Rozprawy doktorskie na temat „Drosophila Indirect Flight Muscles”

Complex behaviors using wings have facilitated the insect evolutionary success and diversification. The Drosophila indirect flight muscles (IFM) have evolved a highly ordered myofilament lattice structure and uses oscillatory contractions by pronounced stretch activation mechanism to drive the wings for high powered flight subject to natural selection. Moreover, the IFM is also utilized during small amplitude wing vibrations for species-specific male courtship song (sine and pulse), an important Drosophila mating behavior subject to sexual selection. Unlike flight, the contractile mechanism and contribution of any muscle gene in courtship song is not known. To gain insight into how separate selection regimes are manifested at the molecular level, we investigated the effect on flight and mating behaviors of mutations in two contractile proteins essential for IFM functions: an IFM-specific protein, flightin (FLN), known to be essential for structural and mechanical integrity of the IFM, and a ubiquitous muscle protein, myosin regulatory light chain (MLC2), known to enhance IFM stretch activation. Comparison of FLN sequences across Drosophila spp., reveal a dual nature with the N-terminal region (63 aa) evolving faster (dN/dS=0.4) than the rest of the protein (dN/dS=0.08). A deletion of the N-terminal region (fln�N62) resulted in reduced IFM fiber stiffness, oscillatory work and power output leading to a decreased flight ability (flight score: 2.8±0.1 vs 4.2±0.4 for fln+ rescued control) despite a normal wing beat frequency. Interestingly, the FLN N-terminal deletion reduced myofilament lattice spacing and order suggesting that this region is required to improve IFM lattice for enhancing power output and flight performance. Moreover, fln�N62 males sing the pulse song abnormally with a longer interpulse interval (IPI, 56±2.5 vs 37±0.7 ms for fln+) and a reduced pulse duty cycle (PDC, 2.6±0.2 vs 7.3±0.2 % for fln+) resulting in a 92% reduction in their courtship success. This suggested that FLN N-terminal region fine-tunes sexually selected song parameters in D. melanogaster, possibly explaining its hypervariability under positive selection. That FLN N-terminal region is not essential but required to optimize IFM functions of both flight and song, indicate that FLN could be an evolutionary innovation for IFM-driven behaviors, possibly through its role in lattice improvement. Mutations of the highly conserved MLC2 [N-terminal 46 aa deletion (Ext), disruption of myosin light chain kinase phosphorylations (Phos), and the two mutations put together (Dual)] are known to impair or abolish flight through severe reductions in acto-myosin contractile kinetics and magnitude of the stretch activation response. Unlike FLN, these MLC2 mutations do not show a pleitropic effect on flight and song. Flight abolished Phos and Dual mutants are capable of singing suggesting that these mutations affect song minimally compared to flight. Moreover, unlike FLN, none of these mutations affect interpulse interval, the most critical sexually selected song parameter in Drosophila. Also, in contrary to the known additive effects of Ext and Phos in the Dual mutant on flight wing beat frequency, a subtractive effect on sine song frequency is found in this study. That mutations in MLC2 are manifested differently for song and flight suggest that stretch activation plays a minimal or no role in song production. The results in this study suggest that the conserved regions of FLN and MLC2 are essential to support underlying IFM contractile structure and function necessary for flight, whereas the fast evolving FLN N-terminal region optimizes IFM's biological performance in flight and species-specific song possibly under positive selection regime.

Human thin filament myopathies are a group of skeletal muscle diseases caused by mutations in thin filament protein genes. Over 170 mutations within the human skeletal Cl-actin gene, ACTA1, cause congenital actin myopathies (CAM). These are dominant, often lethal mutations resulting in death at birth or shortly after. Several mutations have been identified in the genes encoding for Troponin I and Troponin T proteins, which cause arthrogryposis. The aim of this work was to see if the Drosophila Indirect Flight Muscles can be used as a genetic model system, with which to study the ACTA1 and arthrogryposis disorders and understand their aetiology. Six different mutations in the Drosophila Act88F gene, GI5R, I136M, DI54N, VI63L, VI63M and D292V, homologous to the human CAM actin mutations were transgenically expressed in Drosophila Indirect Flight Muscles (lFM) as wild type heterozygotes. All the mutants were dominant and with some myofibrillar defects similar to those seen in humans. Certain mutations resulted in intranuclear rods, similar to those found in humans and split Z-discs. The mutations varied in severity and matched that of the human mutations. An extra copy of wild type actin rescued the phenotype of all the heterozygote mutants, suggesting that upregulation of expression of the wild type actin gene might be a future prospect for therapy. Atypically, flies heterozygous for the R372H Act88F mutation complete normal IFM myogenesis and young flies can fly, but later become flightless and by day 7 show the Drosophila equivalent of the human nemaline phenotype. Electron microscopy revealed progressive loss of muscle structure. From the ultrastructure, the phenotypic requirement for muscle usage and the known α-actinin binding sites on the actin monomer, the R372H mutation is proposed to reduce the strength of F-actin/α-actinin binding, leading to muscle damage during use and breakdown of muscle structure. Binding studies confirmed a I3-fold reduction in u-actinin binding for R372H actin. The GAL4/UAS system was employed for the study of arthrogryposis mutations. The wild-type TnT and TnI IFM isoforms were transgenically expressed to rescue the TnT and TnI IFM nulls, respectively. Only the TnI null was rescued. The TnI arthrogryposis mutants were transgenically expressed and resulted in hypercontracted muscles.

