Early events in annelid regeneration: a cellular perspective.

Integrative and Comparative Biology Integrative and Comparative Biology, volume 54, number 4, pp. 688–699 doi:10.1093/icb/icu109

Society for Integrative and Comparative Biology

SYMPOSIUM

Early Events in Annelid Regeneration: A Cellular Perspective Alexandra E. Bely1 Department of Biology, University of Maryland, College Park, MD 20742, USA From the symposium ‘‘The Cell’s View of Animal Body Plan Evolution’’ presented at the annual meeting of the Society for Integrative and Comparative Biology, January 3–7, 2014 at Austin, Texas. 1

Synopsis The ability to regenerate extensive portions of the body is widespread among the phylum Annelida and this group includes some of the most highly regenerative animals known. Knowledge of the cellular and molecular basis of regeneration in this group is thus important for understanding how regenerative processes have evolved both within the group and across animal phyla. Here, the cellular basis of annelid regeneration is reviewed, with a focus on the earliest steps of regeneration, namely wound-healing and formation of the blastema. Information from a wide range of annelids is compiled in order to identify common and variable elements. There is a large body of valuable older literature on the cellular basis of regeneration in annelids and an effort is made to review this literature in addition to more recent studies. Annelids typically seal the wound through muscular contraction and undergo some autolysis of tissue at the site of the wound. Bodily injury elicits extensive cell migration toward the wound, involving several different types of cells. Some migrating cells form a tissue-clot and phagocytize damaged tissues, whereas others are inferred to contribute to regenerated tissue, specifically mesodermal tissue. In one annelid subgroup, the clitellates, a group of mesodermal cells, sometimes referred to as neoblasts, is inferred to migrate over considerable distances, with cells moving to the wound from several segments away. Epidermis and gut epithelia severed upon amputation typically heal by fusing with like tissue, although not always. After amputation, cellular contacts with the extracellular matrix are disrupted and major changes in cell morphology and adhesion occur within tissues near the wound. Interactions of tissues at the wound appear key for initiating a blastema, with a particularly important role suggested for the ventral nerve cord, although species are variable in this regard; longer-distance effects mediated by the brain are also reported. The anterior–posterior polarity of the blastema can be mis-assigned, leading most commonly to double-headed worms, and the dorsal–ventral polarity of the blastema appears to be induced by the ventral nerve cord. The blastema is thought to arise from contributions of all three tissue layers, with each layer replacing itself in a tissue-specific manner. Blastemal cells originate mostly locally, although some long-distance migration of source-cells is suggested in clitellates. A number of important questions remain about the cellular basis of regeneration in annelids and addressing many of these would be greatly aided by developing approaches to identify and isolate specific cell types and techniques to image and trace cells in vivo.

Introduction Annelids (segmented worms) are a large and diverse phylum that includes many members capable of extensive regeneration (Hyman 1940; Berrill 1952; Herlant-Meewis 1964; Bely 2006). Numerous species can regenerate a complete head, a complete tail, or both at once, in some cases starting from just a small fragment of the original individual. Understanding the cellular and molecular basis of regeneration in annelids can help to address important questions such as: what is the relationship between regeneration and embryogenesis? What processes fail in

poorly regenerating species? How have regenerative abilities and mechanisms evolved within and across phyla? For this reason, regeneration of annelids has been a subject of growing interest. Regeneration in annelids has been studied for well over a century and in a variety of representatives from across the phylum (Randolph 1892; Stephenson 1930; Hyman 1940; Herlant-Meewis 1964; Goss 1969; Bely 2006). Recent studies are focusing increasingly at the molecular level but there is a large, mostly older, literature that focuses at the cellular level. Understanding the cellular and

