wings up A: Biological Overview | References
Gene name - wings up A
Synonyms - Troponin I, Held-up
Cytological map position - 16F7-16F7
Function - cytoskeleton
Symbol - wupA
FlyBase ID: FBgn0004028
Genetic map position - X: 17,999,586..18,010,855 [-]
Classification - Troponin
Cellular location - cytoplasmic, nuclear
|Recent literature||Casas-Tinto, S. and Ferrus, A. (2019). Troponin-I localizes selected apico-basal cell polarity signals. J Cell Sci. PubMed ID: 30872455
Beyond its role in muscle contraction, Drosophila Troponin I (TnI) is expressed in epithelial cells where it controls proliferation. TnI traffics between nucleus and cytoplasm through a sumoylation-dependent mechanism. This study addresses the TnI role in the cytoplasm. TnI accumulates apically in epidermal cells and neuroblasts. TnI co-immunoprecipitates with Par-3/Bazooka and Disc large (Dlg), two apico-basal polarity components. By contrast, Scribbled is not altered by TnI depletion. In neuroblasts, TnI contributes to the polar localization of Miranda while non-polar Dlg is not affected. Vertebrate PI3K contributes to apico-basal polarity of epithelia but Drosophila PI3K depletion alters neither apical TnI or Par3/Bazooka, nor basal Dlg. Nevertheless, overexpressing PI3K prevents TnI depletion defects. TnI loss-of-function disrupts cytoskeletal beta-Catenin, E-Cadherin and gamma-Tubulin localization, along with gammaH2Av revealed DNA damage. The TnI-dependent apoptosis is suppressible upregulating Sparc or downregulating Dronc. Rescue from apoptosis by p35 does not prevent DNA damage demonstrating that both features are mechanistically independent. Thus, TnI binds certain apico-basal polarity signals in a cell type dependent context, and it unveils a hereto unsuspected diversity of mechanisms to allocate cell polarity factors.
|Weismann, C. G., Blice-Baum, A., Tong, T., Li, J., Huang, B. K., Jonas, S. M., Cammarato, A. and Choma, M. A. (2019). Multi-modal and multiscale imaging approaches reveal novel cardiovascular pathophysiology in Drosophila melanogaster. Biol Open 8(8). PubMed ID: 31455664
This study reports a comprehensive assessmentment of cardiovascular physiology in Drosophila. High-speed angiography, optical coherence tomography (OCT) and confocal microscopy were employed to reveal functional and structural abnormalities in the hdp(2) mutant, pre-pupal heart tube and aorta relative to controls. hdp(2) harbors a mutation in wupA, which encodes an ortholog of human troponin I (TNNI3). TNNI3 variants frequently engender cardiomyopathy. The hdp(2) aortic and cardiac muscle walls are disrupted and that shorter sarcomeres are associated with smaller, stiffer aortas, which consequently result in increased flow and pulse wave velocities. The mutant hearts also displayed diastolic and latent systolic dysfunction. It is concluded that hdp(2) pre-pupal hearts are exposed to increased afterload due to aortic hypoplasia. This may in turn contribute to diastolic and subtle systolic dysfunction via vascular-heart tube interaction, which describes the effect of the arterial loading system on cardiac function. This study demonstrates that complex and dynamic micro- and mesoscopic phenotypes can be mechanistically explained in a gene sequence- and molecular-specific manner.
|Nikonova, E., Mukherjee, A., Kamble, K., Barz, C., Nongthomba, U. and Spletter, M. L. (2022). Rbfox1 is required for myofibril development and maintaining fiber type-specific isoform expression in Drosophila muscles. Life Sci Alliance 5(4). PubMed ID: 34996845
Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. This study reports that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type-specific gene and splice isoform expression, notably loss of an indirect flight muscle-specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. It was further shown that Rbfox1 directly binds the 3'-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type-specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type-specific splicing and expression dynamics of identity genes and structural proteins.
The Troponin-Tropomyosin (Tn-Tm) complex regulates muscle contraction through a series of Ca(2+)-dependent conformational changes that control actin-myosin interactions (see video). Members of this complex in Drosophila include the actin-binding protein Troponin I (TnI), and two Tropomyosins (Tm1 and Tm2), which are thought to form heterodimers. Troponin itself contains three subunits, Ca2+ binding (TnC; TpnC47D and TpnC73F in Drosophila), inhibitory (TnI; Wings up A in Drosophila), and tropomyosin binding (TnT; Upheld in Drosophila).
Pre-cellular embryos of TnI, Tm1 and Tm2 mutants exhibit abnormal nuclear divisions with frequent loss of chromosome fragments. During cellularization, apico-basal polarity is also disrupted as revealed by the defective location of Discs large (Dlg) and its ligand Rapsynoid (Raps; also known as Partner of Inscuteable, Pins). In agreement with these phenotypes in early development, on the basis of RT-PCR assays of unfertilized eggs and germ line mosaics of TnI mutants, it was also shown that TnI is part of the maternal deposit during oogenesis. In cultures of the S2 cell line, native TnI is immunodetected within the nucleus and immunoprecipitated from nuclear extracts. SUMOylation at an identified site (see SUMO) is required for the nuclear translocation. These data illustrate, for the first time, a role for TnI in the nucleus and/or the cytoskeleton of non-muscle cells. It is proposed that the Tn-Tm complex plays a novel function as regulator of motor systems required to maintain nuclear integrity and apico-basal polarity during early Drosophila embryogenesis (Sahota, 2009).
Troponin I (TnI) and Tropomyosin (Tm) are actin-binding proteins that regulate muscle sarcomere contraction. The Tn-Tm complex contains three different Troponin polypeptides, C, T and I, and it regulates acto-myosin interactions in response to the rise of free calcium (Clark, 2002). Mammals have three genes expressing TnI known as slow twitch (TNNI1), fast twitch (TNNI2) and cardiac (TNNI3). In humans, mutations in TNNI2 and TNNI3 cause distal arthrogryposis type 2B (Sung, 2003) and familial hypertrophic cardiomyopathy (Kimura, 1997), respectively. In Drosophila, viable mutations in the single gene expressing TnI, wings up A (wupA) [also known as held up (hdp)], result in hypercontraction and degeneration of the indirect flight muscles of the thorax due to recessive hypomorphic point mutations (Prado, 1995). However, studies on lack of function mutations for this gene have been hampered by the fact that null alleles are dominant lethals (Prado, 1999). Mammals contain four tropomyosin genes, TPM1-4, while Drosophila has two, Tm1 and Tm2. In humans, mutant TPM1 is thought to be responsible for type 3 familial hypertrophic cardiomyopathy, whereas TPM2 is involved in nemaline myopathy and TPM3 has been linked to dominant nemaline myopathy. TPM1 has also been identified as a suppressor of malignant transformation as it is downregulated in mammalian transformed cells, and its expression is abolished in human breast tumors. Indeed, it is widely accepted that actin regulation plays a crucial role in cell motility, which is a key feature in metastatic cancers (Sahota, 2009).
Although some of these pathological phenotypes appear unrelated to muscle biology, several lines of evidence indicate that these muscle-specific proteins could have a role in other cell types and processes. For instance, Tm1 is part of the maternal deposit during Drosophila oogenesis, it is required to localize the oskar mRNA at the posterior pole of the oocyte (Erdelyi, 1995), and later in development it localizes to various cell types including the gut, brain and epidermis (Hales, 1994). Also, this study demonstrates that TnI RNA is detected in mature unfertilized eggs, which suggests a role in early embryogenesis. Thus, this study set out to analyze early development phenotypes and their mechanisms in TnI and Tm mutants (Sahota, 2009).
This study shows a novel function for the Tn-Tm complex in regulating nuclear divisions during early embryogenesis in Drosophila. Evidence is provided that TnI is required for maintaining stable chromosomal integrity, which was also show for Tm1 and Tm2. Importantly, the three genes seem required for correct epithelial apico-basal polarity; mutant phenotypes include cellularization defects that mislocalize the polarity markers Discs large (Dlg) and its ligand Rapsynoid (Raps) [also known as Partner of Inscuteable (Pins)]. Consistent with the function of these genes in cellularization and spindle integrity, defects in mitosis and chromosome segregation are observed. In a stable cell line, S2, TnI can be detected within the nucleus. Furthermore, the translocation of TnI to the nucleus is dependent upon a mechanism involving SUMOylation. Taken together, these data implicate the Tn-Tm complex in regulating nuclear functions. Moreover, the results suggest that the Tn-Tm complex is required to maintain correct segregation of chromosomes, as disruption of this complex leads to aberrations including chromosome fragment losses. This is the first evidence that the Tn-Tm complex can regulate both nuclear divisions and cell polarity in Drosophila. This is likely to have important implications in cancer progression since chromosomal instability and the generation of aneuploidies are characteristic hallmarks of many cancers (Sahota, 2009).
