**1. Introduction**

One particular challenge in regenerative biology concerns the development of reconstructive strategies after muscle-related injuries, but also the treatments of degenerative myopathies for which no reliable clinical strategy exists such as muscle dystrophy, sarcopenia, cachexia, to mention just a few [1,2]. In mammals regenerative capacities are restricted to only a small number of organs [3], yet, in other metazoans, the ability to respond to environmental injuries ranges from "simple" wound

healing to complete anatomical and functional restoration of the lost or damaged part of the body, including muscles [4]. The musculature is a tissue specialized in contraction and cells with contractile functions are present in almost all metazoans but, despite their structural similarity, the origin of muscles is considered to be the outcome of a process of convergent evolution [5]. Indeed, typical muscle protein core sets are present even in unicellular organisms and in early diverged organisms like sponges, which lacks a proper tissue organization and therefore "true" muscles, and in cnidarians, where muscle-like cells are present but lack almost all molecular hallmarks of bilaterian striated muscles thus suggesting evolution from cells with ancient contractile machinery [5]. The processes of myogenesis and muscle homeostasis have also various degrees of conservation among different clades, and so is the extent of muscle regenerative capabilities [5–10].

Animals adopt different basic strategies of regeneration that include the activation of adult stem cells, the dedifferentiation of preexisting cells, and/or the proliferation of differentiated cells. This diversity of mechanisms is still widely understudied and underexploited for biomedical applications.

In this review, we provide an outline of main animals' clades (see Figure 1), muscle types, their development, homeostasis, and regeneration abilities highlighting what is known of their molecular mechanisms. We emphasize some potential contributions of comparative studies into the biomedical fields, therefore advocating deeper employment of 'non-canonical' animals as models for muscle regeneration studies.

**Figure 1.** Tree of the animals' clades treated in this review (in brackets the corresponding section numbers).

Understanding the molecular pathways and mechanistic underlying regenerative events may offer insights into potential methods to unlock regeneration in animals where the regenerating capabilities are more restricted, e.g., mammals. Indeed, regeneration is greatly attenuated in mammals although portions of major organs, such as the liver, retain event-triggered regenerative potentials for the entire animal's life [2]. Interestingly, recent pieces of evidence suggest that regeneration can be induced even in non-regenerating species by altering specific signaling pathways [11–13]. This might be also the case for mammals and thus the principles underlying the induction of regeneration in non-regenerating species may be transferred to humans to trigger regeneration [3,14].

Elucidating muscle regeneration in metazoans also provides opportunities to 'model' a complex biological process relevant to human health and offers a window into fundamental principles underpinning this important response.

#### **2. Porifera: Low Body Complexity with High Regenerative Capabilities**

The phylum Porifera includes mainly sponges, aquatic multicellular organisms with relatively simple anatomy, lacking an organization of tissues and organs.

They have a very simple functioning relying on water circulating throughout a system of canals and chambers, called a water-current system. Circulatory, digestive, nervous, and muscular systems are completely absent. Their body is composed of a few types of cells [15,16]. For instance, in demosponges we found pinacocytes (the skin cells), mesenchymal cells, choanocytes (lining in the interior body walls), archaeocytes (totipotent cells), sclerocytes, myocytes, and porocytes (surrounding canal openings). Two types of contractile cells can be identified: the pinacocytes, and the myocytes. Pinacocytes form a functional contractile epithelium. They are composed of actin networks and actin-dense plaques allowing a coordinated contraction in adjacent pinacocytes but their mechanism of contractility remains to be further elucidated [17].

Most sponge species have an extraordinary capacity to regenerate lost body parts. Four cell types have been identified as stem-cell-like in sponge: choanocytes and archaeocytes, also referred to as adult stem cells (ASCs), pinacocytes, and particular ameboid vacuolar cells [18,19].