Orientador: Carminda da Cruz-Landim
Banca: Ana Maria Costa Leonardo
Banca: Flávio Henrique Caetano
Banca: Zilá Luz Paulino Simões
Banca: José Eduardo Serrão
Resumo: Apini e Meliponini são tribos compostas por espécies de abelhas classificadas como eussociais avançadas e, portanto, apresentam divisão de trabalho reprodutivo entre as castas femininas e complexas adaptações comportamentais, adquiridas durante a evolução pelas operárias, para desempenhar as tarefas relativas à manutenção da colônia. A capacidade de voar dos adultos destes insetos está intrinsecamente ligada à maioria de suas atividades como o vôo nupcial para o acasalamento no caso das rainhas e machos e a exploração de novo habitat, fontes de alimentos e estabelecimento de novos ninhos no caso das operárias. Tanto em Apis mellifera, quanto em Scaptotrigona postica, o vôo é realizado por músculos denominados músculos indiretos do vôo por não apresentarem ligação direta com as asas. A contração desses músculos produz mudanças de volume no tórax e indiretamente, o movimento das asas. O objetivo deste projeto foi realizar medidas das fibras desse músculo em cada indivíduo e em cada fase da vida, aplicando aos resultados teste estatístico apropriado para verificar possíveis diferenças de desenvolvimento que possam ser relacionadas à função muscular e comparar a ultraestrutura e citoquímica da musculatura do vôo das castas femininas (rainhas e operárias) e machos em diferentes fases da vida, tendo em vista as diferenças comportamentais e fisiológicas entre as classes de indivíduos das duas espécies. O exame da musculatura do vôo, tanto com microscopia de luz como com microscopia eletrônica de varredura e transmissão, mostrou que o arranjo e a morfologia dos feixes musculares e das fibras que os compõe são similares nas duas espécies, no entanto os feixes musculares de Apis mellifera são formados por número maior de fibras. Medições das larguras das fibras mostraram diferenças estatisticamente significante entre as fases da vida... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Apini and Meliponini are tribes composed of species of advanced eusocial bees and therefore present division of reproductive labor between females and complex behavioral adaptations, acquired during the evolution by workers, to attend the responsibilities for the maintenance of the colony. The ability of adults to fly is intrinsically linked to most of their activities as the nuptial flight for mating in the case of queens and males and exploitation of new habitat, sources of food and establishment of new nests in the case of workers. Both in Apis mellifera, as in Scaptotrigona postica, the flight is accomplished by muscles called indirect flight muscles by not make a direct connection with the wings. The contraction of muscles produces changes in volume in the torax and indirectly, movement of the wings. The objective of this project was to perform measurements of muscle fibers from every individual in every stage of life, applying the appropriate statistical test to results in order determine possible differences in development that may be related to muscle function. Alsoo compare the ultra-structure of and cytochemistry of workers, queens and males flight muscle at different stages of life, with the behavioral and physiological differences between the classes of individuals of the two species. The examination of the muscles of the flight, both with light microscopy, and with scanning and transmission electron microscopy, showed that the arrangement and morphology of the muscle fibers bundles arrangement is similar in the two species, however the muscle bundles of Apis mellifera are formed by larger number of musclefibres. Measurements of the width of the fibers showed statistically significant differences between the life phases of the colonies components and between species. Similarly the ultra-structural examination showed that workers of both species emerge with... (Complete abstract click electronic access below)
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Apini e Meliponini são tribos compostas por espécies de abelhas classificadas como eussociais avançadas e, portanto, apresentam divisão de trabalho reprodutivo entre as castas femininas e complexas adaptações comportamentais, adquiridas durante a evolução pelas operárias, para desempenhar as tarefas relativas à manutenção da colônia. A capacidade de voar dos adultos destes insetos está intrinsecamente ligada à maioria de suas atividades como o vôo nupcial para o acasalamento no caso das rainhas e machos e a exploração de novo habitat, fontes de alimentos e estabelecimento de novos ninhos no caso das operárias. Tanto em Apis mellifera, quanto em Scaptotrigona postica, o vôo é realizado por músculos denominados músculos indiretos do vôo por não apresentarem ligação direta com as asas. A contração desses músculos produz mudanças de volume no tórax e indiretamente, o movimento das asas. O objetivo deste projeto foi realizar medidas das fibras desse músculo em cada indivíduo e em cada fase da vida, aplicando aos resultados teste estatístico apropriado para verificar possíveis diferenças de desenvolvimento que possam ser relacionadas à função muscular e comparar a ultraestrutura e citoquímica da musculatura do vôo das castas femininas (rainhas e operárias) e machos em diferentes fases da vida, tendo em vista as diferenças comportamentais e fisiológicas entre as classes de indivíduos das duas espécies. O exame da musculatura do vôo, tanto com microscopia de luz como com microscopia eletrônica de varredura e transmissão, mostrou que o arranjo e a morfologia dos feixes musculares e das fibras que os compõe são similares nas duas espécies, no entanto os feixes musculares de Apis mellifera são formados por número maior de fibras. Medições das larguras das fibras mostraram diferenças estatisticamente significante entre as fases da vida...
Apini and Meliponini are tribes composed of species of advanced eusocial bees and therefore present division of reproductive labor between females and complex behavioral adaptations, acquired during the evolution by workers, to attend the responsibilities for the maintenance of the colony. The ability of adults to fly is intrinsically linked to most of their activities as the nuptial flight for mating in the case of queens and males and exploitation of new habitat, sources of food and establishment of new nests in the case of workers. Both in Apis mellifera, as in Scaptotrigona postica, the flight is accomplished by muscles called indirect flight muscles by not make a direct connection with the wings. The contraction of muscles produces changes in volume in the torax and indirectly, movement of the wings. The objective of this project was to perform measurements of muscle fibers from every individual in every stage of life, applying the appropriate statistical test to results in order determine possible differences in development that may be related to muscle function. Alsoo compare the ultra-structure of and cytochemistry of workers, queens and males flight muscle at different stages of life, with the behavioral and physiological differences between the classes of individuals of the two species. The examination of the muscles of the flight, both with light microscopy, and with scanning and transmission electron microscopy, showed that the arrangement and morphology of the muscle fibers bundles arrangement is similar in the two species, however the muscle bundles of Apis mellifera are formed by larger number of musclefibres. Measurements of the width of the fibers showed statistically significant differences between the life phases of the colonies components and between species. Similarly the ultra-structural examination showed that workers of both species emerge with... (Complete abstract click electronic access below)

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.