Embryonic lethal insertion lines located near the wupA locus called PL87 and PG31 are lacZ reporter and Gal4 lines, respectively. Both insertion sites were located upstream of the promoter region by means of plasmid rescue experiments (Marin, 2004). A third mutant, Df(1)23437, deletes 2 Kb of the promoter region and is also an embryonic lethal. Quantitative RT-PCR data had shown severely reduced levels of TnI RNA expression in these three mutants, with 23437 showing the most reduction, followed by PL87 and then PG31 (Marin, 2004). This study confirmed that their lethal mutant phenotypes were caused by the TnI gene, as opposed to another gene putatively affected by these chromosomal rearrangements. To this end transgenic lines were generated using the embryonic L9/wupRA isoform of wupA cDNA, under the control of upstream activating sequences (UAS). This isoform was sufficient to completely rescue the embryonic lethality of all three alleles when driven by the general Gal4 driver LL7 [inserted at the αtubulin84B (tub) gene]. Thus, the PL87 and PG31 alleles represent bona fide mutants for TnI, and the TnI L9/wupRA isoform encodes all functions required for correct embryogenesis. Rescued adults were fertile and able to fly, although 40% of pupae failed to emerge and showed a cryptocephalic phenotyp. This phenotype is consistent with the reported downregulation of the TnI gene during metamorphosis (Furlong, 2001). Given that there are adult isoforms of the TnI gene that contain an additional exon, it is unlikely that the L9/wupRA isoform is able to completely replace the functionality of the adult isoforms. This was confirmed by the failed attempt to rescue the wings held up phenotype of viable wupA alleles when driving the L9/wupRA isoform in the adult indirect flight muscles. Indeed, the wupA gene can produce a repertoire of cDNA isoforms, several of which are embryo-specific whereas others are adult-specific. Because embryonic TnI gene expression has been detected before the onset of myogenesis (Prado, 1999), the embryonic lethal mutants were used to look for defects during early embryogenesis. These data demonstrate that the embryonic lethality of the TnI mutants can be rescued to viability using the earliest expressed TnI isoform, and that the phenotypes associated with the three TnI alleles are due to the absence of the TnI gene products (Sahota, 2009).
The Tn-Tm complex has been well studied in the context of muscle contraction (Boussouf, 2007). This study shows that members of the complex also play an earlier role in development to maintain nuclear integrity. The nuclear defects observed in mutants for the three proteins TnI, Tm1 and Tm2 suggest that the whole Tn-Tm complex is required to maintain nuclear integrity. Embryonic lethal mutants are currently not available for the remaining components, mainly TnC and TnT (Sahota, 2009).
Several sarcomere proteins have been reported to play nuclear functions. Titin, a large protein spanning the sarcomere length between Z bands is required for chromosome integrity and control of gene expression through one of its kinase domains. However, the chromosomal effects are not likely to result from a direct binding of titin to chromosomes because a thorough search for proteins associated to metaphase chromosomes of HeLa cells failed to identify it. The issue, however, seems to be still controversial since a titin domain has been identified within the cell nucleus playing a role in proliferation. Zyxin, another actin-associated protein, acts as a tumor suppressor gene in Ewing tumor cells on the basis of its DNA-binding LIM domain and localizes to the nucleus to regulate gene transcription (Sahota, 2009).
This study has immunolocalized TnI to the nucleus and shown nuclear phenotypes in the mutants. It should be noted, however, that the nuclear localization, either in the syncitial embryo or the regular S2 cells, seems dependent on the physiological state of the cell and nucleus. Also, with the techniques used in this study, it cannot be determined whether TnI is bound directly to the chromosomes or through intervening proteins. Because the repertoire of HeLa metaphase chromosome-associated proteins does not include TnI, nor other muscle proteins, the observed effects on chromosome integrity might be produced through indirect links. Nevertheless, one should realize that the referred repertoire is also subject to the technical constrains of the purification methods used in the study of HeLa cells (Sahota, 2009).
This study has also shown that the required nuclear translocation is achieved by SUMOylation, at least in the case of TnI. The putative SUMOylation sequence in exon 10 is required for nuclear import. This site, VKEE, is found in the C-termini of all TnI isoforms because it can be incorporated into the protein sequence, either from exon 9 or exon 10. Thus, all TnI isoforms could be tagged for their function. Other putative SUMOylation sites, if actually used for SUMOylation, could provide further functional diversity for TnI. This mechanism for tagging TnI in Drosophila is likely to be conserved in mammals since the VKEE motif is present in the three TnI gene types (slow twitch, fast twitch and cardiac). Although not addressed in this study, it is possible that a similar mechanism might be used to import Tm1 and Tm2 into the nucleus since they contain suitable motifs in the three isoforms of Tm2 and in one of the two isoforms of Tm1 (Sahota, 2009).
This work on the Tn-Tm complex provides an insight into how DNA aberrations and cellularization defects can be linked, and how this complex is crucially required for both DNA and cellular stability. Given that the Tn-Tm complex is also involved in muscle contraction, it appears likely that there may be other processes where disruption of this complex may be detrimental to the development of the organism. In support of this, it has been shown that mutant TnI allele 23437 displays severe defects in axon guidance and fasciculation and that the TnI L9/wupRA isoform rescues these defects. Considering the role of the Tn-Tm complex in sarcomere contraction and the range of phenotypes described in this study, it seems reasonable to propose that TnI, Tm1 and Tm2 are components of a force-generating complex within the nucleus and in the cytoplasm. However, this remains to be determined since the TnI-associated partners have not being investigated in this study (Sahota, 2009).
Being an actin-binding protein, TnI should perform its nuclear functions in association with actin. This protein is known to help RNA polymerase to move during gene transcription (Ye, 2008). It is currently a matter of debate whether this function requires actin in a globular or a filament structure. However, a recent study reports the interaction of vertebrate fast skeletal TnI with the estrogen receptor during transcription (Li, 2008). By analogy to the role that TnI plays in the sarcomere, where the Tn-Tm complex interacts with the actin filaments, it seems likely that during transcription actin has a filament structure, as in the sarcomere thin filament. Actin is also important for morphogenesis of cells and organs in the early embryo, ranging from nuclear divisions and chromosomal segregation in conjunction with myosin, to the regulation of cell shape and movements. All these processes are also relevant to the formation and progression of tumors. In addition, chromosomal instability, mitotic defects and cell polarity defects are characteristic features of many cancers. The fact that TnI, Tm1 and Tm2 all regulate actin strengthens the argument that they execute this regulation as a complex. Defects in all three genes give rise to similar DNA defects, and also to similar defects in apico-basal cell polarity. These common features provide the basis for a mechanism leading to aneuploidy and aberrant cell signaling. That is, molecules that ensure proper actin function during nuclear divisions also ensure that actin correctly regulates cell polarity, which, in turn, is important in proliferation and growth. The tubulin spindle was also affected in the three mutants, indicating that the integrity of the cytoskeletal network may be compromised when any of these molecules are depleted (Sahota, 2009).
In addition to the cytoskeletal network, the localization of Dlg and Pins were also shown to be disrupted in TnI-Tm mutants. Dlg has been described as a neoplastic tumor suppressor and disruption of polarity is a hallmark of cancer progression. The Pins protein is involved in orientation of asymmetric cell divisions, which is important for specifying cell fate. Consistent with the altered Pins expression, spindle orientation defects are observed in the three mutants. Also, spindle orientation is particularly important for specifying neuronal identity in Drosophila neuroblasts. The recycling of molecules for distinct processes is a recurrent theme in development. Indeed, many actin-binding proteins were first identified for their effects on axon guidance and growth, and were subsequently shown to play important roles during cellularization. Also, Dlg was associated with synaptogenesis before its role in cellularization was determined. The novel function for the Tn-Tm complex uncovered in this study might have opened the way to reveal requirements in other actin-associated events. It was observed that TnI, as well as Tm1 and Tm2, are crucial for the correct development of the central nervous system. Further studies on the role of the Tn-Tm complex during nuclear divisions seem appropriate towards understanding how these proteins affect cell proliferation, and might provide novel targets for controlling cell divisions (Sahota, 2009).
