Drosophila Myogenesis Research Articles

LanB1 Cooperates With Kon-Tiki During Embryonic Muscle Migration in Drosophila.

Muscle development is a multistep process that involves cell specification, myoblast fusion, myotube migration, and attachment to the tendons. In spite of great efforts trying to understand the basis of these events, little is known about the molecular mechanisms underlying myotube migration. Knowledge of the few molecular cues that guide this migration comes mainly from studies in Drosophila. The migratory process of Drosophila embryonic muscles involves a first phase of migration, where muscle progenitors migrate relative to each other, and a second phase, where myotubes migrate searching for their future attachment sites. During this phase, myotubes form extensive filopodia at their ends oriented preferentially toward their attachment sites. This myotube migration and the subsequent muscle attachment establishment are regulated by cell adhesion receptors, such as the conserved proteoglycan Kon-tiki/Perdido. Laminins have been shown to regulate the migratory behavior of many cell populations, but their role in myotube migration remains largely unexplored. Here, we show that laminins, previously implicated in muscle attachment, are indeed required for muscle migration to tendon cells. Furthermore, we find that laminins genetically interact with kon-tiki/perdido to control both myotube migration and attachment. All together, our results uncover a new role for the interaction between laminins and Kon-tiki/Perdido during Drosophila myogenesis. The identification of new players and molecular interactions underlying myotube migration broadens our understanding of muscle development and disease.

An insight on Drosophila myogenesis and its assessment techniques.

Movement assisted by muscles forms the basis of various behavioural traits seen in Drosophila. Myogenesis involves developmental processes like cellular specification, differentiation, migration, fusion, adherence to tendons and neuronal innervation in a series of coordinated event well defined in body space and time. Gene regulatory networks are switched on-off, fine tuning at the right developmental stage to assist each cellular event. Drosophila is a holometabolous organism that undergoes myogenesis waves at two developmental stages, and is ideal for comparative analysis of the role of genes and genetic pathways conserved across phyla. In this review we have summarized myogenic events from the embryo to adult focussing on the somatic muscle development during the early embryonic stage and then on indirect flight muscles (IFM) formation required for adult life, emphasizing on recent trends of analysing muscle mutants and advances in Drosophila muscle biology.

Morphogenesis of the somatic musculature in Drosophila melanogaster.

In Drosophila melanogaster, the somatic muscle system is first formed during embryogenesis, giving rise to the larval musculature. Later during metamorphosis, this system is destroyed and replaced by an entirely new set of muscles in the adult fly. Proper formation of the larval and adult muscles is critical for basic survival functions such as hatching and crawling (in the larva), walking and flying (in the adult), and feeding (at both larval and adult stages). Myogenesis, from mononucleated muscle precursor cells to multinucleated functional muscles, is driven by a number of cellular processes that have begun to be mechanistically defined. Once the mesodermal cells destined for the myogenic lineage have been specified, individual myoblasts fuse together iteratively to form syncytial myofibers. Combining cytoplasmic contents demands a level of intracellular reorganization that, most notably, leads to redistribution of the myonuclei to maximize internuclear distance. Signaling from extending myofibers induces terminal tendon cell differentiation in the ectoderm, which results in secure muscle-tendon attachments that are critical for muscle contraction. Simultaneously, muscles become innervated and undergo sarcomerogenesis to establish the contractile apparatus that will facilitate movement. The cellular mechanisms governing these morphogenetic events share numerous parallels to mammalian development, and the basic unit of all muscle, the myofiber, is conserved from flies to mammals. Thus, studies of Drosophila myogenesis and comparisons to muscle development in other systems highlight conserved regulatory programs of biomedical relevance to general muscle biology and studies of muscle disease.