Muscle development and function are highly synchronized processes that involve simultaneous action of several transcription factors, signalling cascades, kinases and phosphatases, ion channels, structural proteins and secondary messengers. Several aspects of muscle biology including contraction, development, growth, regeneration etc. have been extensively studied using model organisms like Mus musculus, C. elegans, Drosophila melanogaster, Danio rerio and Xenopus. Chapter 1 provides an in-detail account of work done previously in the field of muscle biology that includes literature about different types of muscles in vertebrates, their localization and function in the body, arrangement of thick and thin filaments in different types of muscles and their development. Of the several model organisms available, Drosophila has long been a favored model organism for muscle biologists. The indirect flight muscles (IFMs) of Drosophila are structurally similar to vertebrate skeletal muscles and physiologically to cardiac muscles and have been extensively used as a model to study vertebrate muscle development and function. Chapter 1 provides an overview of literature on development and function of IFMs in Drosophila. An essential and probably most important second messenger utilized by muscles for their function is Calcium. Muscles harbor an intricate and elaborate machinery of proteins that can sense calcium, transport calcium in and out of the cell, bind calcium and regulate gene expression and so on. Chapter 1 briefly explains the studies done previously with respect to calcium signalling in muscles. Background, which led to hypotheses for present study, has been described in the chapter. Chapter 2 includes details about all the techniques that have been used to conduct the experiments. Calcium signalling plays an important role not just in functioning of muscles but also in development, growth, injury and regeneration of the tissue. Cells have an elaborate machinery of calcium handling proteins that work in synchrony to achieve a desired state of function. Details of this have been included in Chapter 4. Calcium handling machinery is comprised of ion channels like voltage gated calcium channels, calcium activated potassium channels, calcium binding proteins, structural proteins etc. Chapter 3 talks about one of the calcium binding protein, Calcineurin, whose function has been implicated in fibre type switching in vertebrate muscles. Its expression in the muscles increases during exercise or weightlifting suggesting its role in conversion of fast glycolytic fibres to slow oxidative fibres that are required for maintaining sustained force in the muscles. Calcineurin works in concert with several transcription factors to bring about changes in transcription under conditions of stress. It is known to interact with NFAT transcription factor to regulate fibre type switching. It is also known to work along with Mef2 transcription factor to regulate expression of genes. Current study shows that the reduction in levels of Calcineurin-A subunit in muscles does not affect function or structure of muscles, but over-expression of the protein causes premature death of majority of the organisms and flight lessness in the escaper flies. On the contrary, loss of regulatory subunit of the protein, Calcineurin-B2, causes muscle hypercontraction in IFMs of Drosophila, suggesting crucial role of the protein in IFM development. The slow, progressive degeneration of IFMs in calcineurin-B2 (canB2) mutants is reminiscent of the muscle hypercontracted phenotype observed in mutants of Myosin heavy chain and the Troponin T and Troponin I proteins. Genetic studies of calcineurin with mutants of Troponin T and I show a synergistic interaction between Troponin T mutant up101 and calcineurin-B2. The Drosophila Troponin T mutation, up101, is equivalent to human cardiomyopathy Troponin T mutations, R92Q and I79N. The contractile machinery of the Troponin T mutants shows increased sensitivity towards calcium and can contract at the calcium concentrations below the threshold level. Current study (Chapter 3) highlights the importance of calcineurin in maintaining calcium homeostasis in muscles. Loss of calcineurin leads to arrhythmic spontaneous calcium oscillations in IFMs, which means that the average time for which the contraction machinery remains in contact with calcium is higher in canB2 mutant than control (frequency of oscillations is higher in canB2 mutants than control) and this probably contributes to the enhanced hypercontraction phenotype in a calcium sensitive mutant of Troponin T. Arrhythmicity in the calcium oscillations is observed as early as 50hrs after puparium formation (APF), well before the muscle degeneration phenotype is manifested in canB2 mutant flies. This reflects towards the importance of calcineurin in maintenance of calcium homeostasis in muscles. Chapter 4 describes study of spontaneous calcium oscillations during IFM development. Calcium is a highly versatile signal that works at different time points to regulate several cellular processes. Spontaneous calcium oscillations have been extensively studied in striated muscles, both the cardiac and skeletal muscles. There are different types of oscillations to which muscles respond. Long duration calcium transients (LDTs) have been identified in Xenopus myocytes and they predominantly occur prior to myofibrillogenesis, whereas SDTs (Short duration calcium transients) are spontaneous calcium oscillations of short duration (2- 3sec) that originate in subsarcolemmal space and are ryanodine sensitive, insensitive to changes in membrane potential and are independent of extracellular calcium. Similar to vertebrate muscles spontaneous calcium oscillations are also observed in IFMs of Drosophila throughout development. These oscillations were not reported previously in this system. We observe that the calcium oscillations in IFMs start as early as 34hrs APF, coinciding with the initiation of myofibrillogenesis process. There were no evident oscillations before 34hrs APF (i.e. from 0- 34hrs; the time point that involves processes of muscle splitting and myoblast fusion). The nature of these oscillations is still obscure. These oscillations vary in frequency, peak width and peak area across development. Previous reports have shown that cells often respond to changes in stimulus by varying frequency of calcium waves. These frequency changes are decoded by sophisticated molecular machines that include calcium sensitive proteins like calcium/calmodulin dependent protein kinase II and protein kinase C. The difference in peak frequency observed in the developing IFM could be due to differential expression of ion channels and structural proteins at these stages. Indeed, our results show that channels like Ryanodine, STIM and cacophony are transcriptionally regulated, and their transcripts are expressed only in adults whereas transcripts of channels like SERCA and slowpoke (Calcium gated potassium channels) are detected strongly throughout the development. Current study shows that spontaneous calcium oscillations in IFMs are sensitive to the levels of SERCA channels. These channels localize to the endoplasmic reticulum and are required for transportation of calcium from cytosol to endoplasmic reticulum. SERCA calcium pump and its function of calcium sequestration is essential for both development and functioning of the muscles because majority of the animals with reduced expression of SERCA do not survive till adult stage, rather they die in early larval or early pupal stages. Knockdown of SERCA in IFMs leads to increase in peak area and peak width of the calcium oscillations, which suggests the defect in calcium sequestration ability of the muscles. This abnormality in the calcium quenching could result in irregular muscle contraction in adult flies, which is shown by the contracted state of the adult muscles in escaper flies. Spontaneous oscillations are also sensitive to the changes in intracellular calcium levels. Reduction in the levels of intracellular calcium by over-expression of calcium binding protein, Parvalbumin, reduces the frequency of oscillations in developing IFM. These flies show defects in their flight ability suggesting that calcium is utmost important for the functioning of the muscles. Taken together, these studies reflect upon importance of calcium signalling in muscle development and function.

