*1.1. General Overview*

*Drosophila melanogaster*, a holometabolic insect with a short lifespan, has served as a simple model to study myogenesis [1,2] and contractile proteins [3] for decades. Myogenesis in *Drosophila* occurs in two waves, one during the embryonic stage that gives rise to the larval body wall or somatic muscles and the second during pupal development that gives rise to adult flight, leg, and abdominal muscles [4]. All these muscles are voluntary, syncytial (multinucleate), and striated making them similar to vertebrate skeletal muscles [5]. Multiple signaling pathways, genes, and processes are conserved from *Drosophila* to vertebrates [6,7]. Muscles provide force to ensure various locomotory behaviors such as crawling, walking, jumping, and flying in *Drosophila*. Thus, they need to carry high levels of a mechanical load and are subject to constant strains, which can potentially disrupt homeostasis. Muscle movements need to be precise and coordinated, where communication with other tissues such as the nervous system provides critical inputs [8]. Muscles are the major reservoir for amino acids in the body that contribute to muscle mass and protein homeostasis [9]. All muscle functionalities require that they are correctly formed in the first place to attain a homeostatic state in which they are physiologically active and stable. Muscle intrinsic signaling as well as signaling from external organs contribute to muscle homeostasis. Muscles display a high degree of plasticity or flexibility at the signaling, metabolic, myonuclear, mitochondrial, and stem cell levels.

This review is divided into three parts. The first part presents an overview of the mechanisms of muscle contraction in *Drosophila*. The second part focuses on the development of the larval and adult muscles. In the third part, we discuss the maintenance of muscle homeostasis in normal conditions and the adverse effects of the loss of this homeostasis in pathological conditions. Throughout the review, the focus is on sarcomeres, which are the basic contractile units of the muscle.

#### *1.2. Major Structural Components of the Drosophila Muscle and Their Vertebrate Counterparts*

In *Drosophila*, muscle function is coordinated by sensory, excitatory, and mechanical inputs by its connection to the nervous system via neuromuscular junctions and to the epidermis via myotendinous junctions akin to vertebrate systems though they present differences, some of which are outlined below.

#### 1.2.1. Sarcomeres

Sarcomeres are the basic contractile units of the muscle and provide the force for contraction during movements (Figure 1). They are repetitively arranged in a regular pattern that gives a striated appearance under the microscope to vertebrate skeletal muscles as well as *Drosophila* somatic, flight, and leg muscles [10,11]. Sarcomeric length, functional domains, and many component proteins are conserved between invertebrates and vertebrates, although studies also point to interesting differences among species, which appear to be adaptations to individual muscle function [12–15]. Despite structural differences in *Drosophila* sarcomeric proteins in comparison to vertebrate counterparts, they have similar functional interactions and possess conserved functional domains; for example, the PEVK domain of the *Drosophila* titin, Sallimus (Sls) confers elasticity similar to vertebrates [16]. Thus, the sarcomere provides an example of nature reusing and repurposing components across evolution.

#### 1.2.2. Myotendinous Junctions (MTJs)

In *Drosophila*, the MTJ is an attachment formed between the muscle and specialized groups of tendon-like cells of ectodermal origin called tendon cells, also known as apodemes (Figure 1a). Unlike vertebrates, *Drosophila* does not have an internal skeleton and tendon cells help anchor the muscles firmly to the cuticular exoskeleton instead, which helps transmit the contractile forces to the body to generate motion. This makes them functionally similar to vertebrate tendons despite their distinct embryological origins, mesodermal for vertebrates and ectodermal for *Drosophila* [17,18]. The formation and maintenance of the MTJ is mediated through the ECM by specific integrin heterodimers on the muscle and tendon ends in *Drosophila* similar to vertebrates [19–22].