#### *Muscle-Like Cells*

Myocytes are spindle-shaped, smooth muscle-like cells containing microtubules lying parallel to peripheral microfilaments. They contract similarly to muscle cells thanks to a non-muscle myosin type II with high homology to that found in bilaterians and vertebrates. They allow the sponge to change shape and expel sediments even without the presence of a nervous system as their contraction is entirely controlled at a cellular level through variation of calcium (Ca2<sup>+</sup>) concentration [20,21]. In particular, channels located at the plasma membrane allow the control of intracellular Ca2<sup>+</sup> concentration that, in turn, regulates cell contractility. This mechanism is believed to be rely on the activation of type II myosin by Ca2+-dependent protein kinases [20,22]. Myocytes allow only movement internal to the sponge but these animals remain essentially sessile. Predation and physical injuries are events very common during the entire adult life of sessile organisms that had to develop efficient strategies of repair and replacement of lost structures to survive [23,24].

Very limited information is currently available on the molecular pathways involved in body regeneration. The activity of ADP-ribosyl cyclase (ADPRC) is related to physiological activities in sponges, such as stem cell duplication and regeneration events [25]. Sponges regenerate using diverse and complex morphogenetic mechanisms involving different cell sources depending on the species. Regeneration can occur through epimorphosis and morphallaxis. The first process involves the formation of a mass of undifferentiated cells (blastema) at the wound site. Pluripotent cells such as archaeocytes and choanocytes from sites adjacent to the injury, undergo a process of epithelial-to-mesenchymal transition (EMT) and migrate to the injured area. Here they actively proliferate and form a typical blastema with dedifferentiated cells. Thereafter a process of mesenchymal-to-epithelial transition (MET) re-establish the differentiated cell identity. Different members of the transforming growth factor (TGF) superfamily are also involved in these processes [26]. In morphallaxis, of particular importance is cell transdifferentiation, the conversion of a differentiated cell to another type of differentiated cell [18]. During this process, spreading and fusion of the epithelia surrounding the wound is accompanied by the transdifferentiation of the choanocytes and exopinacocytes without the formation of a blastema. This supports the hypothesis that these cells combine properties of somatic and stem cells.

Taken together, Porifera represents, for their exceptional regenerative capacities and low body complexity, a promising model for investigating mechanisms of cell recognition, adhesion, migration, and cell type transition during regeneration.

### **3. Cnidarians: The Starlet Sea Anemone,** *Nematostella vectensis* **(Anthozoa)**

Cnidarians (*Hydra*, jellyfish, corals, sea anemones) are aquatic animals that are the sister group to the bilaterian clade [27] and hold a key phylogenetic position for understanding the evolution of common biological processes and mechanisms [28]. The two main groups of the phylum Cnidaria are Medusozoa (jellyfish, hydroids, *Hydra*) and Anthozoa (corals, sea anemones) (Figure 2A). Cnidarians are structurally simple animals with remarkable regeneration capacity. They can regenerate amputated head, foot, and intact animals can even regenerate grouping single dissociated cells [29].

**Figure 2.** The epitheliomuscular system and regenerative capacity of the anthozoan cnidarian *Nematostella vectensis*. (**A**) Schematic representation of the relationship between the main cnidarian lineages and the phylogenetic position of *Nematostella vectensis* (Anthozoa, Hexacorallia). (**B**) The upper

panel shows the muscle network of nematostella in a fixed MyHC1::mCherry transgene [30] labeling the retractor muscles, co-stained with phalloidin thus showing the entire muscle network in green. (ten) tentacles, (\*) mouth, (pha) pharynx, (bc) body column, (ph) physa, (m) mesenteries, (rm) retractor muscles, (pm) parietal muscles. (**B'**) Magnification of a body column region to highlight the orientation of the muscle fibers. (tmf) transverse muscle fibers, (lmf) longitudinal muscle fibers. (**C**) Three epitheliomuscular cell types have been identified in nematostella; they vary in their apical and basal cell junctions as well as their localizations within the body [31]. (**D**) Overview of the known bilateral myogenic factors identified in nematostella. (Present) indicates that the gene has been identified in the genome, (Role) indicates a myogenic role (or not) of this gene in nematostella; (pot. yes), indicates evidence of a myogenic role based on functional experiments or gene expression. (pot. not), indicates evidence of a non-myogenic role based on functional experiments or gene expression. (n/a) data not available. References cited: (a) [32], (b) [33], (c) [34], (d) [35], (e) [36], (f) [37], (g) [38], (h) [39], (i) [40]. (**E**) Oral regeneration of lost body parts after sub-pharyngeal amputation (red dashed line) is completed after 120 h post amputation and reforms a fully functional organism. Animals were fixed at various time points during regeneration and stained with phalloidin to show f-actin filaments (black). Elements of the figure are extracted from [28,41].