Muscle development is a complex and multifactorial process involving assembly of thousands of proteins in a precise and synchronized manner over the course of development of an organism. Multiple genes and signalling pathways have been identified to have pivotal roles in the development and function of a healthy and functional muscle. However, the role of many genes in muscle development remains unknown. One such gene are the BCL11A/B genes. BCL11A and BCL11B are paralogous genes which belong to the kruppel-like C2H2 type zinc finger transcription factors. The BCL11A and BCL11B protein sequences are 58% identical and 68% similar to each other. Both, BCL11A and BCL11B mainly have non-overlapping functions in neurogenesis and immune cell development. Recent studies have reported mutations in BCL11A to be associated with muscle-related defects like hypotonia, speech disorder and gross motor impairments, while mutations in BCL11B have been shown to be associated with muscle-related defects like hypertrophic cardiomyopathy and aortic stiffness. However, there are no studies which have addressed the molecular function of BCL11A and BCL11B in muscle development and function. CG9650 is the ortholog of BCL11A and BCL11B in Drosophila melanogaster. Overall, CG9650 bears 84% similarity to the vertebrate BCL11A and BCL11B proteins. The Indirect Flight Muscles (IFMs) of Drosophila melanogaster occupy a majority of the thorax of the adult fly and are responsible for powering the wing stroke during flight. These muscles consist of two opposing sets of muscles, namely the dorso-longitudinal muscles (DLMs; six in number) oriented anterior to posterior, and the dorso-ventral muscles (DVMs; three in number) oriented in a dorsal -to-ventral manner. The DLMs are formed by fusion of myoblasts with 3 pre-existing templates called Larval Oblique Muscles (LOMs), and subsequent splitting to form the 6 DLMs. The DVMs are formed by de novo fusion of myoblasts. Due to various advantages like the spatially and temporally distinct time-course of the development, dispensability to survival, similarity in development of the IFMs and vertebrate myogenesis, etc, the Indirect Flight Muscles (IFMs) of Drosophila melanogaster are an excellent model system to study the function of genes in muscle development. In this study, we have attempted to determine the role of CG9650 in development of the IFMs of Drosophila melanogaster. Our experiments show CG9650 is expressed during the specification (embryonic), proliferation (larval), and migration & fusion (pupal) stages of IFM development and depletion of CG9650 leads to a defect in the pattern of the DLMs (i.e. reduced number of DLMs). The expression of CG9650 during the proliferation, migration and fusion stages is crucial for patterning of the DLMs. This patterning defect could be rescued by transgenic expression of CG9650 during IFM development. The CG9650-depleted flies were compromised in their flight ability; walking ability of these flies remained unaffected. At the fascicular level, these DLMs have a larger cross-sectional area, more fibers per fascicle, but a decreased packing density of myofibrils compared to control flies. The sarcomeres of CG9650-depleted flies are thinner and longer, and expressed of the Z-disc protein Actinin was reduced compared to that control flies. These fascicular and sarcomeric defects are believed to cause reduced flight ability of the CG9650-depleted flies. CG9650 is also shown to affect Notch signalling in a context-dependant manner. Loss of CG9650 leads to upregulation of Notch signalling in myoblasts at the proliferation (larval) stage of IFM development. This leads to increased proliferation of the myoblasts. Additionally, loss of CG9650 leads to reduced Armadillo levels, which in turn leads to reduced wingless signalling, thus the stratification of myoblasts on the notum region of the wing disc. During the migration & fusion stages of IFM development, SnS, a (Ig-domain containing protein) is required for fusion of the migrating myoblast with the developing fiber. Migrating myobalsts show a diffuse expression of SnS. Notch signalling induces the formation of SnS puncta required for fusion of the myoblast with developing fiber. Perturbation of CG9650 during IFM development leads to decreased Notch signalling and a decrease in the formation of SnS puncta. This leads to decreased myoblast fusion and hence, arrested splitting of the developing LOMs. A transcriptomics approach was used to determine the genes/pathways regulated by CG9650. An RNA-seq using DLMs depleted for CG9650 revealed misregulation of genes mainly involved in myogenesis and neurogenesis. Aret, Act88F, thor, CanA, myofilin, Mlp60A, Zasp52 were among the differentially regulated genes with known functions in myogenesis. These genes have functions during the myofibrillogenesis stage of IFM development and their differential expression could account for the fascicular and sarcomeric defects seen in CG9650-depleted DLMs. Notably, expression of Hibris (a gene known to function in conjunction with SnS to regulate myoblast fusion) was also differentially regulated. Multiple genes known to be targets of Notch signalling were also found to be differentially regulated, thus confirming CG9650 as a regulator of the Notch pathway. Differential regulation of expression of genes regulating in cell division, channel proteins, immunity, cuticle formation and gene expression was also observed. In summary, our study highlights CG9650 as a novel modulator of Notch signalling output and novel regulator of DLM patterning during IFM development.

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.

Muscle development involves concerted action of a repertoire of mechanisms governing myoblast proliferation, migration, fusion and differentiation. Subsequently, there are cellular events administrating proper muscle function and maintenance of muscle integrity. Chapter 1 covers what is known about muscle development, building up of mass and maintenance in vertebrates and Drosophila, highlighting the myogenic programs and factors that play a role in them. The formation of vertebrate skeletal muscles can be recapitulated in Drosophila indirect flight muscles (IFMs), making IFMs an interesting model to dissect and understand the mechanisms of muscle development and maintenance. The cellular and developmental events that occur during IFM development have been discussed in detail along with their genetic control which encompasses both cell autonomous and cell non-autonomous mechanisms. The fly resources and tools used for experimentations have been described in Chapter 2. One of the hallmark events during muscle development is myoblast fusion. Myoblasts are kept in undifferentiated state until they fuse through a balanced action of anti-differentiation and pro-differentiation factors. The swarming myoblasts are in semi-differentiated state and just prior to fusion should exit cell cycle to achieve terminal differentiation. The mechanisms of cyclin/CDK complexes and their regulation via CKI (CDK inhibitor) are known in a cell. However, tissue specific factors exerting additional