Gotowa bibliografia na temat „Drosophila Troponin I”

The indirect flight muscles (IFMs) of Lethocerus (giant water bug) and Drosophila (fruitfly) are asynchronous: oscillatory contractions are produced by periodic stretches in the presence of a Ca2+ concentration that does not fully activate the muscle. The troponin complex on thin filaments regulates contraction in striated muscle. The complex in IFM has subunits that are specific to this muscle type, and stretch activation may act through troponin. Lethocerus and Drosophila have an unusual isoform of the Ca2+-binding subunit of troponin, troponin C (TnC), with a single Ca2+-binding site near the C-terminus (domain IV); this isoform is only in IFMs, together with a minor isoform with an additional Ca2+-binding site in the N-terminal region (domain II). Lethocerus has another TnC isoform in leg muscle which also has two Ca2+-binding sites. Ca2+ binds more strongly to domain IV than to domain II in two-site isoforms. There are four isoforms in Drosophila and Anopheles (malarial mosquito), three of which are also in adult Lethocerus. A larval isoform has not been identified in Lethocerus. Different TnC isoforms are expressed in the embryonic, larval, pupal and adult stages of Drosophila; the expression of the two IFM isoforms is increased in the pupal stage. Immunoelectron microscopy shows the distribution of the major IFM isoform with one Ca2+-binding site is uniform along Lethocerus thin filaments. We suggest that initial activation of IFM is by Ca2+ binding to troponin with the two-site TnC, and full activation is through the action of stretch on the complex with the one-site isoform.

We have cloned and characterized the troponin C gene, pat-10 of the nematode Caenorhabditis elegans. At the amino acid level nematode troponin C is most similar to troponin C of Drosophila (45% identity) and cardiac troponin C of vertebrates. Expression studies demonstrate that this troponin is expressed in body wall muscle throughout the life of the animal. Later, vulval muscles and anal muscles also express this troponin C isoform. The structural gene for this troponin is pat-10 and mutations in this gene lead to animals that arrest as twofold paralyzed embryos late in development. We have sequenced two of the mutations in pat-10 and both had identical two mutations in the gene; one changes D64 to N and the other changes W153 to a termination site. The missense alteration affects a calcium-binding site and eliminates calcium binding, whereas the second mutation eliminates binding to troponin I. These combined biochemical and in vivo studies of mutant animals demonstrate that this troponin is essential for proper muscle function during development.

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, we have focused our 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. Here, we propose 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, we have discovered a similar modular organization of phylogenetically conserved domains.

We have investigated the molecular bases of muscle abnormalities in four Drosophila melanogaster heldup mutants. We find that the heldup gene encodes troponin-I, one of the principal regulatory proteins associated with skeletal muscle thin filaments. heldup3, heldup4, and heldup5 mutants, all of which have grossly abnormal flight muscle myofibrils, lack mRNAs encoding one or more troponin-I isoforms. In contrast, heldup2, an especially interesting mutant wherein flight muscles are atrophic, synthesizes the complete mRNA complement. By sequencing mutant troponin-I cDNAs we demonstrate that the molecular basis for muscle degeneration in heldup2 is conversion of an invariant alanine residue to valine. We finally show that degeneration of heldup2 thin filament/Z-disc networks can be prevented by eliminating thick filaments from flight muscles using a null allele of the sarcomeric myosin heavy chain gene. This latter observation suggests that actomyosin interactions exacerbate the structural or functional defect resulting from the troponin-I mutation.

Abstract The indirect flight muscles (IFM) of Drosophila melanogaster provide a good genetic system with which to investigate muscle function. Flight muscle contraction is regulated by both stretch and Ca2+-induced thin filament (actin + tropomyosin + troponin complex) activation. Some mutants in troponin-I (TnI) and troponin-T (TnT) genes cause a “hypercontraction” muscle phenotype, suggesting that this condition arises from defects in Ca2+ regulation and actomyosin-generated tension. We have tested the hypothesis that missense mutations of the myosin heavy chain gene, Mhc, which suppress the hypercontraction of the TnI mutant held-up2 (hdp2), do so by reducing actomyosin force production. Here we show that a “headless” Mhc transgenic fly construct that reduces the myosin head concentration in the muscle thick filaments acts as a dose-dependent suppressor of hypercontracting alleles of TnI, TnT, Mhc, and flightin genes. The data suggest that most, if not all, mutants causing hypercontraction require actomyosin-produced forces to do so. Whether all Mhc suppressors act simply by reducing the force production of the thick filament is discussed with respect to current models of myosin function and thin filament activation by the binding of calcium to the troponin complex.