The present section focuses on the sea anemone *Nematostella vectensis* that belongs to the Anthozoa, mostly sessile cnidarians that are represented by individual or colony-forming polyps.

The sea anemone *Nematostella vectensis* (Anthozoa, Figure 2B), was initially employed for studying the evolution of embryonic developmental mechanisms [42] and is now emerging as a novel complementary whole-body regeneration model [41]. Nematostella possesses a range of fundamental advantages, such as the access to biological material, a relatively short life-cycle, an annotated genome that revealed astonishing similarities with the one from vertebrates [34], a wealth of -omics data [43,44] and well developed functional genomics and genome editing approaches [45–47].

Nematostella is a rather small sea anemone (juveniles ~0.5 mm, adults ~3 cm), translucent, and well suited for imaging purposes (Figure 2B). It is a diploblastic animal formed by a bifunctional internal endomesoderm and an outer ectoderm. On the oral extremity are the tentacles that surround the mouth and the so-called physa on the opposite. Food caught by the tentacles is ingested via a muscular and neuron-rich pharynx and digested within the body cavity. While most of the digestive enzymes are secreted by the mesenteries that also store nutrients [37], these internal structures play another role as they harbor the gonads that are crucial for sexual reproduction [42] and for inducing a regenerative response [48].

#### *3.1. Muscle Types, Organization, and Myogenic Genes*

Cnidarians display a large diversity of muscle types and organizations that are involved in multiple crucial physiological functions such as feeding, locomotion, or defense [28]. Although this group of marine invertebrates lacks a large part of the molecular hallmarks of striated muscles [5], jellyfish present some ultrastructural and functional features (such as striated myonemes, thick and thin myofilaments, desmosomes as well as a mechanism of excitation–contraction coupling based on intracellular calcium stores [49]) resembling the structure and function of striated muscles [50–53].

For a global overview of cnidarian muscle diversity, their development, and regeneration, please refer to [28]. Most anthozoan muscle cells, and nematostella is no exception, are epitheliomuscular; they contain smooth myofilaments [28] forming a transverse and longitudinal muscle fiber network clearly visible using a MyHC1::mCherry transgenic line [30] and phalloidin/actin staining (Figure 2B'). The epitheliomuscular cells, whose actin fibers form more or less condensed muscle fibers are responsible for various functions of the animal such as feeding or locomotion.

A recent study has characterized three epitheliomuscular cell types (Figure 2C); two types (I and II) with elongated cytoplasmic bridges present in the endodermal parietal and retractor muscles (Figure 2B) and one type III corresponding to basiepithelial muscle cells encountered in the ectoderm of the tentacles [31]. While the known bilaterian myogenic regulatory factors (MRFs [54]) are missing in nematostella (e.g., MyoD, MyoR), a large part of the conserved myogenic gene regulatory network (e.g., Pax3, Pax7, Six1 [55]) has been identified (Figure 2D, reviewed in [28]). However, their exact roles in the formation of the epitheliomuscular cells in nematostella are yet to be understood.

#### *3.2. Muscle Regeneration and Role of Epitheliomuscular Cells in the Regenerative Process*

Like cnidarians in general, nematostella possesses proliferation-dependent whole-body regenerative capacities and can regrow fully functional animals from isolated body pieces within less than a week (Figure 2E) [56–59]. In addition, nematostella is very well suited to compare embryonic development and whole-body regeneration within the same organism [43,57,60], one of the long-lasting questions in regenerative biology [61].

By assessing the MyHC1::mCherry transgenic gene expression after bisection, Renfer and colleagues have observed that the retractor muscles retract from the wound site immediately after amputation. In later steps of the regenerative process, mCherry positive cells accumulate in the regenerating body part suggesting active cellular re-organization and differentiation to reform the retractor muscles [30]. However, the cellular and molecular mechanisms underlying retractor muscle regeneration remain unknown.