control on molecules that participate in cell cycle have been proposed but have not been shown in vivo. Chapter 3 uncovers a novel role played by the transcription factor, Erect wing (Ewg) in IFM development and patterning. Despite the fact that Ewg is known to express in fusing myoblasts and nuclei of developing IFMs and has long been used as a nuclear marker for IFMs, the mechanism(s) behind Ewg‟s function has remained enigmatic. Historical perspective of Ewg has been presented in Chapter 1. One set of IFMs; dorsal longitudinal muscles (DLMs) require larval templates for their formation and the other set; dorsal ventral muscles (DVMs) form de novo. Chapter 3 shows that Ewg is required in a spatio-temporal fashion to initiate myoblast fusion process. In the absence of Ewg, the number of fusion events in a given time is reduced. In addition de novo fusion is observed in the region of DLM development just like DVM and overall IFM development is delayed resulting in an aberrant adult IFM pattern. Genetic studies undertaken reveal a requirement for Ewg in exerting a temporal control on myoblast fusion. This is achieved by down-regulating Cyclin E levels, as a result of which the myoblasts exit cell cycle at G1/S stage. Through this study the proposal for DLM development and pattern has been put forth as follows: i) appropriate progression of DLM development commences on synchronous exit of myoblasts from cell cycle. This function is facilitated by Ewg expression in fusing myoblasts assisting symmetrical DLM formation in hemithoraces. ii) DLM pattern of six muscles in each hemithorax is dependent on template survival which requires fusion of enough myoblasts and further subsequent fusion events to support the splitting of three larval templates or presumptive DLM. The muscles that develop should preserve their structural integrity for efficient functional output. Muscles perform extensive activities warranting high energy requirements. IFMs are widely utilised for thorax movements that aid flight. IFMs are exclusively oxidative in nature with upto 40% mass contributed by large mitochondria themselves. Chapter 4 describes yet another novel finding for Ewg function in IFM maintenance. Vertebrate homolog of Ewg is nuclear respiratory factor 1 (NRF1) known for its role in mitochondrial biogenesis. This prompted an investigation on the role of Ewg, if any, in mitochondrial function and IFM maintenance. In this chapter, Ewg null adult IFMs are shown to undergo degeneration. Mitochondria in these muscles show rounder and smaller phenotype. Mitochondria morphology is traced throughout Drosophila pupal DLM development and extensive fusion is observed in last one-fourth of pupal phase. In Ewg null condition transcripts of Opa1-like required for inner mitochondrial membrane fusion is found to be absent, suggesting lack of mitochondrial fusion behind the smaller mitochondrial morphology. This emerged as an intriguing problem since Ewg expression follows until sarcomerogenesis (formation of sarcomeres) initiates at mid pupal stage. Developmentally extending Ewg‟s expression beyond mid pupal stage is not observed to increase Opa1-like levels pointing an indirect regulation by Ewg. However, Opa1-like knock-down beyond mid pupal stage is not observed to result in any muscle or flight defect. It is thus proposed that Ewg expression early during muscle development helps to up-regulate Opa1-like levels needed to support mitochondrial growth and fusion. In addition, this chapter provides additional data on requirement of Opa1-like for maintenance of mitochondrial as well as muscle integrity. This is the first ever report of tissue specific temporal regulation of Opa1-like by Ewg. Chapter 3 and Chapter 4 conclude spatially segregated functional requirements of Ewg which are also mechanistically distinct. Expression in fusing myoblasts channelizes fusing myoblasts to exit cell cycle and undergo timely fusion saving the larval template, subsequent fusion assists template splitting thus forming the appropriate adult DLM pattern. On the other hand expression until mid pupal stage up-regulates Opa1-like expression, facilitating mitochondrial fusion during the late pupal stage. This as a result helps maintain structural integrity of muscles in the adult. Vertebrate skeletal muscle contains multiple muscle fibres that provide appropriate mass and size to muscles. As DLM share similarity in development to that of the vertebrate skeletal muscle, DLM organisation is studied to get insights into the mechanisms which regulate the process. Chapter 5 discusses role of nuclear number and nuclear activity in determining the DLM organisation. In order to alter nuclear number, myoblast population is reduced to amounts lesser than that of the wild type and to alter nuclear activity, two nuclear encoded genes Opa1-like and Marf , involved in inner and outer mitochondrial membrane fusion respectively have been knocked down in IFMs. First, the DLM organisation is established by comparing it to the vertebrate skeletal muscle organisation. This organisation is affected on lowering the number of myoblasts destined to fuse and form IFMs, without affecting the differentiation. On the other hand, when nuclear encoded mitochondrial fusion genes are knocked down, not only DLM organisation but their differentiation is also affected. A proposal for achieving DLM organisation has been presented which should also apply to vertebrate skeletal muscle given their developmental similarity. In conclusion, the studies decipher a novel mechanism by which a transcription factor, Ewg exerts a temporal control on myoblast fusions directly influencing progression of DLM formation, and thereby, symmetry and pattern. Moreover, Ewg is also shown here to regulate mitochondrial fusion during later pupal stages helping muscles to attain greater function and maintain structural integrity. Discovery of such regulatory mechanisms controlling mitochondrial dynamics in vertebrates can open up new avenues to understand and design new therapeutic approaches to tackle mitochondrial diseases. Additionally, myoblast fusion and hence myonuclear number and their efficient functioning are shown to be important determinants of muscle organisation. This has further implications in understanding and using stem cell science in dystrophic or atrophic or ageing related muscle loss and therapy.