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, we show 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.

We investigated the functional overlap of two muscle Troponin C (TpnC) genes that are expressed in the adult fruit fly, Drosophila melanogaster: TpnC4 is predominantly expressed in the indirect flight muscles (IFMs), whereas TpnC41C is the main isoform in the tergal depressor of the trochanter muscle (TDT; jump muscle). Using CRISPR/Cas9, we created a transgenic line with a homozygous deletion of TpnC41C and compared its phenotype to a line lacking functional TpnC4. We found that the removal of either of these genes leads to expression of the other isoform in both muscle types. The switching between isoforms occurs at the transcriptional level and involves minimal enhancers located upstream of the transcription start points of each gene. Functionally, the two TpnC isoforms were not equal. Although ectopic TpnC4 in TDT muscles was able to maintain jumping ability, TpnC41C in IFMs could not effectively support flying. Simultaneous functional disruption of both TpnC genes resulted in jump-defective and flightless phenotypes of the survivors, as well as abnormal sarcomere organization. These results indicated that TpnC is required for myofibril assembly, and that there is functional specialization among TpnC isoforms in Drosophila.

A suppressor mutation, D53, of theheld-up 2 allele of the Drosophila melanogaster Troponin I (wupA) gene is described. D53, a missense mutation, S185F, of the tropomyosin-2,Tm2, gene fully suppresses all the phenotypic effects ofheld-up 2, including the destructive hypercontraction of the indirect flight muscles (IFMs), a lack of jumping, the progressive myopathy of the walking muscles, and reductions in larval crawling and feeding behavior. The suppressor restores normal function of the IFMs, but flight ability decreases with age and correlates with an unusual, progressive structural collapse of the myofibrillar lattice starting at the center. The S185F substitution in Tm2 is close to a troponin T binding site on tropomyosin. Models to explain suppression by D53, derived from current knowledge of the vertebrate troponin-tropomyosin complex structure and functions, are discussed. The effects of S185F are compared with those of two mutations in residues 175 and 180 of human α-tropomyosin 1 which cause familial hypertrophic cardiomyopathy (HCM).

The Myosin heavy chain (Mhc) locus encodes the muscle-specific motor mediating contraction in Drosophila. In a screen for temperature-sensitive behavioral mutants, we have identified two dominant Mhc alleles that lead to a hypercontraction-induced myopathy. These mutants are caused by single point mutations in the ATP binding/hydrolysis domain of Mhc and lead to degeneration of the flight muscles. Electrophysiological analysis in the adult giant fiber flight circuit demonstrates temperature-dependent seizure activity that requires neuronal input, as genetic blockage of neuronal activity suppresses the electrophysiological seizure defects. Intracellular recordings at the third instar neuromuscular junction show spontaneous muscle movements in the absence of neuronal stimulation and extracellular Ca2+, suggesting a dysregulation of intracellular calcium homeostasis within the muscle or an alteration of the Ca2+ dependence of contraction. Characterization of these new Mhc alleles suggests that hypercontraction occurs via a mechanism, which is molecularly distinct from mutants identified previously in troponin I and troponin T.