The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, it was shown that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects (Marin, 2004).
This in vivo study reveals two regulatory regions, URE and IRE, located immediately upstream and downstream to the transcription initiation site and defined by a characteristic array of binding sites for the transcription factors Mef2, Biniou, and Tinman. The regions are qualitatively identical in their effects, but both are required for proper levels of transcription. Given the span of the genomic fragments tested for the reporter expression, it seems that the full set of positive regulatory elements has been identified. Putative repressor sites, however, remain to be identified. Finally, the transvection experiments suggest that, in addition to the cis-requirements, transcription is also dependent on trans-effects occurring probably at a small critical region close to the putative promoter (Marin, 2004).
The genomic fragments analyzed in LacZ reported transgenes allow identifying regions that contain positive regulatory elements that direct expression to specific tissues and developmental stages. These regulatory modules are revealed as overlapping stretches of DNA rather than separate and mutually exclusive units. For example, the modules for somatic and visceral muscles share ~1 kb of sequences. None of the smaller fragments tested that subdivide this 1 kb, however, could reproduce the original somatic or visceral expression patterns. The case has precedents in other genes such as mef2, tubulin, or tropomyosin 2, and illustrate the intimate relationship between specific sequences (i.e., enhancers or repressors) and the topology of the surrounding chromatin in the context of proper gene expression (Marin, 2004 and references therein).
The location of the two regulatory regions could sustain a particular chromatin structure, perhaps of a hair-pin type, for normal transcription. The spacing requirement and the linear order of interference effects shown by the three rearrangements support this speculation. In females, normal transcription requires correct pairing between both IRE + URE complements, and the spacing becomes less critical as long as it is the same in both chromosomes. The transvection effect that takes place when two homologous copies of the gene are present clearly implies enhanced transcription from the trans-homologue, as demonstrated by QRT-PCR assays in genotypes that allow to discriminate the chromosomal origin of some transcripts (Marin, 2004).
The initiation of transcription is clearly dependent on Mef2. Maintenance, however, does not seem to rely exclusively on this transcription factor. The difference between these two types of transcription has been recently documented, and the present case may indicate that it is a general phenomenon. Because the analyzed regions contain canonical binding sites for Tinman and Biniou, it seems puzzling why these factors are not able to drive gene expression in a mef2 null background. One possibility is that Mef2, in addition to its direct DNA binding activity on wupA and its role as a transcription factor, acts also as a trancriptional cofactor for Tinman and Biniou. Also, the role of other transcription factors such as the one encoded in Dmeso18E remains to be integrated into this scenario. Purification and analysis of the corresponding protein complexes would be required to test these speculations (Marin, 2004).
Concerning the Trithorax group of transcriptional cofactors assayed, the data demonstrate that zeste acts as a repressor for wupA. In addition, Su(z)3 is also a repressor and it behaves similarly to zeste with respect to transvection effects. Additional data on a third gene, Trithorax-like, which encodes the GAGA factor yielded similar effects. They represent cis- and trans-requirements for normal transcription. There seems to be, however, a critical domain near the promoter where the effects of these repressors become evident. Based on the immunity of PG31 heterozygotes to Trithorax group mutant backgrounds, this critical region could be defined by the-249 position as the upstream limit. In addition, the perfect transvection in PL87/PG31 heterozygotes suggest also a critical region of pairing for transcription. It is plausible that both critical regions are coincident. Presumably, pairing of these two rearrangements will be facilitated by the common sequences and size of the inserts. Their different site of insertion and the different transgenes, however, most likely will distort pairing to some extent. Thus, the critical region for transvection might be as small as 30 base pairs upstream of the initiation site. Extensive studies in the gene yellow have reached the same conclusion where the critical region for transvection seems to be the TATA box and an initiator element located in cis. The case of wupA, which does not contain TATA box, suggests that the critical region is the promoter per se, independently of its type. These observations should help to direct future in vitro studies with chromatin fragments (Marin, 2004).
It may seem counterintuitive the observation that z and Su(z)3 mutant backgrounds result in an increase of transcription at wupA, whereas the phenotypic effect shows a loss of transvection. Because the transcriptional change has been consistently observed in all genotypes assayed, including the flies sorted by wing position, it is evident that the phenotype does not correlate with the absolute levels of TnI transcripts. Furthermore, in spite of the very low levels of transcription in 23437/hdp3 females, they are viable and muscles are functional, except those involved in flight. The most plausible interpretation of these observations is that the deleterious effect on muscle structure results from changes in the stoichiometry of certain TnI isoforms rather than in their absolute levels. It is likely that, as in TnI isoform replacement experiments with the vertebrate homologues (Metzger, 2003), the unbalance of certain TnI isoforms lead to unsuitable thin filaments in vivo. If this is also the case in humans, certain muscular diseases, particularly those revealed under intense exercise, may result from mutations in regulatory regions, and thus may have escaped detection under standard sequencing procedures. In this context, the conserved gene regulatory array should be useful to guide future mutant screenings in humans (Marin, 2004).
Z discs are thought to be the anchoring points where thin filaments exert force and contract the sarcomere during muscle activity. It is somewhat unexpected that reduced levels or structural modifications of TnI can result in defective Z discs. The aspect and spacing of these Z disk-like structures suggest that a Z disk results from the lining of independent substructures deposited on the thin filaments and latter organized in register. It is worth noting, however, that a thin filament does not necessarily anchor at a Z disk. The quasi-normal sarcomeres from double mutants in troponin I, hdp2, and myosin or tropomyosin, Su(hdp2), show frequent cases of thin filaments extending more than one sarcomere in length. All these abnormal features of Z discs observed in mutants that involve TnI may indicate additional developmental functions of this protein beyond the well known regulatory role in sarcomere mechanics. Alternatively, these features may result from depletion of bona fide Z disk components whose expression is downregulated because of TnI mutations. This possibility would require a coordinated regulation of gene expression among thin filament components (Marin, 2004).
Vertebrate TnI encoding genes are not yet amenable to the in vivo analysis that Drosophila allows. Nevertheless, previous studies on the quail fast and slow TnI genes show functional evidences of a very similar regulatory structure to that described here for wupA (Yutzey, 1989; Banerjee-Basu, 1993; Nakayama, 1996). Equivalent regions to IRE and URE can be revealed by sequence analysis in other TnI members with the exception of the cardiac gene. The array of regulatory regions and their characteristic features seem fairly well conserved in TnI encoding genes of insects and vertebrates. This observation will be relevant toward the design of tools that aim to mimic the native gene expression in otherwise pathological conditions. Beyond this utilitarian use, the conservation of the TnI regulatory landscape in other genes that encode thin filament components might be indicative of common trends that would ensure proper quantitative expression of these components and, eventually, could help to translate gene regulation into physiology (Marin, 2004).
The control of muscle-specific expression is one of the principal mechanisms by which diversity is generated among muscle types. In an attempt to elucidate the regulatory mechanisms that control fiber diversity in any given muscle, this study focused attention on the transcriptional regulation of the Drosophila Troponin T gene. Two, nonredundant, functionally identical, enhancer-like elements activate Troponin T transcription independently in all major muscles of the embryo and larvae as well as in adult somatic and visceral muscles. It is proposed that the differential but concerted interaction of these two elements underlies the mechanism by which a particular muscle-type establish the correct levels of Troponin T expression, adapting these levels to their specific needs. This mechanism is not exclusive to the Troponin T gene, but is also relevant to the muscle-specific Troponin I gene. In conjunction with in vivo transgenic studies, an in silico analysis of the Troponin T enhancer-like sequences revealed that both these elements are organized in a modular manner. Extending this analysis to the Troponin I and Tropomyosin regulatory elements, the two other components of the muscle-regulatory complex, a similar modular organization of phylogenetically conserved domains was discovered (Mas, 2004; Full text of article).