Muscle development involves concerted action of a repertoire of mechanisms governing myoblast proliferation, migration, fusion and differentiation. Subsequently, there are cellular events administrating proper muscle function and maintenance of muscle integrity. Chapter 1 covers what is known about muscle development, building up of mass and maintenance in vertebrates and Drosophila, highlighting the myogenic programs and factors that play a role in them. The formation of vertebrate skeletal muscles can be recapitulated in Drosophila indirect flight muscles (IFMs), making IFMs an interesting model to dissect and understand the mechanisms of muscle development and maintenance. The cellular and developmental events that occur during IFM development have been discussed in detail along with their genetic control which encompasses both cell autonomous and cell non-autonomous mechanisms. The fly resources and tools used for experimentations have been described in Chapter 2. One of the hallmark events during muscle development is myoblast fusion. Myoblasts are kept in undifferentiated state until they fuse through a balanced action of anti-differentiation and pro-differentiation factors. The swarming myoblasts are in semi-differentiated state and just prior to fusion should exit cell cycle to achieve terminal differentiation. The mechanisms of cyclin/CDK complexes and their regulation via CKI (CDK inhibitor) are known in a cell. However, tissue specific factors exerting additional control on molecules that participate in cell cycle have been proposed but have not been shown in vivo. Chapter 3 uncovers a novel role played by the transcription factor, Erect wing (Ewg) in IFM development and patterning. Despite the fact that Ewg is known to express in fusing myoblasts and nuclei of developing IFMs and has long been used as a nuclear marker for IFMs, the mechanism(s) behind Ewg‟s function has remained enigmatic. Historical perspective of Ewg has been presented in Chapter 1. One set of IFMs; dorsal longitudinal muscles (DLMs) require larval templates for their formation and the other set; dorsal ventral muscles (DVMs) form de novo. Chapter 3 shows that Ewg is required in a spatio-temporal fashion to initiate myoblast fusion process. In the absence of Ewg, the number of fusion events in a given time is reduced. In addition de novo fusion is observed in the region of DLM development just like DVM and overall IFM development is delayed resulting in an aberrant adult IFM pattern. Genetic studies undertaken reveal a requirement for Ewg in exerting a temporal control on myoblast fusion. This is achieved by down-regulating Cyclin E levels, as a result of which the myoblasts exit cell cycle at G1/S stage. Through this study the proposal for DLM development and pattern has been put forth as follows: i) appropriate progression of DLM development commences on synchronous exit of myoblasts from cell cycle. This function is facilitated by Ewg expression in fusing myoblasts assisting symmetrical DLM formation in hemithoraces. ii) DLM pattern of six muscles in each hemithorax is dependent on template survival which requires fusion of enough myoblasts and further subsequent fusion events to support the splitting of three larval templates or presumptive DLM. The muscles that develop should preserve their structural integrity for efficient functional output. Muscles perform extensive activities warranting high energy requirements. IFMs are widely utilised for thorax movements that aid flight. IFMs are exclusively oxidative in nature with upto 40% mass contributed by large mitochondria themselves. Chapter 4 describes yet another novel finding for Ewg function in IFM maintenance. Vertebrate homolog of Ewg is nuclear respiratory factor 1 (NRF1) known for its role in mitochondrial biogenesis. This prompted an investigation on the role of Ewg, if any, in mitochondrial function and IFM maintenance. In this chapter, Ewg null adult IFMs are shown to undergo degeneration. Mitochondria in these muscles show rounder and smaller phenotype. Mitochondria morphology is traced throughout Drosophila pupal DLM development and extensive fusion is observed in last one-fourth of pupal phase. In Ewg null condition transcripts of Opa1-like required for inner mitochondrial membrane fusion is found to be absent, suggesting lack of mitochondrial fusion behind the smaller mitochondrial morphology. This emerged as an intriguing problem since Ewg expression follows until sarcomerogenesis (formation of sarcomeres) initiates at mid pupal stage. Developmentally extending Ewg‟s expression beyond mid pupal stage is not observed to increase Opa1-like levels pointing an indirect regulation by Ewg. However, Opa1-like knock-down beyond mid pupal stage is not observed to result in any muscle or flight defect. It is thus proposed that Ewg expression early during muscle development helps to up-regulate Opa1-like levels needed to support mitochondrial growth and fusion. In addition, this chapter provides additional data on requirement of Opa1-like for maintenance of mitochondrial as well as muscle integrity. This is the first ever report of tissue specific temporal regulation of Opa1-like by Ewg. Chapter 3 and Chapter 4 conclude spatially segregated functional requirements of Ewg which are also mechanistically distinct. Expression in fusing myoblasts channelizes fusing myoblasts to exit cell cycle and undergo timely fusion saving the larval template, subsequent fusion assists template splitting thus forming the appropriate adult DLM pattern. On the other hand expression until mid pupal stage up-regulates Opa1-like expression, facilitating mitochondrial fusion during the late pupal stage. This as a result helps maintain structural integrity of muscles in the adult. Vertebrate skeletal muscle contains multiple muscle fibres that provide appropriate mass and size to muscles. As DLM share similarity in development to that of the vertebrate skeletal muscle, DLM organisation is studied to get insights into the mechanisms which regulate the process. Chapter 5 discusses role of nuclear number and nuclear activity in determining the DLM organisation. In order to alter nuclear number, myoblast population is reduced to amounts lesser than that of the wild type and to alter nuclear activity, two nuclear encoded genes Opa1-like and Marf , involved in inner and outer mitochondrial membrane fusion respectively have been knocked down in IFMs. First, the DLM organisation is established by comparing it to the vertebrate skeletal muscle organisation. This organisation is affected on lowering the number of myoblasts destined to fuse and form IFMs, without affecting the differentiation. On the other hand, when nuclear encoded mitochondrial fusion genes are knocked down, not only DLM organisation but their differentiation is also affected. A proposal for achieving DLM organisation has been presented which should also apply to vertebrate skeletal muscle given their developmental similarity. In conclusion, the studies decipher a novel mechanism by which a transcription factor, Ewg exerts a temporal control on myoblast fusions directly influencing progression of DLM formation, and thereby, symmetry and pattern. Moreover, Ewg is also shown here to regulate mitochondrial fusion during later pupal stages helping muscles to attain greater function and maintain structural integrity. Discovery of such regulatory mechanisms controlling mitochondrial dynamics in vertebrates can open up new avenues to understand and design new therapeutic approaches to tackle mitochondrial diseases. Additionally, myoblast fusion and hence myonuclear number and their efficient functioning are shown to be important determinants of muscle organisation. This has further implications in understanding and using stem cell science in dystrophic or atrophic or ageing related muscle loss and therapy.

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.