On the other hand, there are shreds of evidence suggesting an active role of muscle fibers in the regenerative process. Bossert and colleagues have shown that muscular contractions are involved in reducing the epithelia of the wound site and potentially favoring the wound-healing process [62]. In addition, a stereotyped tissue dynamics that may reflect the above-mentioned observation of MyHc1::mCherry positive cells retracting from the amputation site, supports the idea that muscle contractions play also a crucial role during various phases of the regenerative process [59]. A recent study has shown that the retractor-muscle containing mesenteries are fundamental in inducing regeneration in nematostella [48]. Based on data from planarian [63] and mammalian myoepithelial cells [64], one could thus speculate that the epitheliomuscular cells that form the retractor muscle are also involved in the regeneration process via contraction-independent biochemical signals. However, additional work is required to further support those evidences and to determine the cellular and molecular mechanisms involved.

#### **4. Platyhelminthes: The Freshwater Planarian**

Since the beginning of the 21st century, the freshwater, free-living, flatworm planarian, has become a leading model for the study of development and regeneration mechanisms [65]. As a model organism, it possesses a set of clear advantages. 1. Reductionism: although its relative simplicity, planarians exhibits much of the "complexity" of vertebrate systems, including a well-differentiated nervous system, simple eyes, central brain, triploblastic organization, and bilateral symmetry. 2. Planarians are inexpensive and very easy to rear and maintain in the lab, therefore ideal for primary high-throughput screening processes [66]. 3. Planarians are molecularly-tractable model organisms, easily manipulated by RNAi interference [67] and their thin and somewhat transparent body allows whole-mount in situ hybridization in an intact worm [68].

However, there is no doubt that the most astounding feature is its regeneration capability. Planarians are considered as the "Masters of Regeneration". Adult pluripotent stem cells that are called neoblasts and are the only proliferating cells, account for 25–30% of all cells distributed in the planarian body, and give them remarkable regenerative abilities. Whole worms can regenerate from only a small proportion of the adult worm, within 1–2 weeks. Consequently, full results from regeneration experiments are revealed in a relatively short time. For a broad review of planarian as a model system for regeneration see Ivankovic et al. [69].

#### *4.1. Muscle Types*

The planarian's muscle cells combine features common to both skeletal and smooth muscles [63]. The planarian contains two main muscular systems, somatic and pharyngeal, that differ in their myosin

heavy-chain (MHC) muscle isoforms along with their function and location possibly due to their different biological functions [63]. Without a supportive skeleton, the maintenance of body shape, posture, movements, and defense (strength for their soft bodies) depends on the somatic muscular system. Locomotion is mainly executed by ciliary gliding. The muscular body wall, organized beneath the epithelium, is arranged in a grid work of 4 layers of fibers lying in different orientations and linked to an extracellular network of filaments associated with the body's organs [63]. Moreover, recent works [70–72] revealed that in planarian, the muscles also provide patterning signals essential to regeneration and guidance of tissue turnover and regrowth after injury. Interestingly, this resembles what has been suggested to be a function of the connective tissues in vertebrates [73]. The somatic muscular system provides regeneration guidance through the expression of position control genes (PCGs) differing over time, body region, and types of genes expressed.

In addition to the somatic muscular system, planarians possess a separate pharyngeal musculature system. Planarians have an incomplete digestive system with pharynx (proboscis and anus) connected to the intestine duct by its anterior thus providing a single opening that functions as both, anus and mouth. The pharynx is composed of a muscular tube and demonstrates repertoires of movement capabilities. It is extruded from the body center during feeding and can direct itself toward the food by bending and stretching till it reaches the food; it thus swallows the food and transfers it to the intestine by peristaltic movements. The pharynx does not contain neoblasts [74] and therefore is incapable to regenerate the rest of the worm when amputated. However, a worm losing its pharynx can regenerate it in a few days [75]. The pharynx can thus serve as a module of organ regeneration where stem cells differentiate into distinct cell types to form an organ that integrates within the rest of the body [76].