Mutations in calcineurin B2 gene cause the collapse of indirect flight muscles during mid stages of pupal development. Examination of cell fate-specific markers indicates that unlike mutations in genes such as vestigial, calcineurin B2 does not cause a shift in cell fate from indirect flight muscle (IFM) to direct flight muscle (DFM). Genetic and molecular analyses indicate a severe reduction of myosin heavy chain gene expression in calcineurin B2 mutants, which accounts at least in part for the muscle collapse. Myofibrils in calcineurin B2 mutants display a variety of phenotypes, ranging from normal to a lack of sarcomeric structure. Calcineurin B2 also plays a role in the transition to an adult-specific isoform of troponin I during the late pupal stages, although the incompleteness of this transition in calcineurin B2 mutants does not contribute to the phenotype of muscle collapse. Together, these findings suggest a molecular basis for the indirect flight muscle hypercontractility phenotype observed in flies mutant for Drosophila calcineurin B2 (Gajewski, 2005).
This report further characterizes the IFM collapse phenotype of the canB2[EP(2)0774] mutation. Studies of mutations in other loci that produce IFM collapse revealed two major causes for the phenotype: change of cell fate in the adepithelial cells of the 3rd Instar lava, or hypercontraction of the IFM muscle fibers. In mutants that cause a change in cell fate, such as vg[null], a change in muscle cell fate can be clearly demonstrated by the loss of IFM-specific markers, and the ectopic expression of DFM-specific markers. No such changes are observed in canB2 mutant IFM. Unlike vg[null] mutants, the 88Factin-GFP reporter is expressed strongly in canB2 mutants, even after collapse of the IFM. A DFM-specific marker, gD1142.1-lacZ, expressed in a subset of DFM, also showed no alteration of expression pattern in canB2 mutants. The expression of these reporters in the expected places indicates proper fate determination for the precursor cells that form the DFM and IFM (Gajewski, 2005).
Disruption of the myofibrillar structure by mutation of the fli I locus, encoding a member of the gelsolin protein family, involved in the capping, severing, and bundling of actin filaments, partially suppresses the IFM collapse phenotype, pointing to hypercontraction rather than a shift in cell fate as a cause. Addition of two doses of the fli I allele to a canB2 mutant background significantly increases the numbers of uncollapsed DLM. However, the suppression is not complete, and this may be due to the relatively mild effect of the fli I allele. Null alleles of fli I cause lethality in the early embryonic stages. The fli I allele is a less severe mutation, caused by a change of a highly conserved glycine to serine. It is possible that even with the disruptions of the sarcomeric structure, fli I does not completely inhibit IFM contraction (Gajewski, 2005).
A reduction of calcineurin function has a profound effect on the expression of the mhc gene in the IFM. One copy of canB2[EP(2)0774] enhances the severity of IFM defects in flies heterozygous for the antimorphic Mhc allele. mhc transcripts are barely detectable in the IFM of canB2 mutant flies, and many of the mutant myofibrils have greatly reduced or completely absent thick filaments. However, there is no interference with the tissue-specific splicing of the five versions of Mhc exon 11. The reduction of mhc expression is not due to a nonspecific reduction in transcription; the levels of GFP transcript from a reporter driven by mhc upstream sequences (mhc-GFP) are also reduced in a mutant background, but expression of actin88F is unaffected. The simplest explanation is that transcription of mhc is greatly reduced in the absence/reduction of calcineurin function, but further studies will be needed to confirm it (Gajewski, 2005).
The function of calcineurin in transcriptional activation is well documented, for example, its role in regulating transcription factors such as NFAT and Mef2. There are multiple Dmef2 binding sites upstream of the mhc gene, as well as a binding site for the zinc-finger transcription factor CF2. Work in other systems has established that calcineurin can activate Mef2 both directly and indirectly; it is likely that this will also hold true for Drosophila. Whether calcineurin can affect CF2 activity is not yet known, but the phosphorylation state of CF2 has been demonstrated to play a role in its regulation via the EGFR pathway in Drosophila ovaries. Phosphorylated CF2 is found predominantly in the cytoplasm of the anterodorsal follicle cells, where it is fated to be degraded. It is speculated that removal of the phosphate group allows entry in the nucleus CF2 is expressed in all three muscle types of the Drosophila embryo (Bagni, 2002), but it is not yet known if this protein is required for IFM development. It will be of interest to investigate whether CF2 is expressed in the developing IFM, and what effects calcineurin function (or lack thereof) would possibly have on its subcellular localization (Gajewski, 2005).
It is also interesting to note that the lack/reduction of calcineurin has a much more drastic effect on mhc transcript levels in the IFM than it does on the various muscle types of the abdomen. The amount of total mhc transcripts are readily detectable in the mutant abdominal musculature, but not in the mutant IFM under the same PCR conditions. The transcript is not completely missing in the mutant IFM; if extra PCR cycles are done, or extra fly equivalents of cDNA are added for the mutants, a mhc band can be amplified. The reason for greater IFM sensitivity to lack of calcineurin function is unknown, and warrants further investigation. It may be that calcineurin is part of a system to promote maximum expression of mhc. The IFM are the largest muscles in the fly, and their tightly packed hexagonal arrangement of thick and thin filaments (unique in the fly musculature) could require increased expression of myosin and other structural proteins. There are numerous examples of mutations in muscle structural protein genes that result in a flightless phenotype, but do not impair the functions of other types of muscles (Gajewski, 2005).
The myofilament structure of the canB2 mutants reflects the reduction in mhc transcripts. While about 20% of the adult mutant IFM tissue examined resembled wild type, the majority of samples exhibited some degree of defect. Some myofibrils had patches of organized filament structure at the periphery, but have no recognizable structures in focus at the center region. This is likely the result of hypercontraction, which can lead to random myofilament orientation. In the most severely affected myofibrils, no organized structures of any sort could be detected. Examination of longitudinal sections confirmed this range of phenotypes. Some samples resembled the wild type sarcomeric pattern. Mildly affected mutant tissue had broken Z-bands, partial or missing M-lines (indicative of reduced or missing thick filaments), and shorter sarcomeres (indicative of hypercontraction). The most severely affected mutant muscles lacked any Z-bands or M-lines. The mutant pupal samples tended to display the most severe myofibrillar phenotypes. It is likely that using adults for examination selects against the most severe phenotypes; the animals examined in the pupal stage are likely to represent those that would not have successfully eclosed, and a small sample of canB2 mutant pupae could easily display a propensity for the strongest defects. The canB2[EP(2)0774] mutation is semi-lethal; life cycle analysis reveals that many of the animals that die do so in the pupal stages. It may be that the most severe canB2 phenotypes render the animals unable to eclose, although the role, if any, of the IFM is this process is yet to be confirmed. It is also possible that the most severe canB2 phenotypes could impair other muscles (Gajewski, 2005).
The effect of the canB2 mutation on Tn I expression represents a possible novel role for calcineurin, that being in different isoform formation. Although no direct role for calcineurin in the control of RNA splicing has yet been demonstrated, it is interesting to note that phosphorylation status of SR proteins plays a role in their localization within the nucleus, and assembly, disassembly, and activity of the spliceosome may by influenced by a cycle of protein phosphorylation. The degree of phosphorylation is believed to effect protein-protein and protein-RNA interactions in the spliceosomal complexes. The splicing of at least one variant exon of the mouse CD44 gene is coupled to signal transduction via the protein kinase C/ras signaling pathway. Therefore, it is possible that calcineurin helps regulate a system responsible for transition in the pupal IFM from the smaller Tn I isoform to the larger version, by control of the phosphorylation states of one or more proteins in the spliceosome complex that are required for inclusion of the third exon (Gajewski, 2005).
It should be noted that the effects of the canB2 mutation on the relative levels of the two Tn I isoforms in the adult IFM are highly variable. In some PCR experiments, the smaller, exon 3 lacking transcript is predominant, but in others, both forms can be clearly seen. However, the results for the wild type adults are consistent: the larger transcript is clearly present, with little or no smaller form detected, in multiple repeats of the experiment. Thus, it must be considered that the differential formation of the Tn I isoforms may not be a direct result of altered calcineurin control of splicing in the IFM, but an indirect consequence of the physiological status of mutant versus normal muscle. That is, the switch to the larger exon 3 containing isoform may normally occur in a wild type genetic background due to some signal (or muscle state) perceived and transmitted within IFM that is of a proper developmental age and competency. In canB2 mutants, an abnormal cellular environment may exist in some or all IFM that prevents the normal sensing of this signal and subsequent isoform switch. Thus, the variability observed in the relative ratio of the two Tn I mRNA forms may simply reflect a nonequivalent status of collapsed muscles as to their competency to sense and execute this developmental molecular switch (Gajewski, 2005).
