genes share the same basic organization: an Upstream Regulatory Element (URE) and an Intronic Regulatory Element (IRE). We demonstrate that these two elements are functionally similar in each gene as both can independently control muscle gene expression in all Drosophila muscles. Furthermore, both elements must interact through multi-protein complexes to ensure that the correct amounts of protein accumulate in each particular fibre. Indeed, we identified similar clusters of MEF2 and CF2 transcription factor binding sites in these enhancers. Finally, we demonstrate that CF2 is involved in muscle gene expression regulating the stoichiometry of contractile proteins. Here, we propose that through the existence of two equivalent functional enhancers, the transcription of distinct muscle genes can be modulated, thereby adapting their expression to a cell’s specific needs.

2.

Results

2.1. Myosin and tropomyosin 1 and 2 gene expression is promoted by both an Intronic and an Upstream Regulatory Element Previous studies of the Drosophila Mhc and Tm1 and Tm2 genes identified regulatory sequences in intron 1 of each gene that govern their transcription (Hanke and Storti, 1988; Hess et al., 1989, 2007; Karlik and Fyrberg, 1986; Lin et al., 1996). The independent activity of these IREs as regulators of Mhc, Tm1 and Tm2 expression has been well documented in transgenic flies (Hanke and Storti, 1988; Hess et al., 1989, 2007; Karlik and Fyrberg, 1986; Lin et al., 1996; Marin et al., 2004; Mas et al., 2004). These studies demonstrated that a fragment of approximately 100 bp 5 0 to the transcription start site acts as the endogenous basal promoter. However, this minimal promoter alone cannot drive gene transcription and it requires the intronic elements. Moreover, it has also been shown that these elements have a modular organization. We have shown that the interaction of Upstream and Intronic Regulatory Elements is essential for the correct regulation of TnT expression (Mas et al., 2004). Therefore, to determine if other Drosophila muscle genes have the same organization as the TnT gene we have extended our analysis. To investigate whether additional regulatory elements were located in the 5 0 upstream regions of the Mhc and Tm1 and Tm2 genes, we carried out a more detailed functional and computational analysis (Konig et al., 2002). The alignment of sequences from D. melanogaster and D. pseudoobscura, revealed the striking conservation of the 2.3– 2.7 kb sequences upstream of exon 1 (Fig. 1A and Supplementary Fig. 1) in these three genes. Further analysis of these con-

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Fig. 1 – Myosin and tropomyosin 1 and 2 gene expression is governed by an Upstream Regulatory Element (URE). (A) Phylogenetically conserved domains within the 5 0 upstream regions of the D. melanogaster and D. pseudoobscura Mhc, Tm1, Tm2 and TnT genes. Grey squares represent conserved sequences containing stretches of 30–90 bp with more than 75% conservation. Conserved binding sites for the MEF2 and CF2 myogenic transcription factors are indicated by red and blue ovals, respectively. (B) LacZ staining of third instar larvae, visceral muscles, abdomens and sections of the thorax of 1-day-old adult flies transformed with the TnT URE, Mhc URE, Tm1 URE and Tm2 URE constructs. In the larvae, abdomens and thorax sections, anterior is up and posterior is down. The IFM are indicated by arrows, the TDT by asterisks, the abdominal hypodermic muscles are indicated by arrowheads and ‘‘dv’’ is the dorsal vessel.

served regions showed that they contain several putative binding sites for the myogenic transcription factors MEF2 and CF2 (Gogos et al., 1996; Taylor et al., 1995). These sites were contained within stretches of 30–80/90 bp that present a high degree of conservation between the two species (above 75%; grey boxes in the Fig. 1A and Supplementary Fig. 1). Based on these sequence comparisons and to avoid disturbing any functional interactions, we performed classical gene expression assays using fragments that contained the entire regions identified to direct the expression of LacZ reporter gene (2.7 kb for the Tm1 gene, 2.4 kb for the Tm2 gene and

2.3 kb for the Mhc gene: see Section 4). These constructs will be referred to as Tm1 URE, Tm2 URE and Mhc URE. Each of the three URE regions analysed acted as a very strong enhancer, driving high levels of expression in all major embryonic and larval muscle groups (Fig. 1B). Strong expression was also seen in all adult muscles, except in the specialized flight muscles (IFM) in which the Tm1 URE and Tm2 URE constructs only drove weak transgene expression (see arrow in Fig. 1B). Indeed, Tm1 URE activity was almost undetectable in the IFM. However, we were able to detect differences in the strong transgene expression driven

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by the Mhc URE in the TDT and IFM muscles (data not shown). These observations indicate that like the TnT and TnI genes (Fig. 1), there are two distinct enhancer elements that regulate Mhc, Tm1 and Tm2 gene expression, the IRE and URE. Furthermore, as previously described for the IRE elements, the URE elements appear to be capable of independently reproducing the spatio-temporal expression of Mhc, Tm1 and Tm2, with the exception of the expression observed in the IFM muscles.

2.2. Paramyosin gene expression in all muscle types is promoted by an Intronic Regulatory Element Through the use of different promoters and alternative splicing, the PM and mPM proteins are both produced from the PM/mPM gene. PM transcripts are generated through the upstream promoter while mPM expression is driven by an internal promoter located in intron 7 of the gene. The two overlapping transcription units in the PM/mPM gene are transcribed independently and thus, each of the two promoters regulates the expression of one transcript (Arredondo et al., 2001a). In earlier studies, we identified several regulatory regions in the upstream sequences of PM and mPM that govern their spatio-temporal expression (Arredondo et al., 2001a; Marco-Ferreres et al., 2005). However, when the upstream regulatory regions of this gene were used to direct reporter transcription, high levels of expression were detected in all muscles except for the flight muscles. In these flight muscles

