Primer: Drosophila Myogenesis

The skeletal muscle system is the largest organ in motile animals, constituting between 35 to 55% of the human body mass, and up to 75% of the body mass in flying organisms like Drosophila. The flight muscles alone in flying insects comprise up to 65% of total body mass. Not only is the musculature the largest organ system, it is also exquisitely complex, with single muscles existing in different shapes and sizes. These different morphologies allow for such different functions as the high frequency beating of a wing in a hummingbird, the dilation of the pupil in a human eye, or the maintenance of posture in a giraffe’s neck.

Myogenesis, the development of the musculature, has received considerable attention for its unveiling of basic mechanisms including signaling, transcriptional and posttranscriptional control of cell fate, cell-cell fusion, cellular differentiation and cellular syncytium repair. An increased focus in the field is application of these basic mechanisms to congenital muscle diseases, aging, and cancer-induced muscle wasting (cachexia). The fruit fly Drosophila melanogaster has been the model system of choice for many aspects of myogenesis, successfully leading the field by identifying mechanisms for signal integration on specific promoters, the site for myoblast fusion site, the connection of aberrant myonuclear position to muscle function, and how forces sculpt myofibril formation, among many others. These paradigms have provided novel genes and mechanisms and have shaped studies in other model systems. Moreover the fly model system continues to be adapted to address novel challenges, especially in disease modeling, and no doubt will influence and be influenced by other models. Myogenesis research in Drosophila makes use of Drosophila’s short generation span, ease of genetic manipulation, simplicity of the muscle pattern, and optical tractability with the help of fluorescent reporters. Both basic cell biological processes, such as cell-cell fusion and organelle positioning, and systemic processes, such as muscle growth and atrophy, can be effectively studied in this system.

This primer introduces the key steps and notable variations during Drosophila myogenesis, including gastrulation and muscle formation in the embryo, muscle growth in the larva, and stem cell based muscle remodeling in the pupa, to give rise to a walking and flying adult. We will illustrate these relevant processes using the Dorsal Oblique 1 (DO1) muscle through the life of the organism as our example (Figure 1).

Figure 1. Lineage of the Dorsal Oblique Muscle 1.

Schematic of embryonic to adult lineage tracing of DO1. Embryo (upper and lower panels): The P17 premyogenic cluster gives rise to the DO1 FC (magenta) and the Dorsal AMP (DAMP, turquoise). The DO1 FC will develop into the DO1 larval muscle. Larva (upper and lower panels): The DO1 muscle grows significantly in size, the DAMP begins to proliferate and to self-renew at larval stage L2. Pupa (upper and lower panels): The DO1 muscle is histolyzed in the abdomen. The proliferating DAMPs will form the retractor of tergites (RoT, orange). In the 2^nd thoracic segment, DO1 perdures, dedifferentiates, fuses with the DAMPs, and, via longitudinal splitting, will form 2 dorsal longitudinal (indirect flight) muscles (DLM) (magenta/turquoise gradient). The DAMPs, via association with the wing disc, will also give rise to the dorsoventral (indirect flight) muscles (DVM, turquoise) and the direct flight muscles (DFM, yellow). Adult (upper and lower panels): total P17 offspring: abdominal Rot, thoracic DLMs, DVMs and DFMs.

MYOGENESIS

In contrast to mammals, Drosophila, as a holometabolous insect, has 4 distinct stages constituting its lifecycle: the embryo or egg, larva, pupa and adult. Two of these stages are mobile, with the organism occupying different ecological niches as a larva and as an adult. Consequently, two distinct sets of muscles are generated, unlike in vertebrates, and each are perfectly adapted to the lifestyle for those stages of development. The larva has 54 distinct muscle types with a total number of 459 individual muscle fibers. The adult presents with 152 muscle groups, which are generated either from stem cells de novo or through transdifferentiation of larval muscles with the stem cells. Using the tools available in Drosophila, the fate of a single myogenic cell and its progeny can be traced from its birth during gastrulation, through fate determination and myogenesis in the embryo, to the activation of the adult myogenic progenitors during the larval and pupal stages for adult muscle generation. Many cell biological processes of myogenesis are employed twice: once in the embryo and once in the pupa. We will describe each process in detail for the embryo and only address deviations from the embryonic program in the adult sections. The DO1 muscle in the embryo and larva is a singular tubular muscle that will give rise, depending on whether it is in a thoracic or abdominal segment, to either two adult thoracic fibrillar muscles or an adult abdominal tubular muscle.