Myofibrillogenesis is a complex process, which involves assembly of hundreds of structural proteins in a highly ordered manner to form the contractile structural unit of muscle, the sarcomere. Several myopathic conditions reported in humans are caused due to abnormal myofibrillogenesis owing to mutations in the genes coding for many of these structural proteins. These myopathies have highly variable clinical features and time of onset. Since their aetiology is poorly understood, it becomes imperative to have a model system to study the muscle defects. Present study proposes to employ the Indirect Flight Muscle (IFM) system in Drosophila melanogaster as a model to analyse the development/onset of some of these myopathies and resulting pathophysiology. We have carried out a systematic study on mutations in two major proteins of the sarcomere, actin and myosin, to understand the pathophysiology associated with the disease conditions and in turn gain insights into the process of myofibrillogenesis. To verify whether the human muscle phenotypes are observed in flies, we analysed the IFM for functional and structural defects categorised by the presence of aberrant sarcomeric structures. An important question that we have addressed is whether mutants of the Drosophila IFM recapitulate human conditions and whether it can serve as a good genetic model to study the developmental mechanisms of the human skeletal myopathies in vivo. Mutations of the human ACTA1 skeletal actin gene produce seven congenital myopathies – actin myopathy, nemaline rod myopathy, intranuclear rod myopathy, congenital fibre type disproportion, congenital myopathy with core-like areas, cap disease and zebra body myopathy. Four known mutations in Act88F—a Drosophila homologue of ACTA1—occur at the same actin residues mutated in ten ACTA1 nemaline mutations, A138D/P, R256H/L, G268C/D/R/S and R372C/S. These Act88F mutants were examined for muscle phenotypes with nemaline structures. Mutant homozygotes show phenotypes ranging from lack of myofibrils to almost normal sarcomeres at eclosion. Whereas, heterozygotes do allow myofibrillar assembly to certain extent; however, atypical structures are seen adjacent to normal sarcomeres. Aberrant Z disc-like structures and serial Z disc arrays, ‘zebra bodies’, are observed in homozygotes and heterozygotes of all the four Act88F mutants. The electron-dense structures observed in electron micrographs show homologies to human nemaline bodies/rods, but are much smaller than those typically found in the human myopathy. A possible mechanism for the ‘zebra bodies’ is proposed based on this study. Analysis of IFM at early developmental stages shows that in three of the mutants, there is an abnormal myofibril assembly leading to malformed sarcomeres mirrored in the adult stages. In one of the Act88F mutants, normal myofibrils are seen post-eclosion but the IFM show activity dependant progression of muscle degeneration. All the Act88F mutants produce dominant disruption of muscle structure and function which cannot be rescued even by three copies of the wild type Act88F gene implying that the mutants are strong antimorphs. Myosin myopathies are a group of human muscle diseases with heterogeneous clinical features and are caused by mutations in the skeletal muscle myosin heavy chain. We identified two chemical mutagen generated flightless mutants, Ifm(2)RU1 and ifm(2)RU2 that map closely to myosin heavy chain gene (Mhc) region. Since there are no structural proteins predicted in the mapped region, it was likely that these two are Mhc mutations. We show that Ifm(2)RU1 and ifm(2)RU2 are indeed Mhc mutations and the molecular aberrations affect amino acid residues present in the myosin rod region. Human muscle myosin heavy chain (MyHC) mutations that cause Laing early onset distal myopathy and myosin storage myopathy occur in this domain of the protein. Even though mutations lie in the same region of myosin rod, Ifm(2)RU1 is semidominant, whereas ifm(2)RU2 is recessive. Both the mutants show IFM defects and the presence of abnormal myofibrils. Mutant myofibrillar structures can be rescued with an additional wild type Mhc gene copy. However, the restored myofibrillar structure is incapable of rescuing the flight ability of mutants. The muscle phenotypes are due to defects in thick filament assembly which manifest from the early stages of sarcomere development. The MHC protein rod region is an α-helix that forms coiled-coils which further self assemble to form thick filaments or aggregates as observed in in vitro conditions. Biophysical and biochemical analyses reveal that the coiled-coil structure of mutant rods is not affected, however the thermodynamic stability is altered in ifm(2)RU2 mutation. Interestingly, rod aggregate size and stability are not affected in mutant rods. The Drosophila MHC mutant rods were studied along with four MHC mutant rods that harbour human rod mutations to compare the molecular consequences. The Drosophila mutations do not hamper the rod structure and assembly. Therefore, the defects may arise due to altered interactions with myosin rod binding proteins. Flightin is an extensively studied myosin rod binding protein. The amount and phosphorylation status of flightin are an extremely sensitive measure of thick filament assembly. Flightin phosphorylation is affected in the mutants suggesting a functional dependence on MHC and it also indicates MHC instability. In the light of the work done, we have assessed the mutations with respect to their structure-functional implications. The acto-myosin interactions responsible for the defects are also discussed. Formation of unusual myofibrillar structures are analysed with regards to the process of myofibrillogenesis. An understanding of this entire process with the information available from IFM is reviewed in detail. The work so far has helped in understanding the manifestation of myopathies at tissue/cellular levels with insights into the plausible mechanisms of origin of the disease phenotypes. Myopathic condition may arise due to developmental or functional defects. For therapeutic considerations, the fly provides a simple test to inspect the effects of adding extra copies of the wild type gene. We conclude that the Drosophila IFM provide a good model system for the study of human ACTA1 and MyHC myopathies.

Myofibrillogenesis is a complex process, which involves assembly of hundreds of structural proteins in a highly ordered manner to form the contractile structural unit of muscle, the sarcomere. Several myopathic conditions reported in humans are caused due to abnormal myofibrillogenesis owing to mutations in the genes coding for many of these structural proteins. These myopathies have highly variable clinical features and time of onset. Since their aetiology is poorly understood, it becomes imperative to have a model system to study the muscle defects. Present study proposes to employ the Indirect Flight Muscle (IFM) system in Drosophila melanogaster as a model to analyse the development/onset of some of these myopathies and resulting pathophysiology. We have carried out a systematic study on mutations in two major proteins of the sarcomere, actin and myosin, to understand the pathophysiology associated with the disease conditions and in turn gain insights into the process of myofibrillogenesis. To verify whether the human muscle phenotypes are observed in flies, we analysed the IFM for functional and structural defects categorised by the presence of aberrant sarcomeric structures. An important question that we have addressed is whether mutants of the Drosophila IFM recapitulate human conditions and whether it can serve as a good genetic model to study the developmental mechanisms of the human skeletal myopathies in vivo. Mutations of the human ACTA1 skeletal actin gene produce seven congenital myopathies – actin myopathy, nemaline rod myopathy, intranuclear rod myopathy, congenital fibre type disproportion, congenital myopathy with core-like areas, cap disease and zebra body myopathy. Four known mutations in Act88F—a Drosophila homologue of ACTA1—occur at the same actin residues mutated in ten ACTA1 nemaline mutations, A138D/P, R256H/L, G268C/D/R/S and R372C/S. These Act88F mutants were examined for muscle phenotypes with nemaline structures. Mutant homozygotes show phenotypes ranging from lack of myofibrils to almost normal sarcomeres at eclosion. Whereas, heterozygotes do allow myofibrillar assembly to certain extent; however, atypical structures are seen adjacent to normal sarcomeres. Aberrant Z disc-like structures and serial Z disc arrays, ‘zebra bodies’, are observed in homozygotes and heterozygotes of