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reporter expression was only rarely detected (Fig. 2B, (Arredondo et al., 2001a). To identify new regulatory elements in the intronic regions of the PM/mPM gene, we searched for clusters of myogenic transcription factor binding motifs using a regulatory sequence analysis tool (Konig et al., 2002). Interestingly, introns 1 and 2 contained several putative binding sites for myogenic factors such as MEF2 and CF2 (Fig. 2A). Moreover, as in TnT and in Tm1, Tm2 and Mhc, these sites were contained within larger conserved sequences in the PM gene (grey boxes in the Fig. 2A). Since it was previously shown that intron 1 does not drive reporter expression (Arredondo et al., 2001a), we studied a fragment containing exon 1, intron 1, exon 2 and intron 2 of PM/mPM. In terms of driving the expression of a LacZ reporter gene (construct PM IRE; see Section 4), this fragment acted as a very strong enhancer, directing high levels of expression in all major muscle groups of the embryo and larva, as well as in all adult muscles (Fig. 2B). Interestingly, this was the first time that regulatory elements from the PM/mPM gene have been shown to drive high levels of reporter expression in the IFM (Fig. 2B). Thus, two distinct enhancer elements appear to govern PM expression, an URE element located in the upstream region of the gene and an IRE element that covers introns 1 and 2. As for Tn T and I and Mhc, both elements were functionally equivalent in Tm1 and 2. Thus, the URE and IRE of PM were independently capable of reproducing the spatio-temporal expression of PM in a manner indistinguishable from the endogenous gene, with the exception of IFM expression.

Fig. 2 – An intronic enhancer element governs strong PM expression in all muscle types. (A) Phylogenetically conserved domains within the 5 0 upstream and intronic regions of the D. melanogaster and D. pseudoobscura PM genes are presented schematically. Grey squares represent conserved sequences containing stretches of 30–90 bp with more than 75% conservation. Conserved binding sites for the MEF2 and CF2 myogenic transcription factors are indicated with red and blue ovals, respectively. (B) LacZ staining of third instar larvae, visceral muscles and sections of the abdomens and thorax of 1day-old adult flies transformed with the PM IRE and PM URE constructs. In larvae and thoracic sections, anterior is to the left and posterior to the right. The IFM are indicated by arrows, the TDT by asterisks and the abdominal hypodermic muscles are indicated by arrowheads.

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2.3. The structure of the URE and IRE enhancer elements in co-expressed muscle genes reveals several conserved putative MEF2 and CF2 binding sites We examined the functionally equivalent URE and IRE sequences to determine whether they had common motifs or elements between muscle genes. The discovery of new regulatory regions directing gene expression can be efficiently performed by comparative sequence analysis of functionally equivalent regions coupled to large genomic sequence searches at different levels of resolution (Berman et al., 2002a, 2004; Marco-Ferreres et al., 2005; Marin et al., 2004; Mas et al., 2004). Previous sequence comparisons of the TnT and TnI genes revealed the presence of conserved myogenic motifs within the functional URE and IRE regions in both genes, such as MEF2, CF2, E-boxes, TINMAN and GATA binding sites (Marin et al., 2004; Mas et al., 2004). However, a more detailed study of these sites was guided by sequence alignments incorporating the D. pseudoobscura sequence (www.hgsc.bcm.tmc.edu/projects/drosophila). This analysis not only confirmed that MEF2 and CF2 binding sites formed clusters in both regions of the two genes, but also and most importantly, that those clusters were conserved in the two Drosophila species (Fig. 3). Additional clusters were also

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found in D. melanogaster or D. pseudoobscura genes but they were never conserved. In order to confirm the relevance of the MEF-2/CF2 clusters found in our TnT and TnI analysis, we extended our comparative searches to the muscle genes studied (Tm1, Tm2, Mhc and PM/mPM). Therefore, we searched for MEF2 and CF2 binding sites clustered in 700-bp fragments within large genomic sequences using the CIS-ANALYST tool (http://rana.1bl.gov/cisanalyst; Berman et al., 2002a, 2004). This search was restricted by defining the requirement of at least five indistinct MEF2 and/or CF2 sites (Fig. 3). This analysis revealed the presence of MEF2 and CF2 clusters in the upstream and downstream regions of the Tm1 and Tm2, Mhc and PM genes (Fig. 4A). Interestingly, the actin 87E and actin 57B genes (Fyrberg et al., 1983; Tobin et al., 1990) also contained clusters in a region near their transcription initiation site (Fig. 4B and data not shown). All genes in which clusters were found show the same pattern of expression during development, being expressed in embryonic/larval and adult muscles. Moreover, no clusters containing putative MEF2 and CF2 binding sites were found in the mPM or TnC 41C promoter regions, these being sarcomeric proteins that are exclusively expressed in adults (Fig. 4 and data not shown, (Herranz et al., 2004; Mar-

Fig. 3 – Cis-analyst searches revealed the presence of clusters of putative MEF2 and CF2 binding sites in the upstream and downstream regions of the TnT and TnI genes. The CIS-ANALYST bioinformatics tool (Berman et al., 2002b) was used to search for clusters of MEF2 and CF2 transcription factor binding sites in a radius of 25 kb around the TnT (up) and TnI (wup A) genes. The search parameters used are shown in the upper part of the figure. Blue and brown bars indicate MEF2 and CF2 binding sites, respectively. Boxes indicate conserved MEF2 CF2 clusters in URE and IRE regions.

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Fig. 4 – Search for clusters of MEF2 and CF2 binding sites in the vicinity of several muscle and non-muscle genes. CISANALYST bioinformatics tool was used to search for clusters of MEF2 and CF2 transcription factor binding sites in a radius of 20–30 kb around several Drosophila genes. (A) The search performed around PM/mPM, Tm1 and Tm2 and Mhc genes is shown. A red box indicates the lack of clusters in the vicinity of mPM promoter. (B) The data for yellow-e2 (eye) is presented as an example of the screening of a non-muscle gene. No clusters were found in the vicinity of the 5 0 end of this gene. Note that the nearest clusters in those genes are located close to the actin87E gene. Blue and brown bars indicate MEF2 and CF2 binding sites, respectively, while the boxes indicate the conserved MEF2 CF2 clusters in the URE and IRE regions.

co-Ferreres et al., 2005). Furthermore, these clusters were exclusive to genes encoding muscle proteins sharing the same expression pattern, and they were absent from other non-muscle genes. Accordingly, no MEF-2/CF2 clusters were found in the 5 0 region of 40 non-muscles genes analysed, mostly proneural genes (data not shown).