#### *4.2. Muscle Regeneration and Homeostasis*

Upon regeneration, planarian muscle cells, as all other tissue components, arise from the large reservoir of the existent neoblasts population that migrate to the wound area and start proliferate, thus creating a blastema where they differentiate to form the missing body parts. Irradiation protocols applied to the whole body or to specific areas allows neoblasts ablation [77]. Further transplantation of a single pluripotent neoblast can restore regenerative ability and the whole process can be monitored from scratch [78]. Therefore, planarian is an ideal model for deciphering the mystery of stem cell differentiation [79], allowing experimental approaches that are unavailable in any other model organism. Research on planarian muscle regeneration is still limited but provides some interesting perspectives.

One other unique feature of the planarian model (e.g., *Dugesia japonica* and *Schmidtea mediterranea*) is the ability to shift from growing (up to few centimeters) to de-growing (down to few millimeters) by food deprivation and vice versa. The process depends on the balance between cell proliferation and cell death and by keeping stable body shape and proportions through constant remodeling mechanisms [80]. Therefore, it is a perfect model system for the study of tissue homeostasis (for a broad overview of the subject see [81]).

In spite of their exceptional features and their growing popularity as a model for basic research on regeneration, planarians are not yet considered as a conventional organism for studying human pathologies and diseases, maybe it is time to rethink [82].

#### **5. Mollusks: The Cephalopods**

Cephalopods represent one the main and most evolved mollusk class. They are the most intelligent, mobile, and the largest of all mollusks and include very diverse species such as squid, octopus, cuttlefish and the chambered nautilus.

Regeneration is a frequent event occurring during cephalopods' lifetime. Wild animals often lose body parts such as portions of arms and fins and as a consequence, it is common to find signs of traumatic events on their bodies [83,84]. These events can dramatically impair their capacity to swim, capture, and manipulate preys [85], and therefore they can seriously impact their survival in the natural environment. Indeed, cephalopods can regenerate their cornea, peripheral nerves, and body limb (arms and tentacles) [86]. Cephalopod limbs are complex organs composed of a tightly packed three-dimensional array of muscle fibers controlled by a sophisticated peripheral nervous system (PNS). The arm PNS is composed of three distinct parts: the arm nerve cord (composed of axial nerve cords and the ganglionic core), the sucker ganglia, and the intramuscular nerves. This assembly allows the transmission of a large amount of sensory and motor information to and from the brain [87–90]. All of these structures are fully and functionally recovered during regeneration.

#### *5.1. Muscle Types*

Similar to vertebrates, muscle cells in cephalopods can be found in a variety of different organs that differ dramatically in structure and function. Indeed, muscle cells are in the mantle and appendages (arms and tentacles) but also in eyes, hearts, viscera and chromatophores. Such diversity is paralleled by specific adaptations in muscular organization and physiology.

The majority of the musculature of arms and tentacles is composed by uninucleate transverse or obliquely striated muscle fibers with shared morphological and physiological characteristics. When oblique striation is present, this pattern is uniform and continuous among adjacent cells. Generally, these muscle cells do not exceed 8–20 μm in diameter and 0.8–1 mm in length. The nucleus is in the central portion of the cell whose transverse section is usually round or polygonal, with a mitochondria-rich core and a contractile apparatus in the cortical zone. The contractile apparatus lies along the main axis of the fiber and is organized in sarcomeres with identifiable acto-myosin striations. Cephalopod muscle actin and myosin heavy chain, show strong sequence identity to other invertebrates and vertebrate gene orthologs suggesting a similar contraction mechanism [91,92]. On the contrary, regulatory proteins are very cephalopod-specific [93] suggesting that specific control kinetics and cross-bridge cycle regulation might be developed in cephalopods (for a review see [94,95]). Different from typical skeletal muscles, cephalopod arm muscle cells do not possess a proper T-tubules system, but smaller sarcoplasmic structures named "terminal cisternae" that take contacts with plasma membrane invaginations thus forming "dyads" at the level of the Z-disks. In contrast to the muscle cells of other invertebrates, they are isopotential, and thus each synaptic input can control the membrane potential of the entire muscle cell (for a review see [94]).

Among cephalopods, muscle cells can differ in their activation properties. As an example, in octopus arm, muscle action potentials rely on Ca2<sup>+</sup> spikes [96,97] generating a massive entrance of Ca2<sup>+</sup> that activates a calcium-induced calcium release (CICR) process from the internal stores [98]. An intriguing analogy here can be found with vertebrate cardiac muscle cells that represent an important target of regeneration medicine [99,100].