Muscle contraction is a highly fine-tuned process that requires the precise and timely construction of large protein sub-assemblies to form sarcomeres, the individual contractile units. Mutations in many of the genes encoding constituent proteins of this macromolecular machine result in defective functioning of the muscle tissue, and in humans, often lead to myopathic conditions like cardiomyopathies and muscular dystrophies, which affect a considerable number of people the world over. As more information regarding causative mutations becomes available, it becomes imperative to explore mechanisms of muscle development, maintenance and pathology. In striated muscles, contraction is regulated by the thin filament-specific tropomyosin (Tm) – troponin (Tn) complex (Ca2+-binding troponin-C, inhibitory troponin-I and tropomyosin-binding troponin-T). These troponin subunits are present in 1:1:1 ratio on thin filaments, with 1 Tm-Tn complex present on every 7th actin molecule. This stoichiometry is tightly regulated, and disturbances have been associated with functional defects. Each of these proteins has multiple isoforms, whose expression is controlled both spatially and temporally. The expression of specific combination of isoforms confers specific contractile properties to each muscle subtype. Drosophila melanogaster has been a preferred model of choice to study various aspects of muscle development for decades. In this study, the Indirect Flight Muscles (IFMs) of Drosophila have been used to investigate developmental and functional roles of two temporally regulated isoforms of a vital structural and regulatory component of the sarcomere – Troponin T (TnT). On a larger scale, whole genome expression profiles of mutants that are null for major myofbrillar proteins have also been discussed. IFMs serve as an excellent model system to address these questions, owing to the extreme ease of genetic manipulability in this system, and high degree of homology between mammalian and Dipteran cytoskeletal proteins. Chapter 1 covers basics of muscle biology, and the role of TnT in muscle contraction. Phenomena responsible for generating diversity in genes encoding muscle proteins – alternative splicing and isoform switching – have also been discussed. These mechanisms are highly conserved, as are patterns of TnT splicing and isoform expression across phyla. Mutations leading to altered splicing patterns lead to myopathic conditions, and the importance of model systems to study muscle biology has been emphasized. The advantages of studying Drosophila IFMs and a comprehensive overview of IFM development has been covered. The resources and experimental tools used have been described in Chapter 2. Two isoforms of TnT are alternatively spliced in the Drosophila thorax – one containing alternative exon 10a (expressed in adult IFMs and jump muscle); and one containing alternative exon 10b (expressed in pupae and newly eclosed flies). These exons are spliced in a mutually exclusive manner, and defects in splicing have been reported to cause uncontrolled, auto-destructive contractions. In Chapter 3, a splice mutant of TnT, up1, has been discussed, with respect to its developmental profile. Transgenic rescue experiments with two separate isoforms demonstrate rescue at the structural as well as functional level. Transgenic over-expression, however, leads to functional abnormalities, highlighting the importance of stoichiometry in multi-protein complexes. In Chapter 4, molecular signals that bring about the developmentally regulated TnT isoform switch are discussed. A splicing factor, Muscleblind, has been transgenically knocked down in normal and mutant IFMs to study effects on muscle function. Chapter 5 looks at whole genome transcriptional alterations in muscles null for either actin or myosin. All significant expression changes have been classified into categories based on different biological processes, and an attempt to differentiate generic muscle responses from filament-specific responses has been made. In conclusion, the studies have highlighted the importance of TnT isoform switching, and that extended expression of a pupal stage-specific isoform can partially compensate for loss of the adult isoform. Also, in the absence of major myofibrillar proteins, stress response pathways like heat shock response and protein degradation pathways are activated, along with a subset of metabolic responses that are unique to the thin or thick filament systems.