2.4. The CF2 transcription factor regulates the levels of coexpressed muscle gene expression during Drosophila development Enhancers that contain clusters with at least five putative MEF2 and CF2 binding sites appear to be widely conserved

among muscle genes. Hence, these enhancer regions are likely to be important for the co-ordinated regulation of this group of muscle genes. Drosophila MEF2 is expressed in the precursors of all muscle lineages early in development and its expression persists as the descendants of these cells differentiate. Indeed, this transcription factor is essential for the differentiation of skeletal, cardiac and visceral muscles (Bour et al., 1995; Cripps et al., 2004; Lilly et al., 1994; Junion et al., 2005; Sandmann et al., 2006). By contrast, the Drosophila CF2 zinc finger protein was first identified through its role in dorso-ventral patterning during oogenesis (Hsu et al., 1996, 1992). However, more recently this transcription factor has also been shown to be expressed in the developing muscles

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of the embryo where it first appears at stage 12, the time of skeletal myoblast fusion, as well as later on in all muscle lineages (Bagni et al., 2002). The expression of CF2 in skeletal, visceral and cardiac muscle cells resembles that of the DMEF2 protein, although the onset of the D-MEF2 is slightly earlier (Bour et al., 1995; Cripps et al., 2004; Lilly et al., 1994). However, the specific role of the CF2 transcription factor in regulating muscle gene expression in Drosophila remains unknown. To gain insight into the functional role of CF2 in muscles we have studied two different homozygous Drosophila CF2 mutants (see Fig. 5A and Section 4). The CF2 14778 and CF2 14439 mutants contain a P element insertion into intron 1 and exon 1 of the CF2 gene, respectively. In both mutants, the homozygous animals reach adulthood although there was a reduced CF2 protein in embryos, larvae, pupae and adults when compared with the yw controls (Fig. 5B). Slightly more protein appeared to accumulate in CF2 14778 mutants than in CF2 14439. Having shown that several putative CF2 binding sites are located in the regulatory elements controlling the expression of muscle genes, we assessed whether the reduction in CF2 levels affected the expression of these genes by Quantitative RT-PCR. At different developmental stages, we investigated the expression of the TnT and TnI genes as representative genes from thin filaments, and of the PM gene as a representative thick filament gene. In CF2 14778 embryos, the depletion of CF2 diminished the number of TnT and TnI transcripts and strongly increased the expression of PM relative to control yw embryos (Fig. 6A). In late pupae and adult flies, the imbalance between TnT and TnI relative to PM gene expression augmented. Thus, in late pupae TnT and TnI expression had fallen by approximately 25% whereas in the adults, a 60% decrease was observed. In contrast, the increase in PM gene expression ranged from 65% to 75%, becoming

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more pronounced in the adult musculature. Similar results were obtained for CF2 14439 mutants in late pupae and adults with but not in embryonic samples (Fig. 6B). In the last, TnT, TnI and PM transcripts decreased relative to control yw. This discrepancy is not easy to explain since the precise mechanisms involved in the regulation of these genes by CF2 are unknown. CF2 protein changes in the mutants may explain this effect. It may be possible that since the two mutants analysed express different levels of CF2 protein, the more severe depletion of CF2 protein in the 14439 mutants has a stronger effect on the Paramyosin promoter at embryonic stages. In summary, the decrease of CF2 protein disrupted the expression of thin and thick filament components, altering the stoichiometry of contractile proteins. In this context, changes in the levels of gene expression within any functional group may be critical, and will most probably affect myofibril assembly and correct function of each particular muscle.

2.5. CF2. transcription factor specialized flight and jump muscles

depletion

disrupts

We tested whether the decrease in CF2 expression impaired muscle function in larval and adult mutants. No impairment was observed in mutant larvae in either mutant as both CF2 14778 and CF2 14439 larvae were able to crawl in the same way as yw larvae. However, CF2 14439 flies were completely unable to fly (data not shown) and flight was severely impaired in CF2 14778 flies, indicating an important malfunction in the flight muscles (Fig. 7A). This effect was exacerbated with age in the CF2 14778 flies (Fig. 7B) and we also observed some jumping defects in both lines. To determine whether structural abnormalities were behind the defects in muscle function, we analysed the ultrastructure of the IFM and TDT muscles from mutant flies using

Fig. 5 – There is a clear reduction in CF2 protein during development in CF2 14778 and CF2 14439 mutants. (A) Schematic representation of the Drosophila CF2 gene showing the P element insertions that produced the CF2 mutants. (B) Western blot for CF2 of whole extracts from yw flies and homozygous CF2 mutants at different developmental stages. Loading control is shown in lower panel. Brackets indicate the CF2 isoforms.

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Fig. 6 – Stage-specific transcriptional changes of thin (TnT and TnI) and thick filament (PM) protein components in CF2 mutants. (A) Relative mRNA expression of TnT, TnI and PM in late embryos, late pupae and adults of Drosophila CF2 14778 mutants. (B) Relative mRNA expression of TnT, TnI and PM at the same stages of Drosophila development of CF2 14439 mutants. The horizontal black line (relative expression 1) indicates the levels of transcript expression in the corresponding wild-types.

transmission electron microscopy (Fig. 8). The ultra-structure of the IFM and TDT from wild-type flies is extremely regular. The myofibrils are circular in cross-section and exhibit regular sarcomeric units that are aligned in parallel between adjacent myofibrils in longitudinal section (Fig. 8A). No differences in the ultra-structure of the IFM and TDT muscles were observed in 1-day-old mutant flies when compared to the yw muscles and all the muscles analysed exhibited regular sarcomeric units (data not shown). However, the ultrastructure of the TDT muscles in 10-day-old CF2 14778 and CF2 14439 flies was severely disrupted. The TDT fibres were wavy and/or irregular, and the Z-discs were incompletely assembled or broken (arrows in Fig. 8B and C). Indeed, a large percentage of myofibrils displayed incomplete Z-discs and Mlines. This age-dependent muscle degeneration varied in penetrance and in aged flies, some muscles were only moderately disrupted whereas other muscles in the same fly were severely affected. No severe abnormalities were evident in the ultra-structure of IFM myofibrils from CF2 14778 and CF2 14439 flies (data not shown).

3.