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Annelid regeneration

studied in a broader sampling of species. Cellular studies of regeneration have been carried out in representatives from both major annelid clades, the Errantia (many ‘‘polychaetes’’) and the Sedentaria (many ‘‘polychaetes’’ and all clitellates, including ‘‘oligochaetes,’’ leeches, and relatives), as well as basal annelid lineages (Struck et al. 2011; Weigert et al. 2014). Most of the data discussed here were obtained from the following annelids: Errantia clade—Nereis/ Neanthes (Nereididae), Nephtys (Nephtyidae), Eulalia (Phyllodocidae), Eurythoe (Amphinomidae), Dorvillea (Dorvilleidae); Sedentaria clade—Capitella (Capitellidae), Sabella and Branchiomma (Sabellidae), Hydroides and Salmacina (Serpulidae); Lumbriculus (Clitellata: Lumbriculidae), Eisenia and Allolobophora (Clitellata: Lumbricidae), Enchytraeus (Clitellata: Enchytraeidae), Tubifex, Limnodrilus, Dero, and Pristina (Clitellata: Naididae, including former Tubificidae), Hirudo (Clitellata: Hirudinidae); and basal annelids—Owenia (Oweniidae) and Chaetopterus (Chaetopteridae). For brevity, and because species-level taxonomic revisions are not uncommon, we refer only to generic names in this review.

Wound-healing Muscle contraction Following transverse amputation, nearly all annelids investigated can seal the body effectively by rapid muscular contraction (Hyman 1940; HerlantMeewis 1964; Bilej 1994). Observations of amputations on live specimens of many species indicate that constriction of circular body muscles, and in some cases slight extrusion of gut tissue, quickly closes the wound, stemming the loss of body fluids and presumably protecting against infection. Even annelids that cannot regenerate following transverse amputation often effectively seal such an injury by muscular constriction and later re-epithelialization of tissue (Bilej 1994; Bely 2006; Bely and Sikes 2010). However, at least a few annelids cannot accomplish this; acanthobdellidans (relatives of leeches) have a stiff outer cuticle and fail to seal wounds following transverse amputation, dying shortly after any serious breach of the body wall (Bely 2006). In some species, transverse amputation is accompanied by, and even may be preceded by, autotomy (self-amputation) effected by muscular contraction. In a species of Enchytraeus, transverse amputation typically is followed by autotomy at a stereotypical intra-segmental plane, corresponding to the position of one of the segmental nerve rings (Yoshida-Noro et al. 2000; Kawamoto et al. 2005). Autotomy thus leads to regeneration being initiated from a


tissue-level dynamics that occur during regeneration in annelids is key for interpreting the emerging molecular data and, more broadly, to provide a complete understanding of regenerative processes in annelids. Here, I review literature on cellular and tissuelevel processes that occur during regeneration in annelids. I focus on early phases of regeneration, specifically wound-healing and formation of the blastema (the mass of seemingly undifferentiated cells that forms at the site of the wound and differentiates into the new structures), as these early phases are likely to be most relevant for addressing the kinds of questions outlined above; many studies describe later phases of tissue morphogenesis but these are not reviewed here. Within the wound-healing section, I review information on muscle contraction, autolysis, cell migration, re-epithelialization, and remodeling of tissue and the extracellular matrix. Within the blastema-formation section, I review information on the initiation, polarity, and cellular origins of the blastema. Although the major events of early regeneration are discussed in a rough temporal sequence, it should be noted that many of these events are interconnected and many occur simultaneously. Throughout, I focus on regeneration by epimorphosis (involving addition of new tissue), primarily following transverse amputation; regeneration by morphallaxis (remodeling of existing tissue) is common among annelids (Berrill 1952; HerlantMeewis 1964) but few studies have addressed its cellular underpinnings. This review aims to tie together findings from a range of approaches, including histological and descriptive studies, experimental surgeries, grafting experiments, and proliferation assays. Many of the sources reviewed here represent older literature; it is my hope that by reviewing these works here they will be more broadly known. It should be noted, however, that many of these older studies were performed before the availability of methods to track cells using cell tracers, molecular markers, or timelapse imaging. Conclusions regarding the sources and fates of cells during regeneration or the presence or absence of certain tissues in manipulative experiments often are based on light microscopy and histology of fixed tissue alone. Although these studies have yielded valuable information, their limitations should be borne in mind and their conclusions merit being revisited with modern methods. Findings from a broad range of annelids are reviewed here, with the goal of identifying features of regeneration that appear to be general among annelids, as well as identifying features that merit being