FATE DETERMINATION

All myogenic cells, from fly, mouse or man, derive from the mesoderm, which is laid down during the process of gastrulation in the early embryo. The newly formed mesodermal cells in fly migrate within the coelom, along the ectoderm. As they migrate, these mesodermal cells divide and are exposed to several external signaling cues, including Wingless (Wg) and Decapentaplegic (Dpp). Similar to vertebrates, these signals cooperate to divide the mesoderm into segments and distinct domains, which generate the progenitors for different mesodermally derived tissues. Segmental identity is generated through Hox gene expression.

The first subdivision dissects the mesoderm in the fly according to the segmentation pattern of the animal, followed by a dorsal-ventral and an anterior-posterior compartmentalization. The anterior-posterior division of the mesoderm is achieved through high and low level expression of the transcription factor Twist in each hemisegment. This pattern of Twist determines somatic and cardiac muscle fate, marked by high Twist expression within the anterior portion of the segment, versus visceral musculature and fat body, marked by low Twist expression in the posterior part of the segment. Hence, in our example, the progenitor population for the DO1 muscle is located in the anterior part of the mesoderm, continuing to express the transcription factor Twist at high levels.

Equivalence groups and cell specification

Within the high Twist expressing mesodermal domains, local equivalence groups, marked by expression of Lethal of scute (l’sc), are established through a combination of signaling events including Wg, Dpp and Receptor Tyrosine Kinase (RTK) signaling. There are 18 clusters within each hemisegment, each consisting of 4–6 cells; the DO1 progenitors constitute cluster P17 (Figure 1). Equivalence groups have not yet been detected in other model systems; whether this is due to a lack of appropriate markers, resolution, or different mechanism to allocate myoblasts remains to be investigated as tools and techniques become available. Continuous Ras signaling and lateral inhibition via Notch signaling lead to the specification of 1–2 of these cells within the cluster as either a myogenic or cardiac progenitor. The remaining cells of the cluster that are not specified as progenitors are fated to become fusion competent myoblasts (FCMs) which will provide nuclei and cell mass to the growing muscle through fusion. Our exemplary DO1 muscle cell is now specified as a pre-myogenic progenitor of the P17 cluster and is expressing, in addition to Twist, the transcription factor l’sc.

Generation of founder cells and adult stem cells

The myogenic progenitors then undergo an asymmetric division via Notch signaling, resulting in either two different muscle founder cells (FC), one founder and one adult muscle progenitor cell (AMP), or one founder cell and one pericardial progenitor. The founder cell acts as a seed for the individual muscles in the embryo. The adult muscle progenitor retains Twist expression and remains quiescent until late larval stages. Single founder cells have not been detected in vertebrate systems whereas “founder” myotubes have. Nevertheless, in our example, a single progenitor from the P17 pro-muscle cluster gives rise to the DO1 FC and the only dorsal AMP in the abdominal hemisegments (Figure 1).

Establishing the transcriptional code

FCs are characterized by distinctive transcriptional profiles, which regulate the final properties of the muscle, such as shape, size, muscle - tendon attachment and innervation. In the absence of cell-cell fusion, these founder cells attempt to generate a correctly oriented muscle, albeit without the necessary cell mass which is provided by the fusion process. This transcriptionally encoded identity is based on the unique expression pattern of sets of transcription factors, known as identity genes, and is reminiscent of neuronal identity specification during neurogenesis in fly and vertebrates or of the cranial musculature in chicken and mice. Changes in the identity gene expression pattern in any given FC will affect final muscle morphology and thus function. In contrast to FC transcriptional diversity, the FCMs appear to have a more uniform transcriptional signature within each hemisegment. It is not well understood whether FCMs harbor additional information for final muscle morphology and function or whether they simply provide cell mass and organelles to the growing myotube.

At this point of myogenesis, the DO1 founder cell expresses, in addition to Twist, its unique combination of identity genes, including Krüppel (Kr), Nautilus (Nau), Tup1/Islet 1 and Muscle specific homeobox. These factors provide the cell with its unique morphological characteristics. The FC is now primed for growth through cell-cell fusion (Figure 1).