Discussion

The results of the study presented here demonstrate that a large group of Drosophila muscle genes that present

the same expression profile during development share a basic regulatory organization. This occurs both in genes encoding thick filament components namely, Mhc and PM, and in the Tm1, Tm2, TnT and TnI genes encoding components of the thin filament. The elements involved are an Upstream Regulatory Element (URE) situated in the 5 0 upstream regions and an Intronic Regulatory Element (IRE) situated in introns 1 and 2 of each gene. Each of these regulatory elements, the URE and IRE, is sufficient to independently activate transcription in all muscle types and hence, we infer that they are functionally equivalent. Moreover, these regulatory elements are mostly conserved among Drosophilidae (Supplementary Fig. 2). However, in each particular muscle, LacZ reporter activity varies depending on whether the URE or the IRE element is directing transgene expression. Thus, PM expression is very high in the IFM where the IRE element governs reporter expression, whereas it is low when directed by the URE element (Arredondo et al., 2001a). In summary, either the URE or IRE elements independently govern transgene expression in all muscle types at all developmental stages. While the URE or IRE elements can dictate when and where these co-regulated genes are expressed during development, on their own they are unable to dictate the correct levels of protein expression in each muscle type.

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Fig. 7 – Flight is severely impaired in CF2 14778 mutant flies. Flight was evaluated by scoring the number of staged flies that were able to fly upwards, that were unable to fly or that glide when released in a cage. While 78% of 1-day-old yw control flies can fly upwards and this decreases to 54% in older flies, only 36% of CF2 14778 young flies can fly upwards, reaching 19% in aged flies. We recently demonstrated that the regulation of TnT transcription depends on the synergistic interaction between these two elements (Mas et al., 2004). Moreover, this synergism varies in each particular muscle type in order to produce the different specific levels of protein required. In conjunction with the data presented here and with that from similar studies performed on the TnI gene (Marin et al., 2004), we conclude that the correct levels of expression of co-activated muscle genes are established in each muscle type through a direct or indirect interaction between the URE and IRE elements. Thus, we consider that this is a general mechanism that co-ordinates the expression of co-regulated muscle genes, establishing the proper levels of protein required by each muscle type and mediating the rapid responses required for muscle formation. In mammals, duplicate enhancer-like elements capable of independently activating transcription in all muscle types

Fig. 8 – The ultra-structure of the TDT muscles of 10-day-old wild-type and CF2 14778 and CF2 14439 mutant flies. (A) Longitudinal sections of adult TDT from wild-type flies. (B and C) Show longitudinal sections of TDT from CF2 14778 and CF2 14439 mutant flies, respectively (Bar, 0.5 lm).

and of directing the correct spatio-temporal expression of the same gene have yet to be identified. However, a number of modules or partial regulatory elements, located either upstream (USE/SURE) or downstream (IRE/FIRE) of the transcription initiation site confer transcriptional specificity to slow (TnIs) and fast (TnIf) genes in mouse muscle C2C12 cells

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(Banerjee-Basu and Buonanno, 1993; Nakayama et al., 1996; Yutzey et al., 1989; Zhu et al., 1995). The importance of upstream and downstream elements in driving fibre specific expression of fast and slow TnI genes has been highlighted in studies of transgenic mice (Calvo et al., 2001; Hallauer and Hastings, 2002; Nakayama et al., 1996). As a result, additional unidentified enhancers were claimed to be necessary to recapitulate the quantitative endogenous expression of the TnI genes. In the context of our data, further studies will be necessary to determine if, as in Drosophila, mammals use duplicate regulatory elements to establish the proper levels of gene expression in each muscle type. Indeed, we have preliminary data suggesting that these mechanisms might be totally or partially conserved (Marco et al., unpublished results). The basic regulatory design described here appears to differ in muscle genes with a more restricted expression profiles. This is the case for mPM, which is almost exclusively expressed in adults (Maroto et al., 1996), as well as for actin 88F or flightin that are flight muscle-specific proteins (Nongthomba et al., 2001; Vigoreaux et al., 1993). Recent functional studies to identify regulatory elements that control mPM expression indicated that the structure and organization of such regulatory elements is completely different to that identified here. The three elements identified in mPM are smaller and none of them contain conserved putative MEF2 or CF2 binding sites (see red box in Fig. 4A) (Marco-Ferreres et al., 2005). The search for similar regulatory elements in actin 88F and flightin genes has been unsuccessful, possibly because these two genes are expressed in distinct muscle subsets than mPM. DNA microarray studies in Drosophila have offered us an overview of genomic activity during development, grouping together thousands of genes with similar expression profiles (Arbeitman et al., 2002; Blais et al., 2005). More recently this technology is being directed towards the understanding of the mechanisms driving simultaneous activation of each of these different groups of genes (Sandmann et al., 2007, 2006). The studies performed here on the structure of the different URE and IRE enhancer-like elements reveal that they share common organizational features. Thus, the systematic comparison of six Drosophila structural muscle genes and the large-genome scans identified clusters of MEF2–CF2 binding sites within 700-bp fragments. Moreover, these clusters were always located both upstream and downstream of the transcription initiation site of each of the genes analysed. Since these co-regulated genes share expression profiles, we focused our attention on other muscle genes that also share similar patterns of gene expression, such as actin 57B or actin 87E. Interestingly, we identified similar clusters of MEF2 and CF2 binding sites close to the transcription initiation sites of these two genes. In fact, it has been recently demonstrated that although both MEF2 and CF2 can individually activate actin gene expression, these two factors collaborate in regulating the actin 57B target gene in vitro and in vivo (Tanaka et al., 2008). However, no clusters were identified in transcriptional units with distinct developmental expression patterns, such as the mPM, actin 88F, flightin or TnC 41C genes, which are expressed almost exclusively in different subsets of the adult musculature. Furthermore, no