### 1.2.3. Neuromuscular Junctions (NMJs)

The NMJ is the point of contact between the motor neurons of the nervous system and the muscle, which enables environmental inputs to be transmitted via synapses to the muscle (Figure 1a). The *Drosophila* larval NMJ is an established model for NMJ formation and function. This NMJ is glutamatergic and responds to the neurotransmitter glutamate unlike vertebrate NMJs that are cholinergic and respond to acetylcholine. However, they are of particular interest owing to their similarity to mammalian brain glutamatergic synapses that express multiple genes orthologous to *Drosophila* genes and the ease with which NMJ assembly can be studied in this model [23–25]. It continues to be an active field of study with focus equally shifting to adult motor neurons formed after metamorphosis [26,27].

**Figure 1.** Schematic representation of the larval body wall or somatic muscle structure and the sliding filament theory of muscle contraction. (**a**) Muscle structure with myofibrils and the network of myonuclei, sarcoplasmic reticulum (SR), T-tubules, and mitochondria. The muscle is connected to the nervous system via the neuromuscular junction (NMJ) and to the epidermis via the myotendinous junction (MTJ). Myofibrils are formed of repetitive contractile units, the sarcomeres. (**b**) The structure of a sarcomere and the mechanism of contraction proposed by the sliding filament theory. Ca2<sup>+</sup> ions released upon neurotransmitter signaling from the NMJ launch a cascade by binding to TroponinC (TnC) on the thin filaments of sarcomeres. This Ca2<sup>+</sup> binding causes a conformation change in Tropomyosin (Tm) bound to actin, exposing actin's myosin binding sites. This permits the activated myosin motor domain to bind to actin and slide against it by utilizing the energy stored in Adenosine Triphosphate (ATP).

#### **2. The Sarcomere and Molecular Mechanisms of Muscle Contraction**

Voluntary muscle contraction is a highly coordinated process that depends on cooperative signaling from sensory neurons via interneurons and motor neurons to the NMJ of the muscle [28–30]. Given that the principal muscle function is to generate movements by contracting, the sarcomeric contractile units are indispensable for muscle function and their maintenance is crucial. The *Drosophila* adult indirect flight muscle (IFM) is established as a model to study sarcomere assembly and the functions of its components [31]. IFMs are built of multiple myofibers and have a stereotypic pattern of sarcomeric proteins forming highly ordered myofibrils similar to human skeletal muscles allowing the study of sarcomere malformations under mutant conditions. The IFM is also a model to study stretch activation (SA) [32]. During SA, there is a high frequency of contraction although the nervous system input frequency is much lower. This is possible due to the delayed increase in tension following muscle stretching. SA is a mechanism found in all muscles though it has particular significance in certain muscle types with rhythmic activity such as human cardiac muscles and the fruit fly flight muscles. In contrast to the multi-fiber IFM muscles of the adult, the somatic muscles in the *Drosophila* embryo and larvae are built of only one muscle fiber per muscle and present a much simpler model to study myofibers.

A sarcomere is a specialized structure adapted for muscle contraction (Figure 1). During myofibrillogenesis, newly formed sarcomeres align in repeating units along the length of a muscle to form a myofibril and multiple myofibrils covered by the plasma membrane form a myofiber. A sarcomere is built of thin-actin and thick-myosin filaments with associated proteins facilitating contraction-relaxation cycles. The thick filaments consist of myosin polymers with each myosin consisting of a myosin tail and two myosin heads, which are capable of attaching to actin during muscle contraction. The two ends of a sarcomere are demarcated by a Z-disc, a huge protein complex that anchors the thin filaments that form I-bands on either side of a sarcomere, while the thick filaments form an A-band in the center (Figure 1). In between the two I-bands is an H-zone lacking myosin heads and in the center of the H-zone is an M-line that corresponds to another large protein complex that anchors the thick filaments [33].