CELL-CELL FUSION

Initial muscle growth during embryonic development relies on cell-cell fusion to provide the necessary cell mass. To date, many fundamental discoveries about myogenic cell fusion in vivo has been made in Drosophila, and these findings form the essential framework for myoblast fusion in many model systems. Indeed, Drosophila continues to be on the forefront of in vivo myogenic and indeed cell fusion research. Myoblast fusion in Drosophila is directional and heterotypic: the FC and the surrounding FCMs recognize each other, establish a cell-cell contact and initiate membrane fusion. Cell fusion and the resulting cytoplasmic continuity is thought to be achieved through invasive filopodia, emanating from the FCM into the FC. This process of cell fusion requires extensive actin cytoskeleton modulations, generating the necessary force, and ultimately the formation of the fusogenic synapse and fusion pore opening. The molecular pathway governing these changes in the actin cytoskeleton is illustrated in Figure 2. A particular number of fusion cycles, which is thought to be dictated by the identity transcriptional regulators expressed in a particular FC, results in different myotube sizes for distinct muscles at the end of the fusion phase. The DO1 muscle, for example, will incorporate 24 nuclei.

Figure 2. Molecular pathway of myogenic cell-cell fusion.

Left: Founder Cell/Myotube, right: Fusion competent myoblast. Receptor interaction (Duf/Rst, Sns/Hbs, blue) and receptor recycling in the FC (rols, yellow) triggers signaling cascades in both cell types involving PIP2 (green), Crk/Dock (turquoise), and, FCM specific, Loner (yellow). The FCM pathway splits in 2 branches via Mbc/Elmo (magenta) to Rac (red) and ultimately Scar/WAVE (red), as well as via Blow/Dwip (orange) to WASp (orange). Both cascades converge onto Arp2/3 (white) resulting in actin branching and invasive podosome formation. PI(4,5)P2 (PIP2) – Diaphanous (Dia) (purple) can inhibit Scar activity. The FC pathway co-opts the PIP2-Myoblast City (Mbc)/Elmo-Rac-Scar axis to build an actin sheath. A second pathway via Dock (turquoise), RhoGTP – Rok (orange) leads to MyoII (brown) activity to provide membrane tension for the fusion process. Bottom picture: fusion event in the embryo, membranes labeled with PIP2 (green), F-actin cytoskeletal structures (white) and RacGTP activity (red).

Recognition

In a first step to mediate cell-cell fusion, the FC has to recognize and make contact with the surrounding FCMs. Recognition and adhesion is mediated by cell specific immunoglobin domain-containing transmembrane receptors, which signal to the actin cytoskeleton (Figure 2). Each cell type expresses two closely related receptors, Dumbfounded (Duf)/Roughest (Rst) in the FC and Sticks and Stones (Sns)/Hibris(Hbs) in the FCM, the first of each pair is necessary and sufficient for fusion and a second that is not. The reasons for this dual receptor presence without complete redundancy are not understood. The FC recognizes a single, neighboring FCM through these receptor interactions, forms a stable interface and initiates fusogenic synapse formation. An open question is whether the first fusion event is different from the subsequent ones or not, and whether the first FCM is randomly chosen or specified to be the founding partner of the syncytium. It is equally unknown in vertebrates how the first fusion partners are primed for syncytium formation. Cell-cell recognition, however, is principally conserved from fly to vertebrates with notable variations in the adhesion proteins employed.

Membrane and actin dyamics – fusion pore formation

Now that the FC has recognized and bound its first fusion partner, the transmembrane receptors signal bidirectionally to the membranes and actin cytoskeletons, resulting in different cellular responses. At this stage, the fusogenic synapse is irrevocably established and the cells are unable to abort the process and to detach in search for an alternative fusion partner. The first detectable intracellular effector in both cells is the Phosphoinositide PI(4,5)P2, which accumulates on the inner leaflet of the apposing membranes at the contact site. The signaling cascade then leads via formin (Diaphanous) and Arp2/3 activity to extensive actin remodeling: the accumulation of an actin sheath in the FC and the formation of an actin focus in the FCM (Figure 2). The actin sheath on the FC side provides the necessary tension for the fusion process, while the FCM sided actin focus is the source of invasive podosomes and force generation. Both cellular processes lead to membrane apposition, fusion pore formation, and ultimately, to cell-cell fusion. The exact events after fusion pore formation are less well understood. It is currently unknown if the incorporation of the FCM’s cellular contents and, crucially its nucleus, is passive or active, potentially guided by actin cytoskeletal processes. Nevertheless, actin remodeling is essential not only for fly myoblast fusion, but also for myoblast fusion in vertebrate syncytium formation. The work in Drosophila was the first to identify the need for actin restructuring and has significantly influenced work in other model organisms and man.