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MEF2–CF2 clusters were found in ‘non-relevant’ genes that are expressed in other organs or tissues, such as yellow-e2 that is expressed in the fly cuticle or proneural genes (Drapeau et al., 2001). These results demonstrate that the enhancer organization identified and the conserved clusters in whole-genome scans will be useful for guiding functional analyses. Indeed, this approach may serve to identify new functional components or members of co-expressed groups of genes, as well as grouping together genes with similar patterns of activity. A study was recently performed combining chromatin immunoprecipitation with microarray analysis to identify the in vivo ‘‘occupied’’ MEF2 binding sites present in the whole Drosophila genome (Sandmann et al., 2006). The authors identified 1015 overlapping genomic fragments that were grouped as 670 independent DNA fragments, containing 1975 predicted MEF2 binding sites. In order to test our hypothesis that MEF2–CF2 clusters play an important role in the transcriptional regulation of a subset of muscle genes, we decided to search for MEF2/CF2 clusters within these fragments. Significantly, we found that 15.30% of the MEF2 binding sites have at least one CF2 binding site less than 200 bp away, a frequency that is 9 times higher than it would be expected for a random distribution. Hence, the location of CF2 sites in the vicinity of MEF2 binding sites appears to be conserved in this group of muscle enhancer-like elements. This conservation strongly suggests that these two transcription factors act together to fulfil a central role in regulating muscle development. It has previously been demonstrated that MEF2 is essential for muscle differentiation in both Drosophila and vertebrates, directly activating structural muscle genes (Arredondo et al., 2001a; Kelly et al., 2002; Lin et al., 1996; Marin et al., 2004; Ranganayakulu et al., 1995). Recent studies demonstrated that MEF2 maintains its own expression in all differentiated muscle cell types, suggesting that it is required for muscle maintenance and growth during myogenesis (Arbeitman et al., 2002; Cripps et al., 2004). In vertebrate skeletal muscle, MEF2 cooperates with the myogenic basic helix-loop-helix (bHLH) transcription factors to activate and maintain the muscle phenotype (Black and Olson, 1998). Indeed, in Drosophila TWIST activates MEF2 expression and its expression declines during myogenesis (Baylies and Bate, 1996; Cripps et al., 1998). To date, no other factors are expressed in all muscles that might help maintain muscle-specific gene transcription and the muscle phenotype. Our functional and computational analysis suggests that CF2 is a good candidate to collaborate with complexes containing MEF2 in maintaining muscle-specific gene transcription. Since no null CF2 mutants are available, we conducted a detailed study of two hypomorph CF2 mutants. Q-RT-PCR assays, flight tests and EM microscopy confirmed that depletion of the CF2 protein disturbs muscle gene expression, particularly the relative expression of thick and thin filament components. At distinct stages of development, a reduction in the expression of thin filament genes was mostly paralleled by an increase of thick filament gene transcription in both CF2 mutants. This imbalance was more pronounced as the flies aged. In summary, a strong reduction of CF2 alters the correct expression of thin and thick filament components

MECHANISMS OF DEVELOPMENT

through an unknown mechanism, affecting the stoichiometry of contractile proteins and consequently, myofibril assembly. We conclude that CF2, together with MEF2 and other unknown factors, is an essential component of the macromolecular multi-protein complexes that bind to the URE and IRE enhancer-like elements. Supporting this hypothesis, a very weak interaction between MEF2 and CF2 proteins has been observed in GST-Pull Down assays (unpublished data). Within this complex MEF2 may maintain the muscle phenotype (Cripps et al., 2004) while CF2 guarantees the correct levels of expression of the genes encoding the protein components of thin and thick filaments. We have demonstrated the presence of functionally similar enhancer-like duplicates in Drosophila muscle genes. Direct or indirect interactions between these elements guarantee rapid and strong responses of structural muscle gene expression in muscle formation within each muscle type. We propose that this mechanism, or a similar one, might be a general mechanism to control the correct levels of gene expression during development of functionally related co-expressed genes in all cell types.

4.

Materials and methods

4.1.

Genetic nomenclature and Drosophila maintenance

Gene and chromosome symbols have been described previously (Lindsley and Zimm, 1992). All flies were grown on Carpenter’s medium at 25 °C (Carpenter, 1950).

4.2. Plasmid vectors, P-transformation and Drosophila transgenic lines The D. melanogaster genomic DNAs for Tm1 and 2, and Mhc were obtained from whole extracts of adult flies following standard protocols (Sambrook and Russell, 2001). The regulatory regions from the Tm1, Tm2 and Mhc genes were amplified by PCR using specific oligonucleotides including appropriate restriction sites to facilitate directed cloning into P-transformation vectors. Fragments containing 2.7 kb of the upstream regulatory sequence of the Tm1 gene, 2.4 kb of Tm2 and 2.3 kb of Mhc gene were subcloned and sequenced as described previously (Mas et al., 2004). All constructs were maintained in their native orientation relative to the basal promoters, including exon 1 and were denominated Mhc URE, Tm1 URE and Tm2 URE. The genomic clones containing exon 1, intron 1, exon 2 and intron 2 of the PM gene were subcloned and sequenced as described previously, and the construct was named PM IRE (Marco-Ferreres et al., 2005). All the fragments were cloned into the pH-Pelican P-transformation vector, except for the Mhc fragment that was cloned into p-Pelican (Barolo et al., 2000). These vectors contained flanking insulator sequences to prevent local enhancer effects on reporter expression. The constructs were all sequenced and one copy of each was inserted into the Drosophila lines analysed. The PM 4 transgenic line used here as a control was renamed PM URE (Arredondo et al., 2001a). The generation of germ line transgenic flies using P element-mediated transformation was essentially as described (Spradling and Rubin, 1982).

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4.3.

627

Embryo, larvae and adult staining

Standard procedures with some minor modifications were used to assay b-galactosidase enzyme activity in larvae and adults of transgenic line (Arredondo et al., 2001a; Sullivan et al., 2000). At least 2/3 different lines were analysed for each construct. Micro-dissected third instar larvae and adult cryostat sections were fixed and stained as previously described (Meredith and Storti, 1993). The activity of b-galactosidase was used as a means to compare the transcriptional efficiency between different constructs. In larval and adult muscles, a rough quantification of the expression levels between different constructs was achieved by visually monitoring the time required for the blue reaction product of the Xgal substrate/b-galactosidase reaction to appear. The complete expression patterns were visualized after 2 h staining in larvae, micro-dissected abdomen and visceral muscles of TnT URE, Tm1 URE and Tm2 URE transgenic lines and after 15 min in Mhc URE, PM IRE and PM URE transgenic lines. Thoracic sections were visualized after 45 min in TnT URE and PM IRE lines, after 16 h in Tm1 URE, Tm2 URE and PM URE lines and after 5 min in Mhc URE lines unless otherwise specified.

4.4.