In contrast to the octopus arm muscles, transverse muscles of squid tentacles show 'graded' Na<sup>+</sup> based action potentials different from the typical 'all or nothing' action potentials of squid giant axon or vertebrate muscle fibers. Interestingly, the transverse muscle of the squid arms lacks Na+-based action potentials [101]. All the above-mentioned characteristics co-evolved with the complex brain to body adaptation and limb specialization [102–104] whose integrity is essential to the animal survival.

Several myogenic genes have been identified in some (but not all) cephalopod species. As an example myoblast-specific Myf5 and MyoD proteins have been identified in *Sepia o*ffi*cinalis* tentacles during late stage development and NK4 is found to be involved in cephalopod striated muscle formation just as in vertebrate cardiac cells [94,105,106]. In addition, an hh-homolog signaling molecule and its receptor Patched (Ptc) have been found to be expressed during myoblast differentiation in *Sepia o*ffi*cinalis* [107].

#### *5.2. Muscle Regeneration*

Cephalopod mollusks are a powerful model of limb regeneration due to their similarities in early arm development to vertebrate models and their fast and efficient regenerative abilities (for a review see [94]) and, among regeneration studies of other body parts, rather ample literature is currently available on the regeneration of their limbs (for a review see [84]). However, very little is known about the molecular pathways controlling the regenerative process.

Hereafter, we will employ the octopus arm as a template to describe the step of a regeneration process. Morphologically, a sequence of events can be identified during arm regeneration: (1) wound healing; (2) formation of a knob at the stump tip; (3) elongation of the knob and formation of a hook-like structure; and (4) elongation of the regenerating arm till complete restoration of a functional structure [85,94,108,109].

At early steps of regeneration, a mixture of extracellular matrix (ECM), vesicles, and mucus are present at the plug region, and only subsequently the connective tissue is deposited by fibrocytes migrating to this region [109]. The presence of ECM and connective fibers might be relevant for the correct reorganization of the regenerating structure, a role that has been also suggested in octopus pallial nerve regeneration [84]. Cephalopods might have evolved fine mechanisms of regulating ECM composition and organization during regeneration that favor the tissue competency to regrow. Interestingly, similar fibrillary elements are the ones limiting vertebrates skeletal muscle regeneration as their accumulation at the injury site negatively interferes with regeneration and drives instead scarring and fibrosis of the tissues [94].

At a cellular level, cells composing the stump are first characterized by a layer of undifferentiated cells together with diffuse vascular components forming a typical blastemal region. This structure then disappears, and cells start differentiating [110]. Cell proliferation remains active throughout the entire regeneration process, but while at an early step is primarily localized at the blastema, at later stages it is present within differentiating tissues such as the axial nerve cord and the musculature.

Unfortunately, no study reported so far could reveal the molecular identity of muscle cell precursor during regeneration. It has been speculated that new muscles and nerve cells can originate from dedifferentiated cells of the same type (for a review see [84]) but due to the lack of species-specific molecular markers, we are currently not able to assess the existence of pluripotent vs. lineage-committed progenitor cells, as well as vertebrate satellite-like cells associated with adult muscles. From a mechanicistic viewpoint it has been shown that after an arm lesion, muscles close to the injury site degenerate fast, and large cells containing little protoplasm and a large nucleus appear within the same area. These cells are supposed to be sarcoblasts that later migrate to the most distal part of the wound and undergo active proliferation. Sarcoblasts will then differentiate into the arm and sucker muscle fibers in different time intervals [108]. This process is possibly paralleled by the recovery of the arm functional capacity.

Few data are available on the molecular pathways underlying muscle formation during regeneration in cephalopods. It is known that cephalopods muscle development rely on MRFs, however, still, no data are available on their expression during muscle regeneration in octopus. Several studies suggested that acetylcholinesterase (AChE), a conserved molecule between vertebrates and invertebrates, may orchestrate the formation of the octopus arm during regeneration [110,111] similarly to what happens in regeneration phenomena occurring in other animal phyla such as Platyhelminthes, Mollusca, Arthropoda, and even Chordata (for a review see [94]).