Coordinated repression and activation of two transcriptional programs stabilizes cell fate during myogenesis

Molecular models of cell fate specification typically focus on the activation of specific lineage programs. However, the concurrent repression of unwanted transcriptional networks is also essential to stabilize certain cellular identities, as shown in a number of diverse systems and phyla. Here, we demonstrate that this dual requirement also holds true in the context of Drosophila myogenesis. By integrating genetics and genomics, we identified a new role for the pleiotropic transcriptional repressor Tramtrack69 in myoblast specification. Drosophila muscles are formed through the fusion of two discrete cell types: founder cells (FCs) and fusion-competent myoblasts (FCMs). When tramtrack69 is removed, FCMs appear to adopt an alternative muscle FC-like fate. Conversely, ectopic expression of this repressor phenocopies muscle defects seen in loss-of-function lame duck mutants, a transcription factor specific to FCMs. This occurs through Tramtrack69-mediated repression in FCMs, whereas Lame duck activates a largely distinct transcriptional program in the same cells. Lineage-specific factors are therefore not sufficient to maintain FCM identity. Instead, their identity appears more plastic, requiring the combination of instructive repressive and activating programs to stabilize cell fate.

Coordinated repression and activation of two transcriptional programs stabilizes cell fate during myogenesis.

Molecular models of cell fate specification typically focus on the activation of specific lineage programs. However, the concurrent repression of unwanted transcriptional networks is also essential to stabilize certain cellular identities, as shown in a number of diverse systems and phyla. Here, we demonstrate that this dual requirement also holds true in the context of Drosophila myogenesis. By integrating genetics and genomics, we identified a new role for the pleiotropic transcriptional repressor Tramtrack69 in myoblast specification. Drosophila muscles are formed through the fusion of two discrete cell types: founder cells (FCs) and fusion-competent myoblasts (FCMs). When tramtrack69 is removed, FCMs appear to adopt an alternative muscle FC-like fate. Conversely, ectopic expression of this repressor phenocopies muscle defects seen in loss-of-function lame duck mutants, a transcription factor specific to FCMs. This occurs through Tramtrack69-mediated repression in FCMs, whereas Lame duck activates a largely distinct transcriptional program in the same cells. Lineage-specific factors are therefore not sufficient to maintain FCM identity. Instead, their identity appears more plastic, requiring the combination of instructive repressive and activating programs to stabilize cell fate.

Identification and characterization of novel Kirrel isoform during myogenesis

Somatic cell fusion is an essential component of skeletal muscle development and growth and repair from injury. Additional cell types such as trophoblasts and osteoclasts also require somatic cell fusion events to perform their physiological functions. Currently we have rudimentary knowledge on molecular mechanisms regulating somatic cell fusion events in mammals. We therefore investigated during in vitro murine myogenesis a mammalian homolog, Kirrel, of the Drosophila Melanogaster genes Roughest (Rst) and Kin of Irre (Kirre) which regulate somatic muscle cell fusion during embryonic development. Our results demonstrate the presence of a novel murine Kirrel isoform containing a truncated cytoplasmic domain which we term Kirrel B. Protein expression levels of Kirrel B are inverse to the occurrence of cell fusion events during in vitro myogenesis which is in stark contrast to the expression profile of Rst and Kirre during myogenesis in Drosophila. Furthermore, chemical inhibition of cell fusion confirmed the inverse expression pattern of Kirrel B protein levels in relation to cell fusion events. The discovery of a novel Kirrel B protein isoform during myogenesis highlights the need for more thorough investigation of the similarities and potential differences between fly and mammals with regards to the muscle cell fusion process.

Akirin Links Twist-Regulated Transcription with the Brahma Chromatin Remodeling Complex during Embryogenesis

The activities of developmentally critical transcription factors are regulated via interactions with cofactors. Such interactions influence transcription factor activity either directly through protein–protein interactions or indirectly by altering the local chromatin environment. Using a yeast double-interaction screen, we identified a highly conserved nuclear protein, Akirin, as a novel cofactor of the key Drosophila melanogaster mesoderm and muscle transcription factor Twist. We find that Akirin interacts genetically and physically with Twist to facilitate expression of some, but not all, Twist-regulated genes during embryonic myogenesis. akirin mutant embryos have muscle defects consistent with altered regulation of a subset of Twist-regulated genes. To regulate transcription, Akirin colocalizes and genetically interacts with subunits of the Brahma SWI/SNF-class chromatin remodeling complex. Our results suggest that, mechanistically, Akirin mediates a novel connection between Twist and a chromatin remodeling complex to facilitate changes in the chromatin environment, leading to the optimal expression of some Twist-regulated genes during Drosophila myogenesis. We propose that this Akirin-mediated link between transcription factors and the Brahma complex represents a novel paradigm for providing tissue and target specificity for transcription factor interactions with the chromatin remodeling machinery.