Genomic analyses

Sequence comparison between Drosophila species and searches for transcription factor binding sites were performed with the Gene Jockey II program (Biosoft, Cambridge). The genomic D. pseudoobscura sequence database was accessed through the Drosophila Genome Project (http://www.hgsc. bcm.tmc.edu/projects/drosophila). The consensus sequences used to search for binding sites were YTAWWWWTAR for MEF2 (Taylor et al., 1995) and RTATATRTA for CF2 (Gogos et al., 1996). The genomic sequences around the Mhc, Tm1, Tm2, TnT and TnI and PM gene and the relative positions of the distinct genes were obtained from the GenBank at NCBI and FlyBase (Drysdale et al., 2005). The search for clusters of MEF2 and CF2 binding sites in genomic sequences was performed using the CIS-ANALYST program (Berman et al., 2002a, 2004), http://rana.lbl.gov/cis-analyst). Grouping of the 1015 overlapping genomic fragments containing in vivo occupied MEF2 binding sites (Sandmann et al., 2006) and the searching of putative MEF2 and CF2 binding sites within these fragments was performed using the Vector NTI program.

4.5.

Fly strains and crosses

The CF2 14439 and CF2 14778 mutants were obtained from the BDGP P element Screen/Gene Disruption Project Database (Bellen et al., 2004). Additional information can be obtained from http://flypush.imgen.bcm.tmc.edu/pscreen.

4.6.

Electron microscopy

Transmission electron microscopy was performed using 1and 10-day-old adult flies as described previously, with minor modifications (O’Donnell and Bernstein, 1988). Samples were infiltrated with Araldite Resin Embedding Kit (Taab) and thin sections (60 nm) were obtained using a Reichert Ultracut S

628

MECHANISMS OF DEVELOPMENT

Ultramicrotome (Leyca). The sections were double-stained with 2% uranyl acetate and lead citrate and they were viewed with a Jeol JEM 1010 transmission electron microscope at 100 kV. Photographs were taken with a Gatan BioScan digital camera.

4.7.

Quantitative RT-PCR assays

RNA was extracted from about 100 lg of embryos and 10 individuals of each genotype with Trizol reagent (Invitrogen, Carlsbad, CA), and cDNA was reverse transcribed from 2 lg of RNA using the High Capacity cDNA Archive Kit according to the manufacturer’s instructions (Applied Biosystems). As an internal control, the eukaryotic 18S rRNA gene was used. Exon-specific oligonucleotides and Taqman-MGB probes (Applied Biosystems) were designed for the three genes tested: TnT exons 6–7, TnI exons 7–8 and PM exons 4–5. PCR was performed using the reverse transcription product as templates with the TaqMan Universal PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. Amplifications were carried out in an ABI PRISM 700 Sequence Detection System (Applied Biosystems) using the following cycles: 2 min 50 °C, 10 min 95 °C, 40 cycles (15 s 95 °C, 1 min 60 °C). To calculate the relative index of gene expression we used the 2DDCt method (Livak and Schmittgen, 2001).

4.8.

Flight and jump testing

Flight-testing was performed on 1- and 10-day-old flies as described previously (Drummond et al., 1991). For each genotype, at least two hundred flies were tested for their ability to fly up towards a light source in several experiments. To probe the statistical significance of the experiment, a contingency test was performed using the graph Pad Prism V.4 program (GraphPad software, Inc., San Diego; USA). Assay of fly jumping ability was performed by placing the flies on a white surface and stimulated to jump by moving a fine paintbrush towards them from the rear. For each genotype, at least a hundred flies were tested for their ability to jump in several experiments.

Acknowledgements We are grateful to Dr. Hsu for the generous gifts of the antibodies against CF2. We thank Drs. A. Cano and M. Manzanares for their critical reading of the manuscript, Covadonga Aguado and Francisco R. Urbano for their expert technical EM assistance, and Vanesa Santos for her laboratory assistance. This research was supported by a grants BFU200405384 and BFU2007-061711 from the DGICYT (Spanish Ministry of Education, Culture and Sports). E. Garcı´a-Zaragoza was a pre-doctoral fellow of the Spanish Ministry of Education and J. Vivar is a pre-doctoral fellow at the Universidad Auto´noma de Madrid.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mod.2008.03.003.

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R E F E R E N C E S

Arbeitman, M.N., Furlong, E.E., Imam, F., Johnson, E., Null, B.H., Baker, B.S., Krasnow, M.A., Scott, M.P., Davis, R.W., White, K.P., 2002. Gene expression during the life cycle of Drosophila melanogaster. Science 297, 2270–2275. Arnone, M.I., Dmochowski, I.J., Gache, C., 2004. Using reporter genes to study cis-regulatory elements. Methods Cell Biol. 74, 621–652. Arredondo, J.J., Marco-Ferreres, R., Maroto, M., Cripps, R.M., Marco, R., Bernstein, S.I., Cervera, M., 2001a. Control of Drosophila paramyosin/miniparamyosin gene expression. Differential regulatory mechanisms for muscle-specific transcription. J. Biol. Chem. 276, 8278–8287. Bagni, C., Bray, S., Gogos, J.A., Kafatos, F.C., Hsu, T., 2002. The Drosophila zinc finger transcription factor CF2 is a myogenic marker downstream of MEF2 during muscle development. Mech. Dev. 117, 265–268. Banerjee-Basu, S., Buonanno, A., 1993. Cis-acting sequences of the rat troponin I slow gene confer tissue- and developmentspecific transcription in cultured muscle cells as well as fiber type specificity in transgenic mice. Mol. Cell Biol. 13, 7019– 7028. Barolo, S., Carver, L.A., Posakony, J.W., 2000. GFP and betagalactosidase transformation vectors for promoter/enhancer analysis in Drosophila. Biotechniques 29, 726. 728, 730, 732. Baylies, M.K., Bate, M., 1996. Twist: a myogenic switch in Drosophila. Science 272, 1481–1484. Baylies, M.K., Bate, M., Ruiz Gomez, M., 1998. Myogenesis: a view from Drosophila. Cell 93, 921–927. Bellen, H.J., Levis, R.W., Liao, G., He, Y., Carlson, J.W., Tsang, G., Evans-Holm, M., Hiesinger, P.R., Schulze, K.L., Rubin, G.M., Hoskins, R.A., Spradling, A.C., 2004. The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics 167, 761–781. Berman, B.P., Nibu, Y., Pfeiffer, B.D., Tomancak, P., Celniker, S.E., Levine, M., Rubin, G.M., Eisen, M.B., 2002a. Exploiting transcription factor binding site clustering to identify cisregulatory modules involved in pattern formation in the Drosophila genome. Proc. Natl. Acad. Sci. USA 99, 757–762. Berman, B.P., Nibu, Y., Pfeiffer, B.D., Tomancak, P., Celniker, S.E., Levine, M., Rubin, G.M., Eisen, M.B., 2002b. Exploiting transcription factor binding site clustering to identify cisregulatory modules involved in pattern formation in the Drosophila genome. Proc. Natl. Acad. Sci. USA 99, 757–762. Berman, P., Bertone, P., Dasgupta, B., Gerstein, M., Kao, M.Y., Snyder, M., 2004. Fast optimal genome tiling with applications to microarray design and homology search. J. Comput. Biol. 11, 766–785. Bernstein, S.I., O’Donnell, P.T., Cripps, R.M., 1993. Molecular genetic analysis of muscle development, structure, and function in Drosophila. Int. Rev. Cytol. 143, 63–152. Black, B.L., Olson, E.N., 1998. Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annu. Rev. Cell Dev. Biol. 14, 167–196. Blais, A., Tsikitis, M., Acosta-Alvear, D., Sharan, R., Kluger, Y., Dynlacht, B.D., 2005. An initial blueprint for myogenic differentiation. Genes Dev. 19, 553–569. 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. 9, 730–741. Buckingham, M., Houzelstein, D., Lyons, G., Ontell, M., Ott, M.O., Sassoon, D., 1992. Expression of muscle genes in the mouse embryo. Symp. Soc. Exp. Biol. 46, 203–217. Buckingham, M.E., 1994. Muscle: the regulation of myogenesis. Curr. Opin. Genet. Dev. 4, 745–751.