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Autolysis In several annelid species, histological analyses of fixed samples have shown that some tissues at the site of the wound undergo autolysis soon after injury. In Limnodrilus, autolysis is evident both in ectoderm and in mesoderm within hours after transverse amputation (Cornec et al. 1987), and in an earthworm both the epidermis and the underlying muscle near that wound are broken down within an hour after injury, inflicted by burning a hole in the body wall (Cameron 1932). Additional studies are needed to determine how common autolysis of tissue is following injury, as well as whether the extent of autolysis depends on the severity or type of the injury. Cell migration: phagocytosis, tissue-plug formation, and source-cells Histological studies of tissues fixed at multiple time points after injury suggest that amputation triggers a major migration of cells in the hours following wounding. Migration of cells to the wound appears to be a general feature of annelids, having been inferred in a range of annelids and following a range of injuries (transverse amputation, breach of the body wall, removal of terminal asegmental structures) (e.g., Stephan-Dubois 1954; Hill 1970; Cornec et al. 1987; Bilej 1994; Tettamanti et al. 2004). The period of extensive movements of cells has been documented within minutes to hours after amputation and typically persists at least through the first day after wounding (Cornec et al. 1987; Bilej 1994;

Huguet and Molinas 1994). Several cell types are inferred to migrate toward the wound and perform different functions. Most broadly, these include cells that migrate to form a tissue plug at the wound, cells that migrate to the wound and phagocytize cellular debris, and largely undifferentiated cells that arrive at the wound and which may contribute to regenerated structures, as described below. It is important to note that homologizing cell types across annelid groups remains challenging: the names used to refer to migratory cells often are inconsistent across annelid groups and when the same names have been applied to cells from different groups those cells typically have been homologized only on the basis of morphology, position, and behavior (Cameron 1932; Stephan-Dubois 1954; Cornec et al. 1987; Bilej 1994; Tettamanti et al. 2004). One of the most detailed analyses of cell migration in annelids is that by Cornec et al. (1987) who investigated cellular and tissue dynamics following posterior amputation in Limnodrilus. Based on observations of fixed histological sections, they inferred that a number of cell types migrate in response to wounding and that these fall into two general categories: phagocytes and dissepimentary cells. Migrating phagocytes themselves are of at least two types: coelomocytes and splanchnopleural cells. Coelomocytes are round cells that are free within the coelom and often have numerous inclusions and vesicles. Within the first few hours after amputation, coelomocytes migrate to the site of the wound and phagocytize damaged tissue, especially degenerating muscle fibers generated by an early wave of autolysis. Coelomocytes extend long pseudopods and engulf these damaged cells. The second type of migrating phagocyte is the splanchnopleural cell. These cells normally surround the gut and dorsal blood vessel but, following amputation, they are thought to be the source of two general kinds of migrating cells. The first are cells that have some inclusions and are frequently observed engulfing other cells at the wound. The second are chloragocytes that have many inclusions and appear to transform into eleocytes (with many dark inclusions), which rarely, if ever, engulf other cells. In addition to coelomocytes and splanchnopleural cells, Cornec et al. (1987) also refer to, but do not further elaborate on, the migration of what they call ‘‘free cells,’’ a possible third type of phagocyte that may correspond to what has been called ‘‘amoebocytes type I’’ in Lumbricillus, ‘‘hyalocytes’’ in Eisenia, and/or ‘‘macrophages’’ in Eisenia. In addition to engulfing cells and cellular debris at the wound, phagocytes also form a mass at the wound that helps to physically plug it.


consistent, predictable intraspecific position. In Lumbriculus, high-speed video analysis indicates that autotomy can even occur prior to severing of the body wall (Lesiuk and Drewes 1999). Severe constriction of the body wall for greater than about 80 ms induces an autotomy reflex that severs the body just anterior to the plane of constriction within a few hundreds of a millisecond after compression. The process is so efficient that no loss of blood is apparent. In both of these annelids, anesthesia inhibits the autotomy response (Lesiuk and Drewes 1999; Kawamoto et al. 2005). Both the Enchytraeus species and the Lumbriculus species investigated reproduce asexually by fragmentation (a type of fission) and thus routinely autotomize to reproduce. It would be valuable to determine how common pre-amputation and post-amputation autotomy are among annelids and whether these processes are found primarily among animals that reproduce by fission.