Muscle contraction is a highly fine-tuned process that requires the precise and timely construction of large protein sub-assemblies to form sarcomeres, the individual contractile units. Mutations in many of the genes encoding constituent proteins of this macromolecular machine result in defective functioning of the muscle tissue, and in humans, often lead to myopathic conditions like cardiomyopathies and muscular dystrophies, which affect a considerable number of people the world over. As more information regarding causative mutations becomes available, it becomes imperative to explore mechanisms of muscle development, maintenance and pathology. In striated muscles, contraction is regulated by the thin filament-specific tropomyosin (Tm) – troponin (Tn) complex (Ca2+-binding troponin-C, inhibitory troponin-I and tropomyosin-binding troponin-T). These troponin subunits are present in 1:1:1 ratio on thin filaments, with 1 Tm-Tn complex present on every 7th actin molecule. This stoichiometry is tightly regulated, and disturbances have been associated with functional defects. Each of these proteins has multiple isoforms, whose expression is controlled both spatially and temporally. The expression of specific combination of isoforms confers specific contractile properties to each muscle subtype. Drosophila melanogaster has been a preferred model of choice to study various aspects of muscle development for decades. In this study, the Indirect Flight Muscles (IFMs) of Drosophila have been used to investigate developmental and functional roles of two temporally regulated isoforms of a vital structural and regulatory component of the sarcomere – Troponin T (TnT). On a larger scale, whole genome expression profiles of mutants that are null for major myofbrillar proteins have also been discussed. IFMs serve as an excellent model system to address these questions, owing to the extreme ease of genetic manipulability in this system, and high degree of homology between mammalian and Dipteran cytoskeletal proteins. Chapter 1 covers basics of muscle biology, and the role of TnT in muscle contraction. Phenomena responsible for generating diversity in genes encoding muscle proteins – alternative splicing and isoform switching – have also been discussed. These mechanisms are highly conserved, as are patterns of TnT splicing and isoform expression across phyla. Mutations leading to altered splicing patterns lead to myopathic conditions, and the importance of model systems to study muscle biology has been emphasized. The advantages of studying Drosophila IFMs and a comprehensive overview of IFM development has been covered. The resources and experimental tools used have been described in Chapter 2. Two isoforms of TnT are alternatively spliced in the Drosophila thorax – one containing alternative exon 10a (expressed in adult IFMs and jump muscle); and one containing alternative exon 10b (expressed in pupae and newly eclosed flies). These exons are spliced in a mutually exclusive manner, and defects in splicing have been reported to cause uncontrolled, auto-destructive contractions. In Chapter 3, a splice mutant of TnT, up1, has been discussed, with respect to its developmental profile. Transgenic rescue experiments with two separate isoforms demonstrate rescue at the structural as well as functional level. Transgenic over-expression, however, leads to functional abnormalities, highlighting the importance of stoichiometry in multi-protein complexes. In Chapter 4, molecular signals that bring about the developmentally regulated TnT isoform switch are discussed. A splicing factor, Muscleblind, has been transgenically knocked down in normal and mutant IFMs to study effects on muscle function. Chapter 5 looks at whole genome transcriptional alterations in muscles null for either actin or myosin. All significant expression changes have been classified into categories based on different biological processes, and an attempt to differentiate generic muscle responses from filament-specific responses has been made. In conclusion, the studies have highlighted the importance of TnT isoform switching, and that extended expression of a pupal stage-specific isoform can partially compensate for loss of the adult isoform. Also, in the absence of major myofibrillar proteins, stress response pathways like heat shock response and protein degradation pathways are activated, along with a subset of metabolic responses that are unique to the thin or thick filament systems.

Foraging behaviour in Drosophila melanogaster larvae is influenced by natural allelic variation in the foraging (for) gene that encodes a cyclic GMP – dependent protein Kinase (PKG), such that rovers (forR) traverse greater distances while foraging than sitters (fors). Foraging behaviour is also influenced by natural allelic variation in the wings up A (wupA) gene that encodes the Troponin-I protein (TnI). Specifically, wupAlow allele suppresses the dominance of the forR allele, turning rovers into sitters. The dominance of the natural wupA alleles and their interactions with allelic combinations in for has not been characterized. I conducted various crosses and found that wupA alleles exhibit incomplete dominance. More importantly, I found that allelic combinations of wupA and for gave rise to a range in larval foraging behaviour. In this study, I propose that this gradient effect in foraging behaviour is due to variation in levels of PKG activity and TnI phosphorylation potential.