Sarcomere function is intricately linked to other organelles such as the mitochondria [34], myonuclei [35], sarcoplasmic reticulum (SR), and T-tubules [10,36]. The efficient function of sarcomeres is closely coupled with the periodic arrangement of the SR and T-tubules around them [10,36–38]. T-tubules are regular tubular invaginations of the plasma membrane at each sarcomere. The membrane organelle SR is linked to the myonuclei and T-tubules to facilitate the exchange of proteins and ions. The SR is the major intracellular reservoir of calcium (Ca2<sup>+</sup>) ions in the muscle, which are essential for muscle contraction. The T-tubule and SR form a specialized triad/dyad structure, which is indispensable for correct muscle functioning by excitation-contraction (EC) coupling. This EC coupling enables the transmission of excitation potentials from the NMJ to the SR, which triggers Ca2<sup>+</sup> release from the SR that in turn initiates sarcomeric sliding movements leading to muscle contraction. Apart from Ca2+, other ions contribute to muscle contraction [39]. The Na+K+-ATPase is a Na+-K<sup>+</sup> pump that can pump Na<sup>+</sup> out of and K<sup>+</sup> into the cells against their normal concentration gradients. In muscles, the concentration of these ions fine-tunes the force of contraction [40]. In *Drosophila*, muscles are one of the major organs that express the Na+K+-ATPase α subunit [41]. One form of the Na+K+-ATPase β subunit, Nrv1 interacts with Dystroglycan (Dg), which is part of a complex that helps transmit forces into the muscle cell [42].

The mechanism of muscle contraction is explained by the sliding filament theory [43,44], reviewed by Hugh Huxley [33]. This theory proposes that the myosin head domain acts as a motor and slides against the actin filament powered by the energy stored in ATP. This sliding of the central myosin along the thin filaments causes the two I bands on either side to come closer to each other. During contraction, environmental inputs are transmitted by the nervous system to the NMJ leading to Ca2<sup>+</sup> binding to the Troponin C (TnC) subunit of the Troponin (Tn) complex. This leads to the Troponin T (TnT) subunit that binds to the actin binding protein Tropomyosin (Tm) triggering a conformational

change in Tm, thus shifting its position on actin and exposing the myosin binding site of actin [45–47]. Myosin that is turned 'on' by a myosin regulatory light chain (Rlc) phosphorylation [48] liberates the motor domains in the myosin head that were folded onto the myosin tail, thus facilitating its binding to actin. Subsequent ATP hydrolysis and energy release, thanks to its ATPase activity, permits it to move along the thin filament to contract the muscle. For the muscle to relax, the Troponin I (TnI) troponin subunit inhibits the actomyosin interaction [49] so that Tm covers the myosin binding site of actin and the myosin is switched 'off' and folded back onto the myosin tail [50,51]. This coordinated key muscle function highlights the importance of ionic and sarcomeric component homeostasis in muscles, which implies the supply and maintenance of the right quantities of the right ions and sarcomeric components at the right time to ensure muscle functionality.

During contraction, the MTJ helps anchor the myofibrils and transmits forces [19,52]. Tight interactions between sarcomeric components ensure myofibrillar integrity and prevent disintegration due to contractile forces. CapZ binds to the actin barbed end and links it to the Z-disc [13] while Z-disc proteins such as the filamin Cher [53], Zasp, and α-actinin anchor the thin filaments [54]. Similarly, the M-line protein Obscurin that associates with the thick filament [55], Muscle LIM protein at 84B (Mlp84B) that cooperates with Sallimus (Sls) known as the *Drosophila* titin [56], integrins [57], and other proteins ensure muscle integrity. Sarcomeres are subject to constant mechanical strain due to the thin and thick filament friction and need to be consistently replenished to ensure their function over a lifetime. Since these muscles are voluntary, they also need to be able to stop contracting at will and go back to their natural state. Defective sarcomeric formation, maintenance, and homeostasis are associated with muscular diseases [15,58].