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wound typically along the ventral nerve cord (Randolph 1891, 1892; Stephan-Dubois 1954; Tadokoro et al. 2006). These cells often have been referred to as ‘‘neoblasts,’’ a term first coined for annelids (Randolph 1891, 1892) to describe these cells in Lumbriculus (and now a term also widely used to refer to a pluripotent type of cell in planarians). Despite efforts to identify these cells in other annelids, dissepimentary cells/‘‘neoblasts’’ that migrate intersegmentally appear to be restricted to ‘‘oligochaetes,’’ or at least are easily recognized only in this clade (Boilly 1969b; Hill 1970; Paulus and Mu¨ller 2006). The inferred function of these cells has long been debated, as discussed in the section ‘‘Cellular origins of the blastema,’’ see below. In summary, a significant post-injury phase of cell migration is evident in annelids, suggesting this to be an important aspect of early regeneration, yet much work remains to be done to characterize the types of cells involved, their behaviors, and their functions. Identifying and homologizing types of cells across different groups of annelids remain persistent challenges. To date, migrating cells have been identified almost exclusively based on morphology, yet different cell types could have similar morphologies, the same type could present different morphologies depending on regenerative stage or context, and a particular type of cell may appear different in different groups of annelids. Efforts to generate markers for specific cell types could allow for important advances in our understanding of cell migration and of the homologies among cell types. Another important goal for future research is to identify the cues that trigger the migration of different types of cells. Finally, almost all of the knowledge about migrating cells is based on inferences from static histological sections. Methods to assess cell migration in vivo are greatly needed. Developing in-vivo imaging would allow direct assessments of cell migration, including the direction and speed of movement; would reveal cells’ behaviors and changes in shape; could reveal less obvious movements of cells (e.g., smaller or less numerous cells and minor migration routes); and, if in-vivo imaging can be coupled with labeling of cells, could prove highly valuable for tracing the fates of migrating cells. Re-epithelialization Transverse amputation severs the epidermal and gut epithelia, both of which must then be healed. Most commonly, the severed edges of the epidermis fuse to each other and the severed edges of the gut epithelium fuse to each other (Herlant-Meewis 1964). This


This temporary wound-plug accumulates by about 1 day post-amputation (dpa) and is then degraded by autolysis once the overlying epidermis is healed, at about 2 dpa. The second general category of migrating cells described by Cornec et al. (1987) is the dissepimentary cells, which are cells originating from the segmental septa. Segmental septa are composed of a thick basal lamina with flattened cells on either side (i.e., on the septum’s anterior and posterior faces). In the ventral and posterior part of the septum are round cells with a large nucleus-to-cytoplasm ratio and inferred to be undifferentiated or weakly differentiated. Within 4 h after posterior amputation, in the 10 or so segments nearest the wound, some of these round septal cells increase considerably in size. Over the next 2 days, spindle-shaped cells with similar cytological properties to those on the septa are seen on the dorsal surface of the ventral nerve cord, often between septa; these cells are inferred to have originated from the septa and then migrated toward the wound. Such cells accumulate at the severed tip of the nerve cord, under the healing epidermis. Cornec et al. (1987) suggest that these cells are likely source mesodermal cells for the regenerate, but this is necessarily a preliminary conclusion, as the limitations of using static images to infer movement and fates of cells are considerable. Consistent with Cornec et al. (1987), in a broad range of annelids injury induces the recruitment of many phagocytic cells that engulf damaged cells and cellular debris and induces the formation of a temporary cellular plug at the wound (Cameron 1932; Burke 1974; Cornec 1984; Bilej 1994; Grdisa 2010). Although phagocytosis mostly has been shown through ultrastructural imaging (e.g., transmission electron microscopy), some studies also have demonstrated phagocytosis directly by following the fate of foreign particles (e.g., carbon particles, beads) introduced into the animal (Cameron 1932). Phagocytosis and the formation of wound-plugs are thus components of the post-injury cell migration response that are probably common to most annelids, and, indeed, some aspects may represent part of a generalized response to injury that may be shared with other animals (Vetvicka et al. 1994). Only in clitellates, however, have cells similar to the dissepimentary cells of Cornec et al. (1987) been documented. In a broad range of ‘‘oligochaetes’’ (namely, non-leech clitellates), spindle-shaped cells that appear to be undifferentiated or weakly differentiated are inferred (based on time-series of fixed material) to originate from the base or surface of the septa, become activated by injury, and migrate to the