Taken together, these results have provided mechanistic insights into the cause of IFM collapse in canB2 mutants. Cell fate changes can be ruled out, as can problems with mhc isoform production. In canB2 mutants, the transition to the adult Tn I splice variant is incomplete at best, but this change occurs after the time when the muscles collapse, so an altered stoichiometry of troponin isoforms cannot contribute to this phenotype. Reduction of calcineurin function in the IFM leads to lower levels of mhc transcripts and a variable reduction in the numbers of thick filaments. This reduction in mhc expression is likely a major contributing factor in the collapse of the canB2 mutant IFM. Heterozygotes of Mhc, which is a null allele, have reduced numbers of thick filaments and partial hypercontraction of the IFM. However, there is a striking difference in the collapse phenotypes of canB2 and various mhc mutations. In canB2 mutants, without fail, the collapse of the IFMs is directed towards the posterior of the thorax. In a number of different mhc mutant alleles, the IFM can bunch to either. The most severe myofibrillar phenotypes also suggest problems with more than just mhc. The strongest canB2 phenotypes had no Z-bands or any semblance of sarcomeric structure, an effect seen in some mutations that cause defects in the thin filaments. In animals homozygous for the Tn I allele heldup (hdp), which is functionally a null in the IFM, pupal myofibrils showed diffuse Z-bands at 42 h APF, and no sarcomeric structures by 46-48 h APF. Since no Z-bands in the most severely affected canB2 mutant pupae, it is possible that Z-bands could form and break down in a manner similar to hdp mutants. Therefore, it is quite likely that expression and/or processing of other muscle structural proteins are regulated by calcineurin activity, and these warrant future investigation (Gajewski, 2005).
Myofibrillar proteins assemble to form the highly ordered repetitive contractile structural unit known as a sarcomere. Studies of myogenesis in vertebrate cell culture and embryonic developmental systems have identified some of the processes involved during sarcomere formation. However, isoform changes during vertebrate muscle development and a lack of mutants have made it difficult to determine how these proteins assemble to form sarcomeres. The indirect flight muscles (IFMs) of Drosophila provide a unique genetic system with which to study myofibrillogenesis in vivo. This paper shows that neither sarcomeric myosin nor actin are required for myoblast fusion or the subsequent morphogenesis of muscle fibres, i.e., fibre morphogenesis does not depend on myofibrillogenesis. However, fibre formation and myofibrillogenesis are very sensitive to the interactions between the sarcomeric proteins. A troponin I (TnI) mutation, hdp3, leads to an absence of TnI in the IFMs and tergal depressor of trochanter (TDT) muscles due to a transcript-splicing defect. Sarcomeres do not form and the muscles degenerate. TnI is part of the thin filament troponin complex which regulates muscle contraction. The effects of the hdp3 mutation are probably caused by unregulated acto-myosin interactions between the thin and thick filaments as they assemble. This proposal was tested by using a transgenic myosin construct to remove the force-producing myosin heads. The defects in sarcomeric organisation and fibre degeneration in hdp3 IFMs are suppressed, although not completely, indicating the need for inhibition of muscle contraction during muscle development. mRNA and translated protein products of all the major thin filament proteins are reduced in hdp3 muscles, and how this and previous studies of thin filament protein mutants indicate a common co-ordinated control mechanism that may be the primary cause of the muscle defects is discussed (Nongthomba, 2004).
In recent years significant progress has been made in understanding how different sarcomeric proteins interact during development, myofibril assembly and muscle contraction. Formation of the highly ordered repetitive muscle cytoarchitecture is a complex process and genetic defects produce different forms of myopathy (reviewed in Clark, 2002; Redwood, 1999). It is well established that muscle contraction is regulated by changes in the concentration of Ca2+. The troponin-tropomyosin (Tn-Tm) complex, which is formed by three different troponin polypeptides, T, I and C (TnT, TnI and TnC), together with tropomyosin (Tm), regulates acto-myosin interactions in response to neurally stimulated intracellular release of Ca2+ ions. In the absence of Ca2+, TnI inhibits the generation of acto-myosin forces during muscle contraction (reviewed in Clark, 2002; Geeves, 1999; Gordon, 2000). Although the role of TnI during muscle contraction is well documented, its role during muscle development has not been studied in detail. Two major reasons for this are that loss of function mutations are lethal to individuals (see nemaline TnI nulls) and in vertebrate models expression of alternate isoforms compensate during early developmental stages. For example, cardiac TnI gene knockout mice (Huang, 1999) develop apparently normal hearts owing to the compensatory fetal TnI isoform but eventually acute heart failure occurs in later stages (Nongthomba, 2004).
In Drosophila the IFMs develop by fusion of myoblasts following their migration from the notum region of the wing imaginal discs during early stages of pupation. One group of IFMs, the dorsal longitudinal muscles (DLM), develop by fusion of these myoblasts to the remnants of larval oblique muscles (LOM) in the thorax region that escape complete histolysis at metamorphosis and serve as templates (TEM), whereas the other IFM group, the dorso ventral muscles (DVM), develop by de novo fusion of the myoblasts. During fusion the IFMs elongate to span completely the developing thorax, attach to the tendon cells and, following a fibre shortening to one-third of the original size, begin to undergo myofibrillogenesis, a stage that involves high-level expression of the adult-specific structural sarcomeric proteins. Initial sarcomere organization occurs during this stage and the muscles elongate again. The tendon cells retract as the muscles increase in length and size until functional myofibres are formed. As flies express many IFM-specific sarcomeric protein isoforms and no isoform changes occur during muscle differentiation or during later developmental stages, the IFMs offer an effective genetic model with which to study the null genotypes. These can be used to further understanding of the roles of many sarcomeric proteins from early myogenesis through to the formation of functional myofibrils. Recent studies show that kettin, a constituent of the sarcomeric Z-disc, and paramyosin, which forms part of the thick filament core, are involved in myoblast fusion. This suggests that other sarcomeric proteins could play important roles during early myogenic processes in addition to their important roles as structural and functional components of the myofibril (Nongthomba, 2004).
The single Drosophila TnI gene produces 10 different isoforms by differential splicing of 13 exons. Of these, isoforms differing at exon 6 (6a1, 6a2, 6b1 and 6b2) are mutually exclusive. The IFMs and TDT muscles express only the exon 6b1 isoform with or without exon 3 (Barbas, 1993; Beall, 1991). hdp3 is a TnI mutation affecting alternative splicing of the IFM-TDT-specific exon 6b1, which results in a failure to produce all exon 6b1-containing isoforms (Barbas, 1993); hdp3 thus acts as an IFM-TDT-specific TnI null mutant. In the IFMs of hdp3 flies the second fibre elongation does not take place (Barthmaier, 1995) and in the adults only a few remnants of muscle tissue are seen (Barbas, 1993; Beall, 1991; Prado, 1999). hdp3 was used to ask questions about the role of TnI in myoblast fusion and in the earliest stages of myofibril development. Using both a headless myosin heavy chain construct and myosin heavy chain mutations the role of acto-myosin interactions in producing the hdp3 phenotype as proposed previously (Beall, 1991) was explored further and it was shown that inhibition of muscle contraction during early development is important. In addition, although removal or reduction of acto-myosin forces during development suppresses the major defects of the hdp3 phenotype, it does not produce the almost normal myofibrillar structure of the headless myosin construct on its own. Another aspect of the hdp3 phenotype is a reduced accumulation of the thin filaments and their proteins, which it discussed here in the light of a possible co-ordinated regulation of the expression of these proteins (Nongthomba, 2004).
Muscle development occurs in two distinct stages. The first involves acquisition of muscle cell fate and fusion to form the syncytial myotubes. The second is the differentiation of the muscles including the intracellular assembly of the sarcomeres which are necessary for muscle contraction (Nongthomba, 2004 and references therein).
The development of normal fibre shape and attachment in the absence of expression of sarcomeric actin or myosin, or both, shows for the first time that fibre differentiation involves two independent processes: fibre morphogenesis and myofibrillogenesis. The mechanisms underlying fibre morphogenesis have not been identified but two major cytoskeletal networks that probably drive it are the non-muscle acto-myosin and dynein/kinesin-microtubule systems. Cytoplasmic actin and non-muscle myosin II are localised at muscle fibre membranes and the growing tips of the developing IFM myotubes extending towards their attachment sites. In developing IFM, expression of tubulins begins during the myoblast fusion stage, and microtubules form rings before the thick-thin filament assembly of the nascent myofibrillar lattice within them (Reedy, 1993). These microtubular structures may be important for fibre morphogenesis in the absence of the sarcomeric thick and thin filaments. In a vertebrate cell culture system it has been shown that the muscle-specific RING-finger protein (MURF), which is associated with microtubules, is required for myoblast differentiation, myotube formation and muscle morphogenesis (Nongthomba, 2004).