all the four Act88F mutants. The electron-dense structures observed in electron micrographs show homologies to human nemaline bodies/rods, but are much smaller than those typically found in the human myopathy. A possible mechanism for the ‘zebra bodies’ is proposed based on this study. Analysis of IFM at early developmental stages shows that in three of the mutants, there is an abnormal myofibril assembly leading to malformed sarcomeres mirrored in the adult stages. In one of the Act88F mutants, normal myofibrils are seen post-eclosion but the IFM show activity dependant progression of muscle degeneration. All the Act88F mutants produce dominant disruption of muscle structure and function which cannot be rescued even by three copies of the wild type Act88F gene implying that the mutants are strong antimorphs. Myosin myopathies are a group of human muscle diseases with heterogeneous clinical features and are caused by mutations in the skeletal muscle myosin heavy chain. We identified two chemical mutagen generated flightless mutants, Ifm(2)RU1 and ifm(2)RU2 that map closely to myosin heavy chain gene (Mhc) region. Since there are no structural proteins predicted in the mapped region, it was likely that these two are Mhc mutations. We show that Ifm(2)RU1 and ifm(2)RU2 are indeed Mhc mutations and the molecular aberrations affect amino acid residues present in the myosin rod region. Human muscle myosin heavy chain (MyHC) mutations that cause Laing early onset distal myopathy and myosin storage myopathy occur in this domain of the protein. Even though mutations lie in the same region of myosin rod, Ifm(2)RU1 is semidominant, whereas ifm(2)RU2 is recessive. Both the mutants show IFM defects and the presence of abnormal myofibrils. Mutant myofibrillar structures can be rescued with an additional wild type Mhc gene copy. However, the restored myofibrillar structure is incapable of rescuing the flight ability of mutants. The muscle phenotypes are due to defects in thick filament assembly which manifest from the early stages of sarcomere development. The MHC protein rod region is an α-helix that forms coiled-coils which further self assemble to form thick filaments or aggregates as observed in in vitro conditions. Biophysical and biochemical analyses reveal that the coiled-coil structure of mutant rods is not affected, however the thermodynamic stability is altered in ifm(2)RU2 mutation. Interestingly, rod aggregate size and stability are not affected in mutant rods. The Drosophila MHC mutant rods were studied along with four MHC mutant rods that harbour human rod mutations to compare the molecular consequences. The Drosophila mutations do not hamper the rod structure and assembly. Therefore, the defects may arise due to altered interactions with myosin rod binding proteins. Flightin is an extensively studied myosin rod binding protein. The amount and phosphorylation status of flightin are an extremely sensitive measure of thick filament assembly. Flightin phosphorylation is affected in the mutants suggesting a functional dependence on MHC and it also indicates MHC instability. In the light of the work done, we have assessed the mutations with respect to their structure-functional implications. The acto-myosin interactions responsible for the defects are also discussed. Formation of unusual myofibrillar structures are analysed with regards to the process of myofibrillogenesis. An understanding of this entire process with the information available from IFM is reviewed in detail. The work so far has helped in understanding the manifestation of myopathies at tissue/cellular levels with insights into the plausible mechanisms of origin of the disease phenotypes. Myopathic condition may arise due to developmental or functional defects. For therapeutic considerations, the fly provides a simple test to inspect the effects of adding extra copies of the wild type gene. We conclude that the Drosophila IFM provide a good model system for the study of human ACTA1 and MyHC myopathies.

Mitochondrial Complex I (CI) is composed of 44 distinct subunits that are assembled with eight Fe- S clusters and a single flavin mononucleotide. Mitochondria is highly enriched in the flight muscles of Drosophila melanogaster, however the assembly mechanism of Drosophila CI has not been described. We report that the mechanism of CI biogenesis in Drosophila flight muscles proceeds via the formation of ~315- , ~550-, and ~815 kDa CI assembly intermediates. Additionally, we define specific roles for several CI subunits in the assembly process. In particular, we show that dNDUFS5 is required for converting the ~700 kDa transient CI assembly intermediate into the ~815 kDa assembly intermediate, by stabilizing or promoting the incorporation of dNDUFA10 into the complex. Our findings highlight the potential values of Drosophila as a suitable model organism and resource to study the CI biogenesis in vivo, and to address questions relevant to CI biogenesis in humans. CI biogenesis is regulated by transient interactors known as CI assembly factors (CIAFs). To date, about half of CI disorders are attributed to the mutations in the CI subunits and the known CIAFs. The cause for the other half remains to be discovered, warranting the investigation for additional regulators of CI biogenesis such as novel CIAFs. To identify novel regulators, we cataloged interactors of a core subunit, NDUFS3, knocked each one down by RNAi in the Drosophila flight muscle, and analyzed its effect in the stability of CI by blue-native PAGE. We identified the Drosophila Fragile X Mental Retardation protein (dFMRP) to destabilize the holoenzyme of CI and cause it to misassemble. Therefore, we report dFMRP as a novel regulator of CI biogenesis, and demonstrate the utilization of Drosophila as an effective model system to uncover the mysteries of CI biogenesis.

Locomotion is essential for animals and it is regulated by crosstalk between neurons and muscles. Although defects in the crosstalk between neurons and muscles are implicated in many mobility related diseases in human, however, the underlying molecular mechanisms have not been well characterized. In an attempt to gain insights into the complex neuro-muscular crosstalk at molecular level, we have used Drosophila melanogaster as model system to elucidate function of a gene, taxi, mutations in the gene give rise to defective flight behavior resulting from a defective neuro-muscular crosstalk. The first chapter of this thesis explains how crosstalk between neurons and muscles control different behaviors of Drosophila melanogaster, particularly locomotion. Apart from locomotion, this chapter also explains how flies perform vision and how they response to various stimuli. Second chapter explains materials and methods used to complete the studies. Third chapter explains genetic and phenotypic characterization of jumper mutant, an allele of taxi gene. Further, based on experimental data, how the mutation affects the taxi at both transcription and translational levels have been discussed. We found that I-element insertion located at 5’UTR of the taxi in jumper is responsible for the defective flight behavior in jumper mutant. The I-element insertion leads to increased expression level of taxi in the head without affecting much of transcription and translation in other body parts. Fourth chapter explains the role of taxi in flight behavior of Drosophila melanogaster. We found that knockdown of taxi in neurons gives rise to compromised flight ability. Spatio-temporal conditional knockdown experiments suggest that taxi’s function is critical at around 12 hours After Puparium Formation (APF). Further, from our RNA sequencing result of taxi null, we found that the genes important for maintaining membrane potential are differentially expressed. As a result, neuronal transmission from brain to indirect flight muscles (IFMs) through peripherally synapsing interneurons (PSI) gets altered, which might lead to reduction in wing beat duration of taxi mutants. We found that one of the main regulators of membrane potential is Adar, a gene that is crucial to many molecular and physiological activities, and it is repressed by taxi. Fifth chapter of this thesis explains how taxi affects life span and phototaxis. We found that over-expression of taxi in neurons beyond threshold shorten life span of flies. It is also observed that phototaxis is abnormal with disruption in structure of ommatidia. Six chapter of this thesis explains conclusion of the study. Overall, in present study we have reported for the first time neuronal function of taxi in flight behavior, life span and phototransduction of Drosophila melanogaster.