Myofibrillogenesis is a complex process involving assembly of many structural proteins in an orchestrated spatio-temporal manner to form a highly ordered contractile sarcomeric unit. Mutations in the proteins involved in muscle contraction and function lead to myopathic conditions in human. Hence, understanding the etiology of these diseases and genes involved may help in accurate diagnosis, prognosis and exploration of possible therapeutics. Molecular players and signaling pathways of myogenesis are highly conserved across phyla, enabling us to exploit indirect flight muscles (IFM) of Drosophila melanogaster as a model to study muscle development and function. IFM is the only fibrillar muscle which has considerable functional similarity to vertebrate cardiac muscles. It also enables the analyses of all stages of muscle development from its earliest stages of fusion of the imaginal myoblasts to fully differentiated muscle with its assembled contractile apparatus. Perturbance of developmental process in IFM leads to flightless flies with dysfunctional muscle. High throughput mutant screens, designed to isolate flightless flies have led to the identification of large number of genetic loci which are involved in muscle patterning and myofibrillogenesis, thus giving useful insights into the structural and functional aspects of fibre formation. One such classical mutant, flightless H (fliH), isolated during mutagenesis screen leads to IFM degeneration after fibres are formed normally. This interesting phenomenon is designated as muscle hypercontraction and is comparable to hypertrophic cardiomyopathies in humans. The muscle hypercontraction phenotype in this mutant was found to be temperature dependent and development of the process initiated at later stages of pupation. Cellular events associated with the IFM hypercontraction were followed up through development using this mutant. Further, interaction of fliH allele with other genetic backgrounds gave valuable insights on mechanisms of causation of muscle hypercontraction. Genetics played a pivotal role in identifying the mutant locus. The mutation was genetically mapped to the regulatory region of the wupA gene which was confirmed by sequencing data. The wupA gene codes for Troponin I (TnI), an inhibitory component of the troponin-tropomyosin complex of thin filaments. The mutation leads to reduced level of TnI transcript and hence reduced amount of protein, as a consequence, troponin complex formation is impeded leading to uninhibited acto-myosin interactions, thus causing muscle fibre breakdown. Our study reveals that fliH is a unique allele which confers temperature sensitive muscle phenotype. This is the first mutation found in the regulatory region of any structural gene which is temperature dependant and leads to muscle hypercontraction. This study also emphasizes that stoichiometry of structural proteins is important for proper functioning of muscle. Apart from mutations in sarcomeric genes, perturbations in calcium signaling also affect muscle functioning and lead to development of cardiac hypertrophy and failure. Hence, the role of calcineurin β-subunit (canB2), a calcium dependant protein phosphatase, in muscle was analyzed. Studies involving overexpression of canB2 in IFM showed that it leads to muscle hypercontraction. In addition, characterization of one of the new allele generated for the present study confirmed presence of muscle tearing and sarcomeric structure abnormality. canB2 alleles genetically interact with other hypercontracting alleles and enhance the hypercontraction phenotype. Overall, present study will help us to understand how genetic predisposition can enhance or suppress muscle hypercontraction. In a reverse genetics approach, role of muscle LIM protein, Beadex (Bx) in IFM was analyzed, as point mutations and loss of function alleles of LIM genes are associated with cardiomyopathies in humans. Immuno-histochemistry showed that Bx is expressed in myoblasts associated with wing imaginal disc which gives rise to IFM. Expression is also seen in developing IFM and in the neurons innervating the IFM. However, unlike the other known LIM proteins in Drosophila, Bx was not adhered to muscle fibre and showed predominant cytosolic localization. Targeted knockout and over-expression in muscles showed fibre rupturing and Z-disc deformities. Our results suggest that Bx may be involved in mechano-sensory stress signaling pathway like the other LIM proteins in humans and proper maintenance of the sarcomeric structure. Thus, present study elucidates the role of three loci namely: wupA, canB2 and Bx in proper muscle development and function. All the three loci code for proteins having orthologues in higher vertebrates and have been implicated in the pathogenesis of cardiomyopathies and/or skeletal myopathies in humans. Overall, such studies involving analyses of genes implicated in muscle development and function will help in exploring disease pathways which may help in derivation of new therapeutic strategies.