This study has shown that fibre morphogenesis and myofibrillogenesis are separable, independent processes, but muscle contraction inevitably affects fibre shape. It is therefore not inconsistent that hypercontracting mutations of sarcomeric proteins can seriously affect and destroy muscle fibre shape and integrity (Nongthomba, 2004).
Although the location and function of most sarcomeric proteins in the mature myofibril are known, a number are expressed in post-mitotic myoblasts and myotubes. Genetic studies have shown that D-titin is required for myoblast fusion. The roles of these proteins in fusion are not yet clear. The completion of fibre morphogenesis in the hdp3 mutant would seem to suggest that TnI is not required for these early myogenic processes. However, the hdp3 mutant is a 'null' in the sense that it affects the splicing and thereby translation of the IFM-specific isoform. The viability and otherwise normal behaviour of hdp3 flies argues that in all other muscles the wupA gene expresses normal or sufficient quantities of TnI (Nongthomba, 2004).
Most heterozygotes for contractile protein null mutants exhibit flight impairment and abnormal IFM muscle structure. Most of these are believed to result from stoichiometric imbalances between sarcomeric proteins as they assemble. It is well established that in the regulation of striated muscle contraction TnI acts as the inhibitory regulatory element for actin-myosin interactions in the absence of the Ca2+ binding to TnC (reviewed in Gordon, 2000). In hdp3 homozygotes the absence of TnI seems to lead to an extreme muscle phenotype in which the IFM do not develop because of an absence of inhibition of muscle contraction. However, hdp3/+ heterozygotes show an IFM hypercontraction phenotype (Nongthomba, 2003), in which the IFM develop normally but subsequently during late pupal stages undergo destructive contractions. These observations suggest that during early IFM development the TnI molecules, expressed from the wild-type wupA gene, assembled into troponin complexes are sufficient to inhibit muscle contraction, but that once the muscles can be activated at around 72-75 hours APF (Nongthomba, 2003) the proportion of TnI-containing troponin complexes along the thin filaments are insufficient to re-establish the 'relaxed' muscle state (Nongthomba, 2004).
In hdp3 homozygotes there is none of the IFM-specific TnI isoform. Myosin heads will be able to interact directly with the myosin-binding surface of nascent F-actin, and cause contraction of the fibre at a time when it is normally extending and thereby prevent assembly of developing sarcomeres. Evidence that this process is driven by unregulated acto-myosin interactions comes from the following observations: (1) that myosin mutants that affect the actin-binding sites or hamper the ATP hydrolysis partially suppress the hdp3 fibre phenotype (Nongthomba, 2003); (2) that removing all the myosin (Beall, 1991) or (3) replacing the myosin with a headless isoform completely suppress the hdp3 fibre phenotype. Ultrastructurally, at the earliest stages of myofibrillogenesis hdp3 IFM show SFLS-like structures, which normally precede sarcomere assembly (Reedy, 1993) but in the mutant they are thicker, rounded Z-bodies. The lack of Z-discs at later stages supports the proposal that unregulated acto-myosin interactions in hdp3 myofibrils prevent assembly of sarcomeres or destroy them as they form (Nongthomba, 2004).
If unregulated acto-myosin interactions in developing hdp3 myofibrils were the root cause of the muscle phenotype then the ultrastructural appearance of hdp3; Mhc10; Y97 myofibrils should be no different from those of Mhc10; Y97 flies. This is not the case. The latter genotype forms respectable IFM sarcomeres and myofibrils, although not with the completely regular structure and integrity of wildtype. This result suggests that the hdp3 phenotype is caused by more than aberrant regulation of thick-thin filament interactions. This study shows that the hdp3 mutation affects the expression levels of mRNA and proteins of other thin filament components but is without effect on expression of the myosin heavy chain mRNA. Previously, similar reductions in the accumulation of associated thin filament proteins have been reported for other IFM thin filament null mutants: TnT (Fyrberg, 1990), Tm2 and Actin88F. The reduction in expression of this group of thin filament genes in hdp3 flies may explain the effects of the mutant on the muscle phenotype that is not suppressible by reducing force production during fibre development (Nongthomba, 2004).
Secondary effects of one thin filament mutant on the expression of other thin filament genes, and the regulatory interactions that this implies, are not restricted to Drosophila IFM. In zebra fish, a mutation of the TnT2 gene (silent heart, sih) has been found to severely reduce not only TnT expression but also TnI3 and Tm-α (Sehnert, 2002), and, in a myoblast cell culture system, a ß-actin gene mutation causes reduced protein and mRNA levels of Tm2 and Tm3 (Schevzov, 1993). Furthermore, relative mRNA output between contractile gene families in humans has been found at different stages of development, and independently of isoform switching, which suggests the existence of some form of communication between these genes. These and recent microarray expression profiling of different developmental stages of Drosophila, where many muscle genes were found to be expressed at the same time, suggest the presence of common regulatory interactions between contractile protein gene families. Further microarray profiling of different IFM developmental stages of wild type and of null mutants of the thick-thin filament protein genes will be required to elucidate these pathways (Nongthomba, 2004).
The mechanism by which the mutant transcripts or proteins affect those of the normal thin filament genes is unclear. The various mutants include nonsense and splicing defects. Transcript splicing and mRNA export are mediated by the exon-junction complex (EJC), which contains several proteins involved in nonsense-mediated decay (NMD). It is likely that after the nuclear translation, a pioneer round of translation occurs and the mutant splice defect isoform is eliminated. Therefore the TnI isoform with the 6b1 exon is never formed, which explains why this isoform was never recovered in previous (Barbas, 1993) and present studies. PCR recovery of low levels of TnI cDNA with the later exons from hdp3 IFM may represent partially processed mRNA in the nucleus. Whether this pathway is somehow linked to reduced expression of other related thin filament genes remains a conjecture (Nongthomba, 2004).
Elucidation of how these genes are coordinately regulated seems likely to be important in understanding human mutations that cause muscle disease. Mutations in human TnI are associated with hypertrophic cardiomyopathy (Kimura, 1997) and distal arthrogryposis (Sung, 2003). In mice a cardiac troponin-I knockout leads to lethality (Huang, 1999). This study has shown that the Drosophila IFM and the wupA gene can provide a model system to explore the function of TnI in normal muscle development and disease. The hdp3 mutation would seem a useful tool for starting an investigation of the regulation of other thin filament protein genes during myofibrillogenesis in vivo (Nongthomba, 2004).
In bilaterians, the main regulator of muscle contraction is the troponin (Tpn) complex, comprising three closely interacting subunits (C, T, and I). To understand how evolutionary forces drive molecular change in protein complexes, the gene structures and expression patterns of Tpn genes were compared in insects. In this class, while TpnC is encoded by multiple genes, TpnT and TpnI are encoded by single genes. Their isoform expression pattern is highly conserved within the Drosophilidae, and single orthologous genes were identified in the sequenced genomes of Drosophila pseudoobscura, Anopheles gambiae, and Apis mellifera. Apis expression patterns also support the equivalence of their exon organization throughout holometabolous insects. All TpnT genes include a previously unidentified indirect flight muscle (IFM)-specific exon (10A) that has evolved an expression pattern similar to that of exon 9 in TpnI. Thus, expression patterns, sequence evolution trends, and structural data indicate that Tpn genes and their isoforms have coevolved, building species- and muscle-specific troponin complexes. Furthermore, a clear case can be made for independent evolution of the IFM-specific isoforms containing alanine/proline-rich sequences. Dipteran genomes contain one tropomyosin gene that encodes one or two high-molecular weight isoforms (TmH) incorporating APPAEGA-rich sequences, specifically expressed in IFM. Corresponding exons do not exist in the Apis tropomyosin gene, but equivalent sequences occur in a high-molecular weight Apis IFM-specific TpnI isoform (TnH). Overall, this approach to comparatively analyze supramolecular complexes reveals coevolutionary trends not only in gene families but in isoforms generated by alternative splicing (Herranz, 2005).