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Remodeling of the extracellular matrix and tissues near the wound Amputation leads to extensive changes in the cellular interactions with the extracellular matrix as well as in the contacts among cells within tissues near the wound. These changes have been most extensively studied in histological sections following anterior amputation in Owenia (Fontes et al. 1983; Coulon et al. 1989; Dupin et al. 1991). A basement membrane composed of extracellular matrix is normally present below the epidermis, separating the epidermis from the underlying muscle layer. In uncut animals, at the base of epidermal cells, where they contact the basement membrane, there is an extensive cytoskeleton network, made of actin, that gives these cells a strong apical-basal polarity. These cells also have many adherence-structures anchoring them to other epidermal cells. Muscle-cells below the basement membrane also make numerous contacts with the basement membrane as well as with each other. Following anterior amputation in Owenia, the wound-healing process brings the edges of the epidermis and underlying muscle layer in direct contact, with no basement membrane between them. In addition, epidermal cells that are near the wound and close to the basement membrane lose contact with the basement membrane. Correlated with the loss of contact with the basement membrane, epidermal

cells lose their basal cytoskeleton network and also lose the adherence-structures anchoring them to other epidermal cells. The muscle layer below also changes extensively; muscle-cells near the wound lose contact with the basement membrane and lose the membranes that anchored them to other musclecells. They then autotomize their contractile apparatus and dedifferentiate to a myoblast-like form. Epidermal and muscle-cells then both begin to proliferate (see below ‘‘Cellular origins of the blastema’’). The important changes both in epidermal and in mesodermal cells, and especially their loss of connections to other cells, presumably allow for rearrangement and migration of cells as well as their proliferation during wound-healing and early stages of regeneration. Within a few days after amputation, the extracellular matrix reforms between the woundepidermis and the underlying mesoderm. A thin basement membrane is evident within about 2 dpa, collagen is strongly upregulated at the wound site by 4–5 dpa, and the basement membrane has fully reformed by about 1 week after amputation. At that time, ectodermal and mesodermal cells both re-develop contacts with the basement membrane and adherence-structures with each other (Dupin et al. 1991). In the leech Hirudo, an annelid that wound-heals and scars but does not regenerate structures, there are also significant changes in cell–cell adhesion, cytoskeletal organization, and the extracellular matrix following injury (Huguet and Molinas 1994, 1996; Tettamanti et al. 2004). Wounds in the body wall are sealed largely by vasocentral (fibroblast) cells; these cells migrate in to form a wound-plug, contract the wound’s edges by modifying their cytoskeleton and their cell–cell contacts, and secrete an extracellular scaffold of proteins, largely collagen, that further seals the wound. Although the cells eventually leave the site of the wound, the collagen scaffold is not destroyed and remains as a scar. Available data thus suggest that the extracellular matrix may play an important role in healing wounds and in tissue-level changes at the wound’s site, but only a few studies have investigated such questions in annelids. Studies of a broader range of annelids are needed to identify the general patterns of change in the extracellular matrix and in the remodeling of tissues. Furthermore, experimental work is needed to determine whether the changes in the extracellular matrix and nearby tissues are merely correlated with each other or whether they are actually causally linked, and, if the latter,


produces a continuous outer epithelium (referred to as a wound-epithelium or wound-epidermis) with no opening; the mouth or anus is then formed secondarily. This type of healing occurs following anterior amputations and usually following posterior amputations. However, some descriptions of posterior regeneration indicate that the severed edges of the epidermis fuse directly to the severed edges of the gut epithelium; when this occurs the anus is reformed directly by the wound-healing process. Wound-healing by fusion of the outer epidermis with the epithelium of the gut has been described following posterior amputation in Sabella (Hill 1970) and Nereis (Boilly 1969a), and in Nephtys, posterior amputation can be healed in either way, suggesting that the mode of healing can vary even within a species (Clark and Clark 1962). Regardless of the type of healing, the re-epithlialization process consistently occurs by rearrangement of cells, and without mitosis (Hill 1970; Paulus and Mu¨ller 2006; Zattara and Bely 2011, 2013). Indeed, re-epithelialization typically is complete well before the first indication of cell proliferation at the wound (typically at about 1–2 dpa).

A. E. Bely


what the cause-and-effect relationships are between them.