#### **3. Muscle Diversification—On the Road to Muscle Homeostasis**

Muscle development is a finely orchestrated, synchronized process that occurs in spatial and temporal coordination with the development of other communicating tissues to finally form a homeostatic muscle. There are similarities as well as differences between *Drosophila* and vertebrate myogenesis [59]. During development, each muscle diversifies to attain an identity tailored to its specific functional requirements. The study of muscle diversification during development is of interest in the context of homeostasis for two primary reasons:


#### *3.1. Embryonic Myogenesis of Larval Muscles*

Embryonic myogenesis gives rise to monofiber larval somatic muscles whose main function is to aid in hatching and the peristaltic, crawling movements of the larvae. The embryonic and larval somatic musculature consists of a stereotypical pattern of muscles in each segment, with 30 muscles in most abdominal hemisegments (A2–A6) (figure in Table 1). There are fewer muscles in the posterior and first abdominal hemisegment and a slightly different set of muscles in the three thoracic hemisegments (T1–T3). Embryonic muscles arise from the mesoderm germ layer and their development requires intrinsic mesodermal cues and extrinsic cues from the adjacent epidermal and neural cells. Thus, they develop in synchrony with the development of muscle-interactors such as tendon cells and motor neurons and need to 'speak a common language' to communicate for coordinated development and maintenance.

Somatic muscle specification and differentiation have been reviewed extensively in the past [1,2,7,60–62] and this review presents complementary as well as new information that emphasizes the role of developmental factors in future muscle homeostasis.

3.1.1. Muscle Diversification by the Specification of Muscle Founder Cells Expressing Identity Transcription Factors (iTFs)

The embryo undergoes gastrulation by invagination [64], which brings the three germ layers, the ectoderm, the somatic muscle forming mesoderm, and endoderm in juxtaposition with each other. This helps provide extrinsic signals to the developing mesoderm. Following this juxtaposition, the mesoderm is divided into domains by morphogenic signaling [65] giving rise to a somatic muscle domain in which the transcription factor (TF) Twist (Twi) provides a myogenic switch [66]. Subsequently, equivalence or promuscular cell clusters expressing the neurogenic gene *lethal of scute* (*l'sc*) form and one muscle progenitor cell is singled out from each cluster by lateral inhibition involving Notch and Ras/MAPK signaling [67,68]. The remaining Notch activated cells in the equivalence groups become fusion competent myoblasts (FCMs). This process is reminiscent and coincides temporally with the specification of neural lineages from the neurectoderm [69,70], which occurs during embryonic stages 8–11, while muscle cell identity specification occurs during stages 9–11.

The singled-out muscle progenitors divide asymmetrically to give rise to founder cells (FCs), which are believed to carry all the information necessary to give rise to the diversity of muscle types. Asymmetric divisions of progenitors can give rise to two FCs, an FC and a Numb negative adult muscle precursor (AMP) or an FC and a cardiac progenitor, which subsequently migrate away from each other [67,71,72]. Each FC contains the information to establish one muscle's identity since it can form correct attachments and be correctly innervated even in the absence of myoblast fusion with surrounding FCMs [73,74]. It expresses its characteristic code of TFs known as muscle identity transcription factors (iTFs) (Figure 2). The expression of a combinatorial code of iTFs in distinct progenitors is the result of their spatial positioning as well as tissue specific convergence of multiple signaling cascades [75]. For example, Wg signaling from the adjacent developing central nervous system (CNS) is implicated in the specification of Slouch (Slou) positive FCs [76] highlighting the importance of coordinated tissue development.

**Figure 2.** Spatial and temporal expression muscle identity transcription factors (iTFs) of the larval lateral transverse (LT) muscles. Sizes are not up to scale. Following the specification of progenitor cells by a lateral inhibition by Notch and low Ras/MAPK activity, founder cells (FCs) expressing muscle specific iTFs are specified for each LT muscle, LT1, LT2, LT3, and LT4 with a contribution from homeobox (Hox) genes to specify thoracic versus abdominal identities. Each iTF has preferential binding abilities to certain enhancers. The iTF expression is followed by the regulation of transcription and modulation of expression of their realisator genes which establish muscle identity over the course of development. The spatial and temporal expression of iTFs coupled with their modulation of realisator genes, which include generic muscle genes, in collaboration with Mef2 begs the question about their contribution to muscle homeostasis. Abbreviations: FCM: Fusion competent myoblasts; FC: Founder cells; LT: Lateral transverse muscles; iTF: Identity transcription factor.