Org-1, the Drosophila ortholog of Tbx1, is a direct activator of known identity genes during muscle specification

Members of the T-Box gene family of transcription factors are important players in regulatory circuits that generate myogenic and cardiogenic lineage diversities in vertebrates. We show that during somatic myogenesis in Drosophila, the single ortholog of vertebrate Tbx1, optomotor-blind-related-gene-1 (org-1), is expressed in a small subset of muscle progenitors, founder cells and adult muscle precursors, where it overlaps with the products of the muscle identity genes ladybird (lb) and slouch (slou). In addition, org-1 is expressed in the lineage of the heart-associated alary muscles. org-1 null mutant embryos lack Lb and Slou expression within the muscle lineages that normally co-express org-1. As a consequence, the respective muscle fibers and adult muscle precursors are either severely malformed or missing, as are the alary muscles. To address the mechanisms that mediate these regulatory interactions between Org-1, Lb and Slou, we characterized distinct enhancers associated with somatic muscle expression of lb and slou. We demonstrate that these lineage- and stage-specific cis-regulatory modules (CRMs) bind Org-1 in vivo, respond to org-1 genetically and require T-box domain binding sites for their activation. In summary, we propose that org-1 is a common and direct upstream regulator of slou and lb in the developmental pathway of these two neighboring muscle lineages. Cross-repression between slou and lb and combinatorial activation of lineage-specific targets by Org-1-Slou and Org-1-Lb, respectively, then leads to the distinction between the two lineages. These findings provide new insights into the regulatory circuits that control the proper pattering of the larval somatic musculature in Drosophila.

Differential requirements for Myocyte Enhancer Factor-2 during adult myogenesis in Drosophila

Identifying the genetic program that leads to formation of functionally and morphologically distinct muscle fibers is one of the major challenges in developmental biology. In Drosophila, the Myocyte Enhancer Factor-2 (MEF2) transcription factor is important for all types of embryonic muscle differentiation. In this study we investigated the role of MEF2 at different stages of adult skeletal muscle formation, where a diverse group of specialized muscles arises. Through stage- and tissue-specific expression of Mef2 RNAi constructs, we demonstrate that MEF2 is critical at the early stages of adult myoblast fusion: mutant myoblasts are attracted normally to their founder cell targets, but are unable to fuse to form myotubes. Interestingly, ablation of Mef2 expression at later stages of development showed MEF2 to be more dispensable for structural gene expression: after myoblast fusion, Mef2 knockdown did not interrupt expression of major structural gene transcripts, and myofibrils were formed. However, the MEF2-depleted fibers showed impaired integrity and a lack of fibrillar organization. When Mef2 RNAi was induced in muscles following eclosion, we found no adverse effects of attenuating Mef2 function. We conclude that in the context of adult myogenesis, MEF2 remains an essential factor, participating in control of myoblast fusion, and myofibrillogenesis in developing myotubes. However, MEF2 does not show a major requirement in the maintenance of muscle structural gene expression. Our findings point to the importance of a diversity of regulatory factors that are required for the formation and function of the distinct muscle fibers found in animals.