It is generally accepted that muscle tissue arose early on in the evolution of bilateria. Muscle structure and its mechanism of function have been retained relatively unchanged since that time, although individual muscle tissues are fine-tuned to reflect their physiological functions. Because the myofibril comprises a series of supramolecular complexes, studying the evolution of an individual protein must be carried out in the context of the complex in which it is a part. This analysis was initiated by focusing on one of the better-characterized substructures, the thin filament, starting with its main regulatory switch, the troponin complex. In this work, a comparative approach has been used to expand the description of how troponin genes have evolved in insects to reach their final tissue-specific isoform repertoires (Herranz, 2005).
Comparing gene sequences in the four drosophilid species has made apparent a new set of isoforms in both troponin I and T genes, and their specific expression has been established in the IFM. Alternative splicing of TpnT exon 10A increases variability at the 3' end of these transcripts. Interestingly, the existence of two variable regions at both the Nt and Ct of the proteins causes the distribution of variability in insect TpnT and TpnI alternatively spliced regions to resemble more closely the situation of the vertebrate genes (Herranz, 2005).
Different transcripts of the three Tpn subunits are coordinately expressed in various drosophilid muscle types. In particular, this analysis exposes the expression pattern coincidences between the TpnI exon 6's and the TpnC genes, and between TpnI exon 9/10 and TpnT exon 10A/B, also observed in Apis. All these expression partners seem to have evolved in a similar way as a result of their interactions in the thin-filament structure. Insect TpnC genes also show a complex evolutionary pattern, involving both divergence and convergence events. For instance, Type I TpnC genes, expressing larval hypodermic isoforms, have evolved independently in holometabolous insects, leading to two recently acquired isoforms in the drosophilid species, only one isoform in Anopheles, and two distantly related isoforms in Apis. TpnI 6a exons, the transcripts of which are mainly expressed in larval hypodermic muscles, may have evolved similarly. There is just one in Anopheles and two 6a exons in the Drosophilidae and Apis that could have been obtained independently in both insect orders. A different case of coupled evolution affects the TpnI exon 9 and TpnT exon 10A pair so that after appearing, probably at the beginning of the holometabolous insect branching, they became coexpressed in the very specialized IFM. In fact, this pair of exons shows higher sequence variability than its counterpart, the TnI exon 10 and TnT exon 10B pair. This higher variability in IFM-expressed isoforms can be related with the strong selection forces under these muscles (Herranz, 2005).
Following these ideas, it was of interest to explore how far a functional basis of this coexpression/coevolutionary trend could be established. Comparing the secondary structure predictions of the insect and human orthologous troponin proteins led to conclusions that, although the sequence conservation between deuterostomes and protostomes troponins is not very high, the structural predictions are conserved. For example the secondary structure predictions of Drosophila IFM TpnI isoform and the human cardiac TpnI are remarkably conserved. This conservation includes the interaction sites of TpnI with TpnC, Tm, TpnT, and actin as previously described in mammals and recently was confirmed in a crystal structure of half of a cardiac troponin complex. In particular, the insect TpnI alternative exon 6b2 sequence is equivalent to that of the cardiac TpnI exon interacting with the TpnC Nt sequence or actin, depending on the calcium concentration. All this information, together with structural data from vertebrates and from the insect Lethocerus, lead to a model for the troponin complex interactions in Drosophila that takes into account possible effects of isoform variability. Complex integrity may be stabilized by the interaction of the TpnI and TpnT variable Ct regions (exon 9/10 and exon 10A/10B, respectively) and TpnI Nt region with the globular Ct domain of TpnC. TpnT interacts with Tm mainly through its central domains, but its Ct polyglutamic tail and its Nt variability region (also polyglutamic rich) probably contribute to stabilize this interaction (Herranz, 2005).
The expression patterns, sequence evolutionary trends, and the secondary structure predictions taken together indicate that the alternatively spliced exons in TpnI and TpnT genes have evolved in a concerted way, a result consistent with the interactions of their products in the troponin complex. However, this has occurred independently in the different insect orders, at least for TpnC genes and for TmH/TpnH. The relationship between the variable regions of the troponin complex proteins and their putative interactions with coexpressed isoforms in different muscles or developmental stages are represented in an evolutionary/functional network for the different insect groups. The interactions among the different isoforms of the troponin-tropomyosin complex components produced by alternative splicing (TpnI and TpnT) or differential gene expression are indicated in the figure. The overall conservation of the relationships of TpnI with TpnC and TpnT can be observed among the insects studied. The main exception lies in the switching of the TmH for TnH in Apis, as discussed below. Furthermore, in the drosophilids, a TpnI exon 3 incorporating a PAANGKA heptad repeat is also only expressed in Drosophila IFM because Anopheles and Apis lack this exon. The reasons for this variability remain to be clarified (Herranz, 2005).
APPAEGA heptad repetitions are encoded in the IFM-specific exons (16 and 17) of the Drosophila tropomyosin Tm1 gene that produces two heavy tropomyosins (TmH 33 and 34) with long Ct extensions. In the Anopheles Tm1 gene, only one exon containing this kind of sequence has been located. Interestingly, in Apis the tropomyosin genes lack these long Ct extensions but a similar region is present in the TpnI gene where it is encoded by exons H1, H2, and H3 spliced together to produce a similarly sized, APPAEGA-rich extension. A transcript was also found containing only the first 30% of the alanine/proline-rich extension in the IFM. The presence of these TpnH extended isoforms in the Apis TpnI gene does not affect the standard repertoire of TpnIs, which are similarly processed and expressed as in dipterans (Herranz, 2005).
The main variability in the isoforms' expression and sequences occurs in the asynchronous IFM. Two ideas are generally accepted in relation to these muscles. First, no single biochemical feature is known that completely correlates with the stretch-activation phenomenon that is also observed in skeletal and cardiac muscles of vertebrates. The phenomenon is much stronger and persistent in insect IFM. Second, the IFM asynchrony has evolved independently several times during insect evolution. Molecular data are no exception. The interspecies-independent but intraspecies-adapted evolution of both the TpnC genes and the TpnT and TpnI genesplicing variants could have played important roles in how the IFMs have achieved their special properties (Herranz, 2005).
Previously published work has addressed the issue of isoform variability and function in relation to its evolutionary conservation a comprehensive analysis of the type performed in this article has not been attempted yet. Together, the higher variability of muscle structures in protostomes and the increasing availability of genome information across taxa open the way to understanding the independent coevolution of genes whose proteins are involved in supramolecular complexes. The approach used reveals coevolutionary trends in components of the complexes sharing tissue expression patterns. The advent of whole-genome sequences from further insect species will help in extending and refining this model for the evolution of the troponin complex. Some splicing isoforms or even complete genes that are weakly expressed or expressed in a very restricted pattern of tissues can be overlooked during a conventional study. What are the functional consequences of the expression of minor isoforms in a supramolecular complex? It could just provide a background repertoire of isoforms to improve and be used in remodeling evolutionary processes but its conservation across different species suggests a more direct role. In the case of insect thin filaments, the tropomyosin-troponin complex has been coevolving to respond to the functional necessities of the asynchronous flight musculature in different orders. Some alanine/proline-rich motifs have appeared and have become associated with different polypeptides in the complex. Different alternatively spliced exons encoding parts of different subunits of the complex, some of them also independently evolved, correlate with the type and function of the muscle. The sequencing effort in different organisms offers new opportunities to test, among other things, gene annotation accuracy, but more importantly the correlation of genotype changes and the functional phenotypic features that have evolved in particular groups of organisms (Herranz, 2005).
To identify proteins that interact in vivo with muscle components a genetic approach was used based on the isolation of suppressors of mutant alleles of known muscle components. This system was applied to the case of troponin I (TnI) in Drosophila and its mutant allele heldup2 (hdp2). This mutation causes an alanine to valine substitution at position 116 after a single nucleotide change in a constitutive exon. Among the isolated suppressors, one of them results from a second site mutation at the TnI gene itself. Muscles endowed with TnI mutated at both sites support nearly normal myofibrillar structure, perform notably well in wing beating and flight tests, and isolated muscle fibers produce active force. The structural and functional recovery in this suppressor does not result from a change in the stoichiometric ratio of TnI isoforms. The second site suppression is due to a leucine to phenylalanine change within a heptameric leucine string motif adjacent to the actin binding domain of TnI. These data evidence a structural and functional role for the heptameric leucine string that is most noticeable, if not specific, in the indirect flight muscle (Prado, 1995; Full text of article).