#### **6. Nematodes: The** *Caenorabditis elegans* **Model**

Nematodes are one of the most diverse animal phyla. They occupy a large variety of environments, and many species are parasitic. Nematodes are relatively small animals (~1 mm long adults), and, given their size, a heart and a closed circulatory system are not required.

*C. elegans* has been employed as a model to study extrinsic and intrinsic factors crucial for axon regeneration and wound healing. In particular it have disclosed important aspects of the mechanisms of wound healing and cellular plasticity, axon regeneration and transdifferentiation in vivo [112].

#### *Muscle Type and Homeostasis*

The majority of muscles of the animal body wall are used for the animal's locomotion [113]. *C. elegans* body wall muscle cells are spindle-shaped mononuclear cells with multiple sarcomeres per cell [114]. Muscle cells are obliquely striated and form body-wall muscles running along the length of the body underneath the epidermis [115,116]. Unlike most other animals, their innervation is unusual in that the nerves do not branch out into the muscles but the muscle cells send extensions (muscle arms) to the nerve cord to receive *en passant* synapses from the motor neurons [117,118].

Embryonic development of body wall muscle is controlled by maternally expressed genes initially, but then there is a switch to control by zygotically expressed genes. Several molecular players (e.g., Wnt/Mitogen-Activated Protein (MAP) kinase signaling, Myogenic regulatory factors (MRFs) as many others) act during muscle development and differentiation. For a detailed description please refer to [119,120].

*C. elegans* muscles lack satellite cells (muscle stem cells) and therefore muscles cannot regenerate. Adult worms only carry post-mitotic body wall muscles [119]. It is interesting to notice that, although lacking an open circulatory system, proteins and structures composing the body wall muscles manifest a high homology with that of human heart muscle. In addition, many molecules involved in sarcomere assembly and maintenance are in common with other animals. A dystrophin ortholog, dys-1 gene, has been identified in *C. elegans* with a key role in the sarcomere structural regulation [121]. The mechanism of assembly of sarcomeres into functional muscles have been extensively investigated in *C. elegans* within the context of repair following activity-induced muscle stress and muscle degeneration [117].

For the reasons listed above, *C. elegans* has become a model study for muscle diseases such as Duchenne's Muscular Dystrophy (DMD) [122] and cardiomyopathies [113]. A more explanatory and detailed list of advantages and disadvantages of this animal as a model of human heart pathologies can be found in [113].

Interestingly, the lack of regeneration capacity of *C. elegans* muscles has been key to the use of this animal as a model of DMD. Indeed, as *C. elegans* adult muscle cells are mono-nucleated and post-mitotic, they can be individually tracked during the process of muscle degeneration and do not undergo fibrosis and chronic inflammation, processes that are common in vertebrate models [121].

#### **7. Artropods: The Insect** *Drosophila melanogaster*

Insects have a reduced lifespan and events related to degeneration/regeneration processes following physical, pathological or aging damage are less frequent. Hence, the establishment of a real physiological regenerative mechanism have been under a lower evolutionary pressure. However insects manifest adult muscle hypertrophy, which can be viewed as a degeneration/regeneration-like process, in response to particular hormones as well as to environmental factors, population density, food availability, or mating [123,124].

*Drosophila melanogaster* is a model organism in which genetic and molecular techniques, coupled with physiological and structural approaches, have been used to unravel specific issues of invertebrates and vertebrates muscle biology, including regeneration processes.

#### *Muscle Type and Homeostasis*

In *D. melanogaster* larvae and adults, three types of muscles can be recognized: (1) Tubular muscles, including most of the adult skeletal muscles. They are striated with a centrally located nucleus and are synchronous muscles because each nerve stimulation evokes calcium release from internal stores which triggers a mechanical contraction of the muscle similarly to vertebrate skeletal muscles [125]. (2) Adult indirect flight muscles, or "fibrillar" muscles. In these muscles individual myofibrils can be identified by light microscopy; they are striated and asynchronous muscles as their mechanical response is activated both by calcium following nerve impulse and by stretch-activation due to the elastic recoil of thorax cuticle [125,126]. (3) Supercontractile striated visceral and heart muscles, and larval body wall

muscles. Supercontractile muscles are called "supercontractile" because they can contract to a length well below 50% of their resting length [127–131]. They contract in response to caffeine also in the absence of external calcium, showing that a functional store of calcium is present in the sarcoplasmic reticulum and that it is sufficient for muscle contraction [132]. Interestingly, a similar activation property has been also found in the octopus arm muscles [98].