Formation of the blastema Initiation of the blastema

cut occurs at an axial position incapable of regeneration, no nerve fibers extend subepidermally (Coulon and Thouveny 1984). Although a considerable amount of data suggests that key aspects of annelid regeneration are dependent on the nerve cord, there also appear to be some components of regeneration that occur independently of this structure as well as some species in which regeneration can still proceed without a ventral nerve cord at the site of the wound. In Eisenia, in the absence of the nerve cord at the wound, ectodermal and mesodermal growth is inhibited but considerable endodermal growth still proceeds (Avel 1961). Indeed, in individuals in which the nerve cord is removed over several segments closest to the site of amputation, the anterior limit of the gut still grows substantially, even though a blastemal outgrowth never forms. Eventually the tip of the gut grows ventrally and then curves posteriorly, extending until it contacts the ventral nerve cord several segments back. Despite the growth and formation of a pharynx anlagen in these animals, pharyngeal differentiation does not occur, suggesting that signals from the nerve cord or the blastema itself are required for the differentiation process. Additional data also suggest a role for tissues other than the nerve cord in initiating regeneration. In Tubifex, it is the severing of the gut at the site of the wound that is implicated in initiating blastemal outgrowth, although comparable studies in Eisenia show no such requirement (reviewed by Avel 1959). Finally, in Nereis, a blastemal outgrowth does form following amputation within a body region in which the nerve cord has been removed (although the resulting blastema is abnormal, having apparently incomplete dorsal–ventral polarity) (Combaz and Boilly-Marer 1976; WattezCombaz 1995), in contrast to the several examples noted in the preceding paragraph in which comparable cuts fail to elicit a blastema. Although the kinds of surgical manipulations described above provide important insights into the regenerative process, they should also be interpreted with some caution. First, excisions of certain tissues (e.g., nerve cord and gut) can alter the spatial relationships among remaining tissues in operated animals; the collapsing of tissues may still allow for some contact between relevant tissues (Fitzharris and Lesh 1969). Second, the degree of inhibition of regeneration can vary even across closely related species. For example, comparable experiments performed by the same experimenter on two species of Eisenia resulted in considerably different outcomes: amputation within a body region in which the ventral nerve cord was removed led to


Studies in a range of annelids indicate that interactions of tissues at the site of the wound can be important for initiating a blastema. In particular, several lines of evidence suggest that the severed end of the ventral nerve cord can elicit regeneration and proliferative outgrowth. First, surgeries in which a severed end of the nerve cord is diverted to a wound in the body wall in Eisenia lead to the formation of an ectopic blastema that differentiates into a lateral head (Avel 1959). Thus, interaction between a cut ventral nerve cord and an injured body wall may be sufficient to initiate formation of a blastema. Second, amputation in a body region that has had the ventral nerve cord removed can inhibit regeneration, often completely (Avel 1961). This effect has been shown to depend both on the timing of removal of the ventral nerve cord and on the distance between the severed nerve cord and the site of the wound. When the ventral nerve cord is removed following amputation in Eisenia, the degree of inhibition decreases as the time between amputation and removal of the nerve cord increases, until, beyond a certain time (4–5 days, or mid-blastemal stage) the negative effect of removing the nerve cord on regeneration disappears. In Eurythoe, extirpation of the ganglion closest to the site of the wound retards regeneration, whereas extirpation of the two nearest ganglia abrogates it completely, leading only to healing (Mu¨ller et al. 2003). Thus, the severed nerve cord appears to be important for initiating and sustaining the early stages of regeneration in a temporally and spatially dependent manner. Third, in several groups of annelids investigated, the earliest cell proliferation detected during regeneration occurs near the severed ventral nerve cord (Coulon and Thouveny 1984; Mu¨ller et al. 2003). Finally, the presence of nerve ganglia or nerve fibers correlates with regenerative ability in several species. In Chaetopterus, excising the portion of a long mid-segment (segment 12) that has the ventral ganglion leads to regeneration, while excising a similar-sized portion of the same segment but that does not contain the ventral ganglion leads to failure to regenerate (Hill 1972). In Owenia, when anterior amputation occurs in a regeneration-competent region of the body, nerve fibers extend from the ventral nerve cord into the site of the wound, with the fibers running between the epidermis and mesoderm. However, when the