Downstream of Identity Genes: Muscle-Type-Specific Regulation of the Fusion Process

In all metazoan organisms, the diversification of cell types involves determination of cell fates and subsequent execution of specific differentiation programs. During Drosophila myogenesis, identity genes specify the fates of founder myoblasts, from which derive all individual larval muscles. Here, to understand how cell fate information residing within founders is translated during differentiation, we focus on three identity genes, eve, lb, and slou, and how they control the size of individual muscles by regulating the number of fusion events. They achieve this by setting expression levels of Mp20, Pax, and mspo, three genes that regulate actin dynamics and cell adhesion and, as we show here, modulate the fusion process in a muscle-specific manner. Thus, these data show how the identity information implemented by transcription factors is translated via target genes into cell-type-specific programs of differentiation.

RacGAP50C directs perinuclear γ-tubulin localization to organize the uniform microtubule array required forDrosophilamyotube extension

The microtubule (MT) cytoskeleton is reorganized during myogenesis as individual myoblasts fuse into multinucleated myotubes. Although this reorganization has long been observed in cell culture, these findings have not been validated during development, and proteins that regulate this process are largely unknown. We have identified a novel postmitotic function for the cytokinesis proteins RacGAP50C (Tumbleweed) and Pavarotti as essential regulators of MT organization during Drosophila myogenesis. We show that the localization of the MT nucleator gamma-tubulin changes from diffuse cytoplasmic staining in mononucleated myoblasts to discrete cytoplasmic puncta at the nuclear periphery in multinucleated myoblasts, and that this change in localization depends on RacGAP50C. RacGAP50C and gamma-tubulin colocalize at perinuclear sites in myotubes, and in RacGAP50C mutants gamma-tubulin remains dispersed throughout the cytoplasm. Furthermore, we show that the mislocalization of RacGAP50C in pavarotti mutants is sufficient to redistribute gamma-tubulin to the muscle fiber ends. Finally, myotubes in RacGAP50C mutants have MTs with non-uniform polarity, resulting in multiple guidance errors. Taken together, these findings provide strong evidence that the reorganization of the MT network that has been observed in vitro plays an important role in myotube extension and muscle patterning in vivo, and also identify two molecules crucial for this process.

Founder cells regulate fiber number but not fiber formation during adult myogenesis in Drosophila

During insect myogenesis, myoblasts are organized into a pre-pattern by specialized organizer cells. In the Drosophila embryo, these cells have been termed founder cells and play important roles in specifying muscle identity and in serving as targets for myoblast fusion. A group of adult muscles, the dorsal longitudinal (flight) muscles, DLMs, is patterned by persistent larval scaffolds; the second set, the dorso-ventral muscles, DVMs is patterned by mono-nucleate founder cells (FCs) that are much larger than the surrounding myoblasts. Both types of organizer cells express Dumbfounded, which is known to regulate fusion during embryonic myogenesis. The role of DVM founder cells as well as the DLM scaffolds was tested in genetic ablation studies using the UAS/Gal4 system of targeted transgene expression. In both cases, removal of organizer cells prior to fusion, causes formation of supernumerary fibers, suggesting that cells in the myoblast pool have the capacity to initiate fiber formation, which is normally inhibited by the organizers. In addition to the large DVM FCs, some (smaller) cells in the myoblast pool also express Dumbfounded. We propose that these cells are responsible for seeding supernumerary fibers, when DVM FCs are eliminated prior to fusion. When these cells are also eliminated, myogenesis fails to occur. In the second set of studies, targeted expression of constitutively active Ras V12 also resulted in the appearance of supernumerary fibers. In this case, the original DVM FCs are present, suggesting alterations in cell fate. Taken together, these data suggest that DVM myoblasts are able to respond to cues other than the original founder cell, to initiate fusion and fiber formation. Thus, the role of the large DVM founder cells is to generate the correct number of fibers, but they are not required for fiber formation itself. We also present evidence that the DVM FCs may arise from the leg imaginal disc.