Reprogramming

The incoming FCM nucleus adopts the transcriptional profile of the FC nucleus and down regulates FCM specific transcription factors such as Lame duck (Lmd). In our example, the FCM nuclei incorporated into the DO1 muscle immediately start expressing Kr and Nau while ceasing the Lmd expression. The developing musculature provides, therefore, a rare opportunity to observe nuclear reprogramming in vivo in real time and will provide valuable insights into this process. The growing syncytium harbors now two nuclei with converging expression profiles and is primed to initiate the fusion process again. It is unknown if a similar process is occurs during vertebrate myogenesis, however, during satellite cell mediated muscle repair, the incoming satellite cell nucleus has to be reprogrammed to fit the expression profile of the repaired fiber. Notably, PAX7, a marker of satellite cells, has to be down regulated post fusion.

Counting

It is important to note that, in Drosophila, each individual muscle incorporates a set number of cells and nuclei, which is thought to be determined by the transcriptional profile of the individual FC. Since myoblast fusion is an iterative event, a mechanism to count the fusion cycles and to terminate the process has been hypothesized to exist. To date, only limited data is available on fusion cycle control and it remains to be seen if this process is exclusively governed FC autonomously or instructed by additional external cues. However, it has been shown that the transcriptional profile directly influences the number of nuclei ultimately incorporated into the growing fiber. In our example, the DO1 muscle will incorporate a set number of cells until it reaches the necessary number of nuclei. During this time, the shape and size of the myotube changes significantly and the environmental interactions lead to the establishment of tendon attachment sites via the myotendenous junction (MTJ), and of neuronal input via the neuromuscular junction (NMJ). In vertebrates, no comparable restriction of myonuclei has been reported. Yet, when considering fiber repair as well as fiber growth upon training, the number of nuclei incorporated during these processes are predetermined. It remains to be seen if the restriction of myonuclei per fiber is transcriptionally instructed, fiber size dependent or both.

SYNCYTIUM MATURATION

Once the muscle cell of a particular identity is specified, has incorporated the necessary number of nuclei into its syncytium, and formed its tendon attachments, several subcellular structural alterations take place to generate the final, contractile, functional unit known as a muscle fiber. This process is once again conserved from Drosophila to higher vertebrates and presents a valuable model system to investigate myofiber maturation in vivo.

Nuclear positioning

In muscle cells of all organisms, a crucial step for myofiber maturation and its final functionality is the redistribution of the cell organelles acquired through cell-cell fusion. While it is unknown whether the cellular contents of the FCMs are incorporated into the syncytium randomly or in accordance to the cellular architecture of the FC, it is known that nuclei are aggregated at the end of fusion. As the myotube matures into a myofiber, these myonuclei and associated organelles are evenly distributed, following characteristic intracellular movements. Recent evidence suggests that this positioning is an active, directed process that depends on the microtubule cytoskeleton and associated motor proteins. Moreover, genetic mutations that alter positioning result in myofibers with decreased contractile ability. Indeed, similar myonuclear positioning phenotypes have been observed in various human myopathies, including Centronuclear myopathies and Myotonic dystrophy. Once again, the genetic and optical capabilities of Drosophila myogenic research provide ample in vivo opportunities to investigate these understudied processes and rare diseases.

For the DO1 myotube, the myonuclei are aggregated in one cluster after fusion but then partition into two distinct clusters while the myotube matures its MTJ. Next, each cluster of myonuclei moves towards the ends of the myotube, in close proximity to the MTJ. Finally, the clusters of myonuclei are redistributed along the length of the muscle cell, as the MTJ finally matures and the NMJ sends coordinated signals from the central nervous system.

Contractile apparatus assembly

Terminal differentiation of the growing fiber also induces the production of proteins necessary to construct the basic unit for muscle contraction: the sarcomere. The sarcomere is conserved from invertebrates to higher vertebrates and is composed of intercalating thick and thin filaments of myosin and actin, respectively. Multiple sarcomeres are assembled, one after the other, into a myofibril, and each muscle fiber has multiple myofibrils. These myofibrils are organized with the transverse tubules (t-tubules), which allow rapid depolarization of the muscle membrane upon neural input, and with the sarcoplasmic reticulum that stores calcium. Upon neural stimulation, calcium is released leading to activation of the machinery necessary for myosin-actin interaction and subsequent contraction of the myofiber. Note, that the myotube is now referred to as a myofiber. The embryonic DO1 muscle is now fully formed and active. Its sister cell, DAMP, is still mononucleate and quiescent. By coordinating the contraction of DO1 with other muscles in the segment, the embryo hatches into the larva.