Search PubMed for articles about Drosophila Troponin
Bagni, C., et al. (2002). The Drosophila zinc finger transcription factor CF2 is a myogenic marker downstream of MEF2 during muscle development. Mech. Dev. 117(1-2): 265-8. PubMed ID: 12204268
Banerjee-Basu, S. and Buonanno, A. (1993). cis-acting sequences of the rat troponin I slow gene confer tissue- and development-specific transcription in cultured muscle cells as well as fiber type specificity in transgenic mice. Mol. Cell. Biol. 13: 7019-7028. PubMed ID: 8413291
Barbas, J. A., Galceran, J., Krah-Jentgens, I., de la Pompa, J. L., Canal, I., Pongs, O. and Ferrus, A. (1991). Troponin I is encoded in the haplolethal region of the shaker gene complex of Drosophila. Genes Dev. 5: 132-140. PubMed ID: 1899228
Barbas, J. A., Galceran, J., Torroja, L., Prado, A. and Ferrus, A. (1993). Abnormal muscle development in hdp3 mutant of Drosophila melanogaster is caused by splicing defect affecting selected troponin-I isoforms. Mol. Cell. Biol. 13: 1433-1439. PubMed ID: 7680094
Barthmaier, P. and Fyrberg, E. (1995). Monitoring development and pathology of Drosophila indirect flight muscles using green fluorescent protein. Dev. Biol. 169: 770-774. PubMed ID: 7781915
Beall, C. J. and Fyrberg, E. (1991). Muscle abnormalities in Drosophila melanogaster heldup mutants are caused by missing or aberrant troponin-I isoforms. J. Cell Biol. 114: 941-951. PubMed ID: 1908472
Boussouf, S. E. and Geeves, M. A. (2007). Tropomyosin and troponin cooperativity on the thin filament. Adv. Exp. Med. Biol. 592: 99-109. PubMed ID: 17278359
Clark, K. A., McElhinny, A. S., Beckerle, M. C. and Gregorio, C. C. (2002). Striated muscle cytoarchitecture: an intricate web of form and function. Annu. Rev. Cell Dev. Biol. 18: 637-706. PubMed ID: 12142273
Erdelyi, M., Michon, A. M., Guichet, A., Glotzer, J. B. and Ephrussi, A. (1995). Requirement for Drosophila cytoplasmic tropomyosin in oskar mRNA localization. Nature 377: 524-527. PubMed ID: 7566149
Furlong, E. E. andersen, E. C., Null, B., White, K. P. and Scott, M. P. (2001). Patterns of gene expression during Drosophila mesoderm development. Science 293: 1629-1633. PubMed ID: 11486054
Fyrberg, E., Fyrberg, C. C., Beall, C. and Saville, D. (1990). Drosophila melanogaster troponin-T mutations engender three distinct syndromes of myofibrillar abnormalities. J. Mol. Biol. 216: 657-675. PubMed ID: 2124273
Gajewski, K. M., Wang, J. and Schulz, R. A. (2005). Calcineurin function is required for myofilament formation and troponin I isoform transition in Drosophila indirect flight muscle. Dev. Biol. 289: 17-29. PubMed ID: 16297904
Geeves, M. A. and Holmes, K. C. (1999). Structural mechanism of muscle contraction. Annu. Rev. Biochem. 68: 687-728. PubMed ID: 10872464
Gordon, A. M., Homsher, E. and Regnier, M. (2000). Regulation of contraction in striated muscle. Physiol. Rev. 80: 853-924. PubMed ID: 10747208
Hales, K. H., Meredith, J. E. and Storti, R. V. (1994). Transcriptional and post-transcriptional regulation of maternal and zygotic cytoskeletal tropomyosin mRNA during Drosophila development correlates with specific morphogenic events. Dev. Biol. 165: 639-653. PubMed ID: 7958428
Herranz, R., et al. (2005). The coevolution of insect muscle TpnT and TpnI gene isoforms. Mol Biol Evol. 22(11): 2231-42. PubMed ID: 16049195
Huang, X., Pi, Y., Lee, K. J., Henkel, A. S., Gregg, R. G., Powers, P. A. and Walker, J. W. (1999). Cardiac troponin I gene knockout a mouse model for myocardial troponin I deficiency. Circ. Res. 84: 1-8. PubMed ID: 9915769
Kimura, A., Harada, H., Park, J. E., Nishi, H., Satoh, M., Takahashi, M., Hiroi, S., Sasaoka, T., Ohbuchi, N., Nakamura, T. et al. (1997). Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy. Nat. Genet. 16: 379-382. PubMed ID: 9241277
Li, Y., Chen, B., Chen, J., Lou, G., Chen, S. and Zhou, D. (2008). Fast skeletal muscle troponin I is a co-activator of estrogen receptor-related receptor alpha. Biochem. Biophys. Res. Commun. 369: 1034-1040. PubMed ID: 18331830
Marin, M. C., Rodriguez, J. R. and Ferrus, A. (2004). Transcription of Drosophila troponin I gene is regulated by two conserved, functionally identical, synergistic elements. Mol. Biol. Cell 15, 1185-1196. PubMed ID: 14718563
Mas, J. A., García-Zaragoza, E. and 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(4): 1931-45. PubMed ID: 14718560
Metzger, J. M., et al. (2003). Sarcomere thin filament regulatory isoforms: evidence of a dominant effect of slow skeletal troponin I on cardiac contraction. J. Biol. Chem. 278: 13118-13123. PubMed ID: 12551900
Nakayama, M., Stauffer, J., Cheng, J., Banerjee-Basu, S., Wawrousek, E. and 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. PubMed ID: 9858575
Nongthomba, U., Cummins, M., Vigoreaux, J. and Sparrow, J. C. (2003). Suppression of muscle hypercontraction by mutations in the myosin heavy chain gene of Drosophila melanogaster. Genetics 164, 209-222. PubMed ID: 12750333
Prado, A., Canal, I., Barbas, J. A., Molloy, J. and Ferrus, A. (1995). Functional recovery of troponin I in a Drosophila heldup mutant after a second site mutation. Mol. Biol. Cell 6: 1433-1441. PubMed ID: 8589447
Prado, A., Canal, I. and Ferrus, A. (1999). The haplolethal region at the 16F gene cluster of Drosophila melanogaster: structure and function. Genetics 151: 163-175. PubMed ID: 9872957
Redwood, C. S., Moolman-Smook, J. C. and Watkins, H. (1999). Properties of mutant contractile proteins that cause hypertrophic cardiomyopathy. Cardiovasc. Res. 44: 20-36. PubMed ID: 10615387
Reedy, M. C. and Beall, C. (1993). Ultrastructure of developing flight muscle in Drosophila. Dev. Biol. 160: 443-465. PubMed ID: 8253277
Sahota, V. K., Grau, B. F., Mansilla, A. and Ferrás, A. (2009). Troponin I and Tropomyosin regulate chromosomal stability and cell polarity. J. Cell Sci. 122(Pt 15): 2623-31. PubMed ID: 19567471
Schevzov, G., Lloyd, C., Hailstones, D. and Gunning, P. (1993). Differential regulation of tropomyosin isoform organization and gene expression in response to altered actin gene expression. J. Cell Biol. 121: 811-821. PubMed ID: 8491774
Sehnert, A. J., Huq, A., Weinstein, B. M., Walker, C., Fishman, M. and Stainier, D. Y. R. (2002). Cardiac troponin T is essential in sarcomere assembly and cardiac contractility. Nat. Genet. 31: 106-110. PubMed ID: 11967535
Sung, S. S., Brassington, A. M., Grannatt, K., Rutherford, A., Whitby, F. G., Krakowiak, P. A., Jorde, L. B., Carey, J. C. and Bamshad, M. (2003). Mutations in genes encoding fast-twitch contractile proteins cause distal arthrogryposis syndromes. Am. J. Hum. Genet. 72: 681-690. PubMed ID: 12592607
Ye, J., Zhao, J., Hoffmann-Rohrer, U. and Grummt, I. (2008). Nuclear myosin I acts in concert with polymeric actin to drive RNA polymerase I transcription. Genes Dev. 22: 322-330. PubMed ID: 18230700
Yutzey, K.E., Kline, R.L. and Konieczny, S.F. (1989). An internal regulatory element controls troponin I gene expression. Mol. Cell. Biol. 9: 1397-1405. PubMed ID: 2725509
date revised: 21 July 2022
Home page: The Interactive Fly © 2009 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.