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Polarity of the blastema Annelid blastemas have either anterior (head) or posterior (tail) axial polarity; no mixed-polarity phenotypes are known (Avel 1959). Indeed, even amputating in a body region in which axial polarity should

differ across the surface of the wound (following a body-wall graft) produces a blastema with a single polarity (reviewed by Avel 1959). Although the cues that assign a blastema its axial polarity remain to be elucidated, blastemas that regenerate with incorrect polarity can be generated, indicating that polarity can be mis-assigned. For example, heads have been formed at posterior cut sites in Sabella following treatment with colchicine (a tubulin-polymerization inhibitor that blocks mitosis, among other effects) (Fitzharris and Lesh 1969), in Dero when tiny fragments (one to three segments) are excised (Hyman 1916), occasionally in Enchytraeus following simple amputations (Myohara et al. 1999), and at relatively high frequencies in this same Enchytraeus species when amputated individuals are treated with an anesthetic that inhibits corrective (post-amputation) autotomy (Kawamoto et al. 2005). Regarding the last of these, corrective autotomy is a post-injury self-amputation that places the site of the wound at a stereotypical intra-segmental plane; thus, in this species, initiating a blastema from the wrong intrasegmental position may be what leads to mis-assigned polarity. Based on available records of biaxial worms, double-headed worms, with a head regenerated at the posterior end, appear to be much more common than double-tailed worms, with a tail that regenerated at the anterior end. The only examples of double-tailed individuals that could be found are in Eisenia (Gates 1949, 1950). The assignment of dorsal–ventral polarity of the blastema has also been investigated using grafting experiments (Combaz and Boilly-Marer 1976; Wattez-Combaz 1995). These studies have been conducted on Nereis, in which a blastemal outgrowth can form even in the absence of the ventral nerve cord. Amputations made in body regions from which the ventral nerve cord had been removed yielded an abnormal blastema that formed a pygidium (posterior tip) and segments, but which were smaller and with fewer structures than normal. To investigate the dorsal–ventral polarity of these ‘‘aneurogenic’’ blastemas, patches of body wall from them were grafted onto normal hosts. Because parapodia (segmental, lateral projections of the body wall) are thought to form at the junction between dorsal and ventral body-wall tissue, ectopic parapodia are expected to form at boundaries between patches of dorsal tissue and ventral tissue, but not between two patches of dorsal tissue or between two patches of ventral tissue. Results showed that ectopic parapodia formed at the edges of grafts when body wall from anywhere in the aneurogenic blastema was grafted onto the ventral region, but not onto the dorsal


regeneration failing in all individuals in one species but resulted in a small, slowly growing blastema in about one quarter of the animals in a second species (Avel 1961). Thus, carefully controlled experiments and investigations in a broader range of annelids are needed to strengthen the conclusions of these studies. In addition to the effect of interactions of tissues at the site of the wound, a number of studies suggest that the nervous system, and the brain in particular, can have long-distance effects on regeneration, possibly through the release of hormones promoting or inhibiting regeneration. The effect of manipulations such as removal of the brain varies considerably among species, however. In Nereis, removal of the brain inhibits posterior regeneration (Golding 1967), yet in a number of other annelids (Branchiomma, Chaetopterus, Eophila, and Eulalia) (Gallissi 1965; Hill 1972; Olive and Moore 1975) posterior regeneration is still possible and may even be promoted by removing the brain. In Allolobophora, the effect of removing the brain on posterior regeneration has been found to depend on the developmental stage of the animal: in juveniles, presence of the brain promotes regeneration, whereas in sexually mature (clitellated) individuals, presence of the brain inhibits regeneration (AlonsoBedate and Sequeros 1985). If these effects on regeneration are indeed caused by hormones emanating from the brain, then hormonal changes at sexual maturity may be mediating these stage-dependent effects. In summary, a large amount of experimental and correlational evidence suggests that the nervous system has a particularly important role in initiating and sustaining at least certain aspects of blastemal development. Many manipulations that have been performed are ‘‘cut-and-paste’’ type experiments and are necessarily rather crude, yet effects on regeneration can be dramatic (e.g., complete failure of regeneration). Replicating some of these experiments in a broader range of annelids would be useful for assessing how general their results are. Determining what molecules are responsible for both short-range and long-range signals that inhibit or promote regeneration is also an important direction of research to pursue.