Larval and adult muscle cells and fibers derive from progenitor cells of the embryonic mesoderm. Signaling crosstalk between ectoderm and mesoderm (for instance Decantaplegic and Wingless) and gene (e.g., *twist*, *even-skipped,* and *floppy-paired*) dynamic temporal expression, regulate the muscle cell fate of these cells [133,134]. Cardiac and visceral muscle cell progenitors are formed from these generic muscle cell progenitors by their compartmentalization in segmental regions with low Twist high Even-skipped domains. High Twist high Sloppy-paired domains are, on the other hand, a key point for the development of somatic cell progenitors [134,135]. Both these muscle cell progenitors undergo then asymmetric cell divisions, which generate low Twist cells that fuse to form embryonic myoblasts and subsequently embryonic muscles. Some, but not all, asymmetric division give rise also to a single founder cell and to an adult muscle precursor (AMP), an adult muscle stem cell that remains in quiescence.

Larval muscles degenerate throughout metamorphosis. In some cases (indirect flight muscles in the thorax) larval muscles are utilized as templates for the formation of adult muscles. In other cases, peripheral nerve fibers and the space between larval muscle fibers drive adult muscle fibers development and differentiation. Adult muscle fibers origin from AMP precursors which proliferate, differentiate, and fuse to form myotubes and then adult fibers. This process was deeply studied in indirect flight muscles that are the main power source for flight. From these studies, however, it was found that only a small number of AMPs descendent stem cells remain associated with the adult differentiated indirect flight muscle fibers. These cells resemble mammalian satellite cells which are associated with the adult skeletal muscle fibers and retain the competence to proliferate and differentiate in myoblasts and then adult myofibers when stimulated (for example following skeletal muscle fiber degeneration). It is interesting to note also that these "fly satellite cells" undergo a proliferation/differentiation program leading to the generation of myoblasts which fuse with a damaged indirect flight muscle fiber. This process, similarly to what was observed in vertebrate satellite cells of skeletal muscle fibers, points to repair the damaged fibers, and it is activated by Notch-Delta signaling [136]. In the absence of tissue damage, satellite cells are maintained, not differentiated, and "quiescent" probably by the transcription factor Zhf1 [137].

Other authors claim that there are probably no "satellite cells" in adult flies' muscles. Indeed, almost all of these studies were investigating the indirect flight muscles (IFMs) that are considered the most similar to mammalian skeletal muscles. In these, regeneration processes are triggered by damage consequent to physical or pathological injuries as well as damages related to aging. Considering fly lifespan, these events are less frequent and therefore they should have exerted a minor pressure from the evolutionary point of view to establish a real regenerative mechanism in adult muscles. Moreover, a regeneration-like process is considered for adult skeletal muscles as regarded as muscle hypertrophy. Again, in flies, differently from other invertebrates, the small dimension of IFMs could have been a factor against a "pressure" from an evolutionary perspective.

#### **8. Echinoderms: A Compendium of Regeneration Strategies**

Echinodermata is a phylum consisting of radially symmetrical marine animals. All larval and adult echinoderms exhibit high regenerative capacities of entire lost parts following predation or traumatic events [138]. Echinoderms manifest all the regenerative strategies identified in other animals, such as epimorphosis and morphallaxis, and have an impressive high genetic homology with Chordates. They can show epimorphic processes, by which a blastemal is formed through active proliferation of migratory undifferentiated cells. They can also show morphallaxis, where cells derive from differentiation, transdifferentiation, or migration of existing tissues. Most classical and

bio-molecular tools currently available have been successfully employed in this animal species giving rise to a large body of literature on Echinoderms regeneration from molecular, cellular, and tissue level. These features make them interesting models in translational research [139].