The muscular system is a highly complex and important system in the body. Proper muscle physiology is critical for locomotion, digestion, circulation, reproduction as well as for metabolic and immune homeostasis. Defects in muscle development, structure or function result in muscle disorders and diseases. Chapter 1 reviews the important events of muscle development and growth as well as the various processes that are involved in the regulation of the same. The muscle disorders that occur due to the mis -regulation of these processes are discussed. Specifically, the significance of microRNAs in muscle development and function in the context of cardiac hypertrophy has been described. Chapter 1 also explains the importance of mitochondrial morphology and function for normal tissue functioning along with the dynamic processes that mediate changes in mitochondrial shape and size, namely fusion and fission. Thus, the first chapter discusses what is known and unknown about the roles played by microRNAs in and the regulators of mitochondrial dynamics during muscle development, and highlights questions being addressed in the present thesis. The advantages of using the Drosophila indirect flight muscle (IFMs) as a model system to address the unanswered questions are also enumerated in this chapter. The main features of IFM development and the similarities with vertebrate muscle development have been highlighted. Chapter 2 details the various Drosophila melanogaster lines, genetic tools and the experimental techniques used in this study. In the context of muscle function, the spatial and temporal regulation of the expression and assembly of structural proteins into structural units (sarcomeres) is crucial. The derailing of this process has been shown to result in number of muscle defects. One among them is cardiac hypertrophy which is characterized by mis-regulation of structural protein levels. Recently, miR-9 was shown to be involved in cardiac hypertrophy, however the role played by miR-9 in the regulation of muscle proteins is not known. Chapter 3 explains the novel findings regarding the role of miR-9a (Drosophila homolog) in the regulation IFM development. Results from IFM-specific over-expression of miR-9a during early muscle development indicate that miR-9a may have a role in repressing the regulators of dorsal longitudinal muscles (DLMs – a subset of the IFMs) patterning. The results discussed in this chapter also reveal that the over-expression of miR-9a exclusively in the IFMs during myofibrillogenesis rendered the flies flightless and the muscles showed hypercontraction -an auto-destructive process resulting from mis-regulated acto-myosin interactions. Bioinformatics analysis predicted 27 putative targets of miR-9a in muscles and Troponin-T (TnT), a structural protein component of the thin filament complex required for regulation of muscle contraction, was identified as putative target of miR-9a . Based on the observations that TnT levels are reduced when miR-9a is over-expressed and that overexpression of TnT, which lacked the miR-9a binding site, resulted in rescue of miR-9a over-expression phenotype, Chapter 3 concludes by stating that Troponin T is a major target of miR-9a in the IFMs. This finding along with the fact that human cardiac Troponin T (TNNT2) possesses a miR-9 binding site indicates that miR-9 could be involved in regulating the Troponin T levels during cardiac hypertrophy. Maintenance of mitochondrial quality and quantity is vital particularly in an energetically active tissue such as muscle. Mutations in the genes encoding regulators of mitochondrial dynamics have been shown to result in degenerative diseases. However, the process of mitochondrial fusion and fission are not well studied in vivo , especially during tissue development. In Chapter 4, the changes in mitochondrial morphology across IFM development have been described for the first time. Since all the major events of myogenesis during IFM development have been well demarcated and can be spatio-temporally tracked, it serves as a good model to investigate the mitochondria dynamics and roles of molecular players. Mitochondrial morphology was observed to be thin and continuous in the early stages of development, circular during mid-pupal phase and large and tubular during late pupal stage, indicating the occurrence of both mitochondrial fusion and fission during myogenesis. Further, Chapter 4 details the effect of knock down of the regulators of mitochondrial fusion and fission, namely Mitochondrial associated regulatory factor (Marf) and Dynamin related protein 1 (Drp1) during development of the IFMs. Genetic studies that revealed the importance of these regulators in mammalian development and human diseases are also mentioned. The results presented in Chapter 4 show that the knock down of Marf during development of the IFMs resulted in abnormal mitochondrial morphology and dysfunctional mitochondria that undergo mitophagy. While, Marf expression was found to be vital during early in IFM development, it did not appear to be as necessary during later in development. Knock down of Marf during the myofibrillogenesis phase of IFM development did not result in any defect in mitochondrial morphology function and myofibril ultrastructure. Importantly, it is shown in Chapter 4 that when Marf was depleted from early in development, adult flies exhibited abnormal sarcomeric structures, were incapable of flight and had greatly reduced life span. On the other hand, knock down of Drp1, the regulator of fission did not affect the mitochondrial morphology, muscle function and myofibril ultrastructure. Therefore, for the first time, this study reports that the spatiotemporal regulation of mitochondrial fusion and not fission appears to be critical for IFM development, maintenance, and function. In conclusion, the present study offers the following novel insights into the regulation of IFM development and the how specific developmental events influence IFM function; I) The major target of miR-9a in IFMs is Troponin T, whose levels must be regulated during myofibrillogenesis in order to achieve stoichiometric balance essential for muscle contraction. II) The expression of Marf, a mediator of mitochondrial fusion is crucial during a window of time in IFM development in order to achieve normal mitochondria morphology and function as well as structurally sound, functional muscle fibres in adult.