Daughterless dictates Twist activity in a context-dependent manner during somatic myogenesis

Somatic myogenesis in Drosophila relies on the reiterative activity of the basic helix–loop–helix transcriptional regulator, Twist (Twi). How Twi directs multiple cell fate decisions over the course of mesoderm and muscle development is unclear. Previous work has shown that Twi is regulated by its dimerization partner: Twi homodimers activate genes necessary for somatic myogenesis, whereas Twi/Daughterless (Da) heterodimers lead to the repression of these genes. Here, we examine the nature of Twi/Da heterodimer repressive activity. Analysis of the Da protein structure revealed a Da repression (REP) domain, which is required for Twi/Da-mediated repression of myogenic genes, such as Dmef2, both in tissue culture and in vivo. This domain is crucial for the allocation of mesodermal cells to distinct fates, such as heart, gut and body wall muscle. By contrast, the REP domain is not required in vivo during later stages of myogenesis, even though Twi activity is required for muscles to achieve their final pattern and morphology. Taken together, we present evidence that the repressive activity of the Twi/Da dimer is dependent on the Da REP domain and that the activity of the REP domain is sensitive to tissue context and developmental timing.

Mind bomb 2, a founder myoblast-specific protein, regulates myoblast fusion and muscle stability

A fundamental step during Drosophila myogenesis is the specification of founder myoblasts (FMs). Founders possess the information required for the acquisition of muscle identity and for the execution of the myogenic programme, whereas fusion-competent myoblasts (FCMs) acquire this information after fusing to founders. Very little is known about genes that implement the execution of the myogenic programme. Here we characterise Mind bomb 2 (Mib2), a protein with putative E3 ubiquitin ligase activity that is exclusive of FMs and necessary for at least two distinct steps of the founder/myotube differentiation programme. Thus, in mib2 mutants, the early process of myoblast fusion is compromised, as FMs undergo a reduced number of rounds of fusion with FCMs. At later stages, with the onset of muscle contraction, many muscles degenerate, display aberrant sarcomeric structure and detach from tendons. The fusion process requires intact E3-RING-finger domains of Mib2 (the putative catalytic sites), probably to eliminate the FCM-specific activator Lmd from nascent myotubes. However, these sites appear dispensable for muscle integrity. This, and the subcellular accumulation of Mib2 in Z and M bands of sarcomeres, plus its physical interaction with nonmuscle myosin (a Z-band-localised protein necessary for the formation of myofibrils), suggest a structural role for Mib2 in maintaining sarcomeric stability. We suggest that Mib2 acts sequentially in myoblast fusion and sarcomeric stability by two separable processes involving distinct functions of Mib2.

Kette and blown fuse interact genetically during the second fusion step of myogenesis in Drosophila.

Drosophila myoblast fusion proceeds in two steps. The first one gives rise to small syncytia, the muscle precursor cells, which then recruit further fusion competent myoblasts to reach the final muscle size. We have identified Kette as an essential component for myoblast fusion. In kette mutants, founder cells and fusion-competent myoblasts are determined correctly and overcome the very first fusion. But then, at the precursor cell stage, fusion is interrupted. At the ultrastructural level, fusion is characterised by cell-cell recognition, alignment, formation of prefusion complexes, electron dense plaques and membrane breakdown. In kette mutants, electron dense plaques of aberrant length accumulate and fusion is interrupted owing to a complete failure of membrane breakdown. Furthermore, we show that kette interacts genetically with blown fuse (blow) which is known to be required to proceed from prefusion complexes to the formation of the electron dense plaques. Interestingly, a surplus of Kette can replace Blow function during myogenesis. We propose a model in which Dumbfounded/Sticks and stones-dependent cell adhesion is mediated over Rolling Pebbles, Myoblast city, Crk, Blown fuse and Kette, and thus induces membrane fusion.