MECHANISMS OF DEVELOPMENT

Calvo, S., Vullhorst, D., Venepally, P., Cheng, J., Karavanova, I., Buonanno, A., 2001. Molecular dissection of DNA sequences and factors involved in slow muscle-specific transcription. Mol. Cell Biol. 21, 8490–8503. Carpenter, J.M., 1950. A new semisynthetic food medium for Drosophila. Drosophila Inform. Service 24, 96–97. Cripps, R.M., Black, B.L., Zhao, B., Lien, C.L., Schulz, R.A., 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., Lovato, T.L., Olson, E.N., 2004. Positive autoregulation of the myocyte enhancer factor-2 myogenic control gene during somatic muscle development in Drosophila. Dev. Biol. 267, 536–547. Drapeau, M.D., 2001. The family of yellow-related Drosophila melangaster proteins. Biochem. Biophys. Res. Comm. 281, 611–613. Drummond, D.R., Hennessey, E.S., Sparrow, J.C., 1991. Characterisation of missense mutations in the Act88F gene of Drosophila melanogaster. Mol. Gen. Genet. 226, 70–80. Drysdale, R.A., Crosby, M.A., Gelbart, W., Campbell, K., Emmert, D., Matthews, B., Russo, S., Schroeder, A., Smutniak, F., Zhang, P., Zhou, P., Zytkovicz, M., Ashburner, M., de Grey, A., Foulger, R., Millburn, G., Sutherland, D., Yamada, C., Kaufman, T., Matthews, K., DeAngelo, A., Cook, R.K., Gilbert, D., Goodman, J., Grumbling, G., Sheth, H., Strelets, V., Rubin, G., Gibson, M., Harris, N., Lewis, S., Misra, S., Shu, S.Q., 2005. FlyBase: genes and gene models. Nucleic Acids Res. 33 (Database Issue), D390–D395. Frith, M.C., Hansen, U., Weng, Z., 2001. Detection of cis-element clusters in higher eukaryotic DNA. Bioinformatics 17, 878–889. Furlong, E.E., 2004. Integrating transcriptional and signalling networks during muscle development. Curr. Opin. Genet. Dev. 14, 343–350. Fyrberg, E.A., Mahaffey, J.W., Bond, B.J., Davidson, N., 1983. Transcripts of the six Drosophila actin genes accumulate in a stage- and tissue-specific manner. Cell 33, 115–123. Gogos, J.A., Jin, J., Wan, H., Kokkinidis, M., Kafatos, F.C., 1996. Recognition of diverse sequences by class I zinc fingers: asymmetries and indirect effects on specificity in the interaction between CF2II and A + T-rich elements. Proc. Natl. Acad. Sci. USA 93, 2159–2164. Hallauer, P.L., Hastings, K.E., 2002. TnIfast IRE enhancer: multistep developmental regulation during skeletal muscle fiber type differentiation. Dev. Dyn. 224, 422–431. Hanke, P.D., Storti, R.V., 1988. The Drosophila melanogaster tropomyosin II gene produces multiple proteins by use of alternative tissue-specific promoters and alternative splicing. Mol. Cell Biol. 8, 3591–3602. Herranz, R., Diaz-Castillo, C., Nguyen, T.P., Lovato, T.L., Cripps, R.M., Marco, R., 2004. Expression patterns of the whole troponin C gene repertoire during Drosophila development. Gene Expr. Patterns 4, 183–190. Hess, N., Kronert, W.A., Bernstein, S.I., 1989. In: Kedes, L., Stockdale, F. (Eds.), Cellular and Molecular Biology of Muscle Development. A.R. Liss, New York, pp. 621–631. Hess, N.K., Singer, P.A., Trinh, K., Nikkhoy, M., Bernstein, S.I., 2007. Transcriptional regulation of the Drosophila melanogaster muscle myosin heavy-chain gene. Gene Expr. Patterns 7, 413– 422. Hsu, T., Bagni, C., Sutherland, J.D., Kafatos, F.C., 1996. The transcriptional factor CF2 is a mediator of EGF-R-activated dorsoventral patterning in Drosophila oogenesis. Genes Dev. 10, 1411–1421. Hsu, T., Gogos, J.A., Kirsh, S.A., Kafatos, F.C., 1992. Multiple zinc finger forms resulting from developmentally regulated alternative splicing of a transcription factor gene. Science 257, 1946–1950.