Growth

The functionality of a muscle fiber is, in part, dependent on its size in all systems. Muscle growth in the Drosophila larva crucially depends on two factors: 1) the incorporation of cytoplasm, nuclei and organelles through iterative rounds of fusion during embryogenesis, as there is no fusion during the larval stages and 2) cell autonomous growth, regulated by Insulin signaling and its downstream effector, Myc, over a 4 day period. Interestingly, while Insulin signaling by itself is sufficient to autonomously affect fiber growth through Akt-mTor, Myc signaling affects nuclear ploidy in the fly larva, which in turn acts on fiber size. The increased ploidy allows each nucleus to transcribe simultaneously from several copies of one gene locus to keep pace with the needed protein production for this intense growth phase. The mechanisms by which the number of nuclei and growth work together to get a muscle of a particular size remain unclear in all organisms. For the DO1 myofiber, each nucleus undergoes several rounds of endoreplication, and DO1 grows several 10-fold in size in accordance with the growth of the overall somatic musculature, for example, the ventral lateral muscles 3 and 4 both grow 25–40-fold, adding to its myofibrils and other organelles to allow the larva to move. Interestingly, during this period the muscles act as an endocrine (myokine) organ, releasing factors that coordinate the growth of other tissues with its growth.

At the end of the third instar stage, the DO1 muscle is nearing the end of its life. However, its sister cell, the quiescent DAMP, is reactivated during larval stage L2 onwards; it proliferates and its progeny migrate within the larva in preparation for the second wave of myogenesis during the pupal stage. A notable difference at this stage of development is apparent in the thoracic versus the abdominal segments. The DAMP progeny migrates towards the wing disc in the 2nd thoracic segment and continues to proliferate, while in the abdominal segments, proliferation is much more limited and the DAMP progeny remains in close contact with the associated motorneuron (Figure 1).

METAMORPHOSIS

At the end of the larval stage, the larva crawls out of the food and finds an elevated, dry place to form a pupa. It is within this pupa, over the next 5 days, that, in the process of metamorphosis, the larval musculature undergoes tissue histolysis, continued stem cell proliferation, and the differentiation and formation of newly formed fibers with different shapes, sizes, attachment sites, innervations and hence functions. In the following sections we will review these steps in more detail.

Cell lysis

During pupariation, the bulk of the larval, striated body wall musculature is histolyzed. The process of histolysis is triggered by ecdysone signaling and is crucial for metamorphosis to progress. In contrast to the cells of the salivary glands and the midgut, the muscle cells do not undergo canonical histolysis but controlled apoptosis. In the abdomen, the majority of muscles, including DO1, degenerate within the first hours of pupa formation. The notable exceptions in the abdomen are the segment border muscle and the dorsal acute muscles 1–3. These muscles perdure for several hours before being also lysed. Remarkably, in the thorax, DO1 is spared completely and serves as template for the dorsal longitudinal flight muscles (DLMs). We will discuss this subset of myofibers and the process of trans-differentiation after the next section.

Stem cell activation and fusion

The adult muscle progenitors (AMPs) born as siblings to the founder cells in the embryo, lay quiescent, in close contact with the myofiber, the motorneurons, and other AMPs through filopodia extensions. At the beginning of the larval stage, the AMPs are activated and start to proliferate, first symmetrically and later asymmetrically to self renew the stem cell pool, similar to what is found in vertebrate systems. The underlying signaling cascade that is responsible for activation and proliferation includes the Insulin – Notch – Myc pathway. These cells differentiate into myoblasts, gaining identity through a similar, though less well investigated, coding mechanism as in the embryo, and populate the developing adult fly body and generate the adult striated musculature through fusion generated syncytia. The fusion process in the pupa relies on the same set of genes as the embryonic fusion process (Figure 2). While actin is required and a F-actin focus is present, whether invadopodia like those observed in the embryo are responsible for fusion remains unknown. Similarly, while F-actin and its regulators are required for vertebrate fusion, it remains to be tested whether invadopodia are present.