Myogenic differentiation of Drosophila Schneider cells by DNA double-strand break-inducing drugs

Drosophila melanogaster has been widely used as a model organism to study various aspects of development. Apart from the whole Drosophila embryo, there are a number of cultured cell lines derived from Drosophila embryo that have also been used for elucidating various aspects of development. Drosophila Schneider line 2 cells were derived from the late stages of the embryo (Schneider, 1972). We found that the Schneider cells undergo myogenic differentiation upon treatment with neocarzinostatin (NCS), DNA double-strand break (DSB)-inducing drug, as indicated by elongated morphology, myosin heavy chain protein expression, multinucleation and exit from the cell cycle. No induction of differentiation was observed when cell proliferation was inhibited with drugs that do not cause DNA DSBs. Pre-treatment of Schneider cells with inhibitors of PKC, PP 1/2A, p38 MAPK, JNK and proteasomes resulted in the inhibition of morphological differentiation induced by NCS. These results indicate that DNA DSBs can turn on the myogenic program in Drosophila Schneider cells and the process is dependent on PK C-, PP 1/2A-, p38 MAPK-, and JNK- mediated signaling and proteasomal activity. The molting hormone, 20-hydroxyecdysone (20-HE), also showed an anti-myogenic effect on the process. This is the first report of insect cells undergoing differentiation by DNA DSB-inducing drugs as far as we know, and it provides a very useful and convenient in vitro system to study various aspects of Drosophila myogenesis.

Notch: a membrane-bound transcription factor.

Notch signaling is both simple and complex. It is simple because no secondary messengers are required as Notch can directly report to the nucleus after contact with ligand at the plasma membrane. Notch acts as a membranebound transcription factor that is released in response to ligand binding by two

Myoblasts incompetent encodes a zinc finger transcription factor required to specify fusion-competent myoblasts in Drosophila.

We report a new gene, myoblasts incompetent, essential for normal myogenesis and myoblast fusion in Drosophila. myoblasts incompetent encodes a putative zinc finger transcription factor related to vertebrate Gli proteins and to Drosophila Cubitus interruptus. myoblasts incompetent is expressed in immature somatic and visceral myoblasts. Expression is predominantly in fusion-competent myoblasts and a loss-of-function mutation in myoblasts incompetent leads to a failure in the normal differentiation of these cells and a complete lack of myoblast fusion. In the mutant embryos, founder myoblasts differentiate normally and form mononucleate muscles, but genes that are specifically expressed in fusion-competent cells are not activated and the normal downregulation of twist expression in these cells fails to occur. In addition, fusion-competent myoblasts fail to express proteins characteristic of the general pathway of myogenesis such as myosin and Dmef2. Thus myoblasts incompetent appears to function specifically in the general pathway of myogenesis to control the differentiation of fusion-competent myoblasts.

Muscle LIM proteins are associated with muscle sarcomeres and require dMEF2 for their expression during Drosophila myogenesis.

A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Previously, we characterized two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, which show restricted expression in differentiating muscle lineages. To extend our analysis of Drosophila Mlps, we characterized the expression of Mlps in mutant backgrounds that disrupt specific aspects of muscle development. We show a genetic requirement for the transcription factor dMEF2 in regulating Mlp expression and an ability of dMEF2 to bind, in vitro, to consensus MEF2 sites derived from those present in Mlp genomic sequences. These data suggest that the Mlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, we analyzed the subcellular distribution of Mlp84B in detail. Immunofluorescent studies revealed the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and alpha-actinin, key components of these structures, also failed to perturb Mlp84B distribution. In conclusion, we have used molecular epistasis analysis to position Mlp function downstream of events involving mesoderm specification and patterning and concomitant with terminal muscle differentiation. Furthermore, our results are consistent with a structural role for Mlps as components of muscle cytoarchitecture.

Muscle pattern diversification in Drosophila: the story of imaginal myogenesis.

There are two phases of somatic muscle formation in Drosophila. During embryonic development, one phase of myogenesis generates larval muscle elements that mediate the relatively simple behavioural repertoire of the larva. During pupal metamorphosis, a diverse pattern of muscle fibres are assembled, and these facilitate the more elaborate behavioural patterns of the adult fly. In this review, we discuss the current status of understanding of the cellular, genetic, and molecular mechanisms of pattern formation during the second phase, imaginal muscle development. We briefly compare aspects of embryonic and adult myogenesis in Drosophila and muscle development in vertebrates and highlight conserved themes and disparities between these diverse myogenic programmes.