1 2 5 ( 2 0 0 8 ) 6 1 7 –6 3 0

629

Junion, G., Jagla, T., Dplant, S., Tapin, R., Da Ponte, J.P., Jagla, K., 2005. Mapping Dmef2-binding regulatory modules by using a ChIP-enriched in silico targets approach. Proc. Natl. Acad. Sci. USA 102, 18479–18484. Karlik, C.C., Fyrberg, E.A., 1986. Two Drosophila melanogaster tropomyosin genes: structural and functional aspects. Mol. Cell Biol. 6, 1965–1973. Kelly, K.K., Meadows, S.M., Cripps, R.M., 2002. Drosophila MEF2 is a direct regulator of actin57B transcription in cardiac, skeletal, and visceral muscle lineages. Mech. Dev. 110, 39–50. Konig, S., Burkman, J., Fitzgerald, J., Mitchell, M., Su, L., Stedman, H., 2002. Modular organization of phylogenetically conserved domains controlling developmental regulation of the human skeletal myosin heavy chain gene family. J. Biol. Chem. 277, 27593–27605. Lilly, B., Galewsky, S., Firulli, A.B., Schulz, R.A., Olson, E.N., 1994. D-MEF2: a MADS box transcription factor expressed in differentiating mesoderm and muscle cell lineages during Drosophila embryogenesis. Proc. Natl. Acad. Sci. USA 91, 5662– 5666. Lin, M.H., Nguyen, H.T., Dybala, C., Storti, R.V., 1996. Myocytespecific enhancer factor 2 acts cooperatively with a muscle activator region to regulate Drosophila tropomyosin gene muscle expression. Proc. Natl. Acad. Sci. USA 93, 4623–4628. Lindsley, D.l., Zimm, G., 1992. The Genome of Drosophila melanogaster. Academic Press, San Diego. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(Delta Delta C(T)) method. Methods 25, 402–408. Marco-Ferreres, R., Vivar, J., Arredondo, J.J., Portillo, F., Cervera, M., 2005. Co-operation between enhancers modulates the quantitative expression from the Drosophila paramyosin/ miniparamyosin gene in different muscle types. Mech. Dev. 122, 681–694. Marin, M.C., Rodriguez, J.R., Ferrus, A., 2004. Transcription of Drosophila troponin I gene is regulated by two conserved, functionally identical, synergistic elements. Mol. Biol. Cell 15, 1185–1196. Maroto, M., Arredondo, J., Goulding, D., Marco, R., Bullard, B., Cervera, M., 1996. Drosophila paramyosin/miniparamyosin gene products show a large diversity in quantity, localization, and isoform pattern: a possible role in muscle maturation and function. J. Cell Biol. 134, 81–92. Mas, J.A., Garcia-Zaragoza, E., Cervera, M., 2004. Two functionally identical modular enhancers in Drosophila troponin T gene establish the correct protein levels in different muscle types. Mol. Biol. Cell 15, 1931–1945. Meredith, J., Storti, R.V., 1993. Developmental regulation of the Drosophila tropomyosin II gene in different muscles is controlled by muscle-type-specific intron enhancer elements and distal and proximal promoter control elements. Dev. Biol. 159, 500–512. Nakayama, M., Stauffer, J., Cheng, J., Banerjee-Basu, S., Wawrousek, E., Buonanno, A., 1996. Common core sequences are found in skeletal muscle slow- and fast-fiber-type-specific regulatory elements. Mol. Cell Biol. 16, 2408–2417. Nongthomba, U., Pasalodos-Sanchez, S., Clark, S., Clayton, J.D., Sparrow, J.C., 2001. Expression and function of the Drosophila ACT88F actin isoform is not restricted to the indirect flight muscles. J. Muscle Res. Cell Motil. 22, 111– 119. O’Donnell, P.T., Bernstein, S.I., 1988. Molecular and ultrastructural defects in a Drosophila myosin heavy chain mutant: differential effects on muscle function produced by similar thick filament abnormalities. J. Cell Biol. 107, 2601–2612. Pennachhio, L.A., Rubin, E.M., 2001. Genomic estrategies to identify mammalian regulatory sequences. Nat. Rev. Genet. 2, 100–109.

630

MECHANISMS OF DEVELOPMENT

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., Russell, D.W., 2001. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory Press. Sandmann, T., Girardot, C., Brehme, M., Tongprasit, W., Stolc, V., Furlong, E.E., 2007. A core transcriptional network for early mesoderm development in Drosophila melanogaster. Genes Dev. 21, 436–449. Sandmann, T., Jensen, L., Jakobsen, J., Karzynski, M.M., Eichenlaub, M., Bork, P., Furlong, E., 2006. A temporal map of transcription factor activity: mef2 directly regulates target genes at all stages of muscle development. Dev. Cell 10, 797– 807. Spradling, A.C., Rubin, G.M., 1982. Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218, 341–347. Sullivan, W., Ashurner, M., Scott Hawley, R., 2000. Drosophila Protocols. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

1 2 5 ( 2 0 0 8 ) 6 1 7 –6 3 0

Tanaka, K.K., Bryantsev, A.L., Cripps, R.M., 2008. Myocyte enhancer factor 2 and chorion factor 2 collaborate in activation of the myogenic program in Drosophila. Mol. Cell Biol. 28, 1616–1629. Taylor, M.V., 1998. Muscle development: a transcriptional pathway in myogenesis. Curr. Biol. 8, R356–R358. 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 primordia and differentiated cells of the embryonic somatic, visceral and heart musculature. Mech. Dev. 50, 29–41. 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. Vigoreaux, J.O., Saide, J.D., Valgeirsdottir, K., Pardue, M.L., 1993. Flightin, a novel myofibrillar protein of Drosophila stretchactivated muscles. J. Cell Biol. 121, 587–598. Yutzey, K.E., Kline, R.L., Konieczny, S.F., 1989. An internal regulatory element controls troponin I gene expression. Mol. Cell Biol. 9, 1397–1405. Zhu, L., Lyons, G.E., Juhasz, O., Joya, J.E., Hardeman, E.C., Wade, R., 1995. Developmental regulation of troponin I isoform genes in striated muscles of transgenic mice. Dev. Biol. 169, 487–503.