In the abdomen, the AMPs start to proliferate at larval stage 2, stay in close contact to the motorneuron which is spared during histolysis, and begin to form the adult abdominal muscles through myoblast fusion with their own progeny. These AMPs do not migrate extensively, but rebuild the musculature at their position, which they occupied since embryogenesis. The 1 dorsal, 2 dorso-lateral, 2 ventro-lateral and 1 ventral AMPs will hence give rise to the respective dorsal, lateral and ventral abdominal muscle groups. The DO1 associated AMP will, for example, give rise to the retractor of tergites (RoT) muscles in each hemisegment.

In the thorax, AMPs start to proliferate earlier and migrate extensively to associate closely with the developing imaginal discs. It is from this stem cell niche at the disc, that further proliferation is initiated and myoblasts migrate out to populate locations within the thorax and the legs. While the exact lineage of embryonic progenitors to the different muscles in the leg and thorax have not been all defined to date, the size and accessibility of these muscles make them an excellent model system for further study.

The equivalent cell population to the fly AMPs in higher vertebrates is the satellite cells which facilitate muscle repair and growth. These cells are set apart from the initial myoblast population and remain quiescent, in close contact with a fiber, until their activation to repair a damaged fiber or for additional growth. Remarkably, the activation of both cell populations relies on Notch-Myc signaling, reiterating the conserved nature of myogenic programs from fly to vertebrates.

Transdifferentiation

An interesting deviation from the de novo generation of striated muscle by stem cell progeny is the generation of the dorsal longitudinal flight muscles (DLM). These muscles are built using a set of larval muscles, the Dorsal Oblique muscles 1–3 of the thoracic hemisegment T2, which escaped histolysis. These myofibers dedifferentiate and abandon their larval specification but do not undergo histolysis. Next, extensive growth through fusion with stem-cell derived myoblasts, followed by splitting of each muscle along its length results in the doubling of the muscle set. In essence, these fibers take the place of AMPs in the abdomen or even of FCs during embryonic development. It remains an open question as to why the fly deviates from the canonical path of myogenesis to generate the DLMs. It is further unknown if the DLMs harbor a transcriptional profile, similar to the identity genes of the embryonic FCs that guide their development.

LEARNING FROM THE FLY

Work on myogenesis in Drosophila has revealed many important cellular processes that directly apply to vertebrate muscle development, maintenance and repair, as well as disease states. Notably, the molecular underpinnings of cell-cell fusion, as discovered and first described in fly, have instructed extensive work in vertebrate systems, confirming the conserved mechanism of myogenic syncytium formation. Similarly, the emerging field of organelle, and specifically nuclear, positioning in the growing, matured or repaired myofiber, is most advanced in the fly and will elucidate many mechanisms of muscle disease and development in humans. While the high degree of transcriptional identity of the fly embryonic and larval muscles have yet to be directly translate-able to higher vertebrate and man, differences in susceptibility of muscle groups to disease and external influences, might be explained on the basis of muscle identity as defined in Drosophila. Moreover, the optical and genetic accessibility of Drosophila tissues allows for unprecedented assessment of muscle with its tendons, motor neurons and sensory neurons in an in vivo context. Muscle contraction and organismal locomotion/flight assays allow the pairing of structural changes to function. This comprehensive view of muscle and its interacting tissues allows for insight to the development of connectivity and coordination of these tissues as well as diseases that affect these tissue interactions. No doubt, these studies with impact those in other model systems and man. Finally, the relatively short life-span, paired with the lack of a satellite cell based repair mechanisms, allows for a variety of aging and maintenance questions that would be challenging in most higher vertebrate model organisms. Given the increasing longevity of humans and the prevalence of a more sedate life-style, understanding the process of muscle aging and wasting will be paramount. The fly provides a suitable, fast and reliable system to investigate these questions.

CONCLUSION

In this primer we have introduced the process of myogenesis and the unique adaptations of myogensis tailored to each developmental stage in Drosophila melanogaster. We followed an exemplary cell and its progeny through the different life stages of the fly and highlighted cell biological processes that are informative for researchers working in other fields or different model organisms. The ease of genetic manipulation, optical tractability and low costs make Drosophila an excellent system to study these processes. This system remains attractive as a model to investigate basic cellular processes relevant to the understanding of basic cellular mechanisms, muscle disease etiology and progression, as well as modeling muscle specific processes. The continuous study of Drosophila myogenesis will address the many open questions in the fields of cell-cell fusion, cell differentiation and dedifferentiation, muscle homeostasis, repair and wasting as well as shed light on the evolutionary pressures that led to the invention of this incredible, syncytial organ system.