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Cellular origins of the blastema In a broad range of annelids, the location of cells in mitosis and the incorporation of thymidine-analogs indicate that ectoderm, mesoderm, and endoderm all proliferate at the wound, consistent with the hypothesis that all three tissue layers contribute to the blastema (Buongiorno-Nardelli and Thouveny 1966; Hill 1970; Coulon and Thouveny 1984; Paulus and Mu¨ller 2006; Zattara and Bely 2011). Consistently, there is also a significant delay (usually on the order of at least a day) between the time of amputation and the earliest evidence of proliferation; this delay suggests that some preparatory steps, such as wound-healing and dedifferentiation, may need to occur prior to blastemal growth. The epidermis of the regenerated body wall, the cell bodies of the nervous system, and the foregut are ectodermal tissues that, based on time-series studies

of fixed tissues, appear to derive from the epidermis at the wound. The epidermis covering the wound and later that of the blastema is highly proliferative and this proliferation appears to be the only source of new epidermal cells (Boilly 1969b; Hill 1970; Zattara and Bely 2011). The ventral epidermis of the blastema in Limnodrilus generates some cells with little cytoplasm that appear to move inwards to form new nerve ganglia. In this and a number of other annelids, the ectoderm thus appears to furnish internal cells that will become neural cells (Cornec et al. 1987; Cornec 1990). In a broad range of annelids, nerve fibers thought to originate from cells in the old ventral nerve cord also invade the blastema and form some of the neural tracts of the new brain and ventral nerve cord (Mu¨ller 2004; Mu¨ller and Henning 2004; Paulus and Mu¨ller 2006; Zattara and Bely 2011). Regarding the foregut, the regenerated buccal cavity appears to originate from invaginated surface ectoderm at the site of the wound (Hyman 1916, 1940; Zattara and Bely 2011). Although data from a number of sources suggest that ectodermal structures consistently arise from the wound’s epidermis, one study does suggest, based on the histology of fixed tissue, that dissepimentary cells may contribute to nerve ganglia (Cornec et al. 1987); this possibility warrants further investigation since a mesodermal source for regenerated neural cells is unexpected. Histological studies of fixed tissue at different stages of regeneration also suggest that the source of new mesoderm is primarily local dedifferentiated muscle-cells and/or dissepimentary cells (cells of the lining of the septa) that migrate to the wound, although endothelial cells (associated with blood vessels) have also been implicated as a source. Muscle regeneration has been studied in some detail during anterior regeneration in Owenia (Fontes et al. 1983). In this annelid, histological studies suggest that longitudinal muscles at the site of the wound dedifferentiate into myoblast-like cells, proliferate, and then redifferentiate into new muscle-cells. During dedifferentiation, the muscle-cells, which are mononucleate, lose their membrane-anchoring structures and autotomize, separating into a nucleated part and contractile part. The shed, enucleated part degenerates in the coelom or is phagocytized by coelomocytes; the nucleated part gives rise to dedifferentiated muscle-cells that migrate to the wound, proliferate there, and redifferentiate. Interestingly, according to this study, this process involves major changes in cellular morphology but little change in molecular expression, suggesting that morphological dedifferentiation of muscle-cells may not alter the terminal


region, of the host. This, and other, experiments (including the grafting of a supernumerary ventral nerve cord) suggest that the body wall of the blastema in Nereis does not have an inherent dorsal– ventral polarity but instead has a default state of dorsal polarity; the ventral nerve cord then imposes ventral polarity in tissue that is in close proximity. Results consistent with these findings were also found in earthworms (Avel 1942; Okada and Kawakami 1943; Avel 1947; cited in WattezCombaz 1995). In these annelids, a blastema with normal dorsal–ventral polarity was produced from a stump that was not expected to have dorsal–ventral polarity in the body wall, a cuff of body-wall tissue with all dorsal or all ventral polarity having been grafted at the site prior to amputation. These experiments suggest that the dorsal–ventral polarity of the blastema does not inherit this polarity solely from the body wall at the site of the wound. In summary, knowledge of the assignment of blastemal polarity remains rudimentary and stems from very few studies. Both anterior–posterior and dorsal– ventral polarity can be mis-assigned through experimental manipulations, but the underlying mechanisms assigning polarity remain to be elucidated. Identifying the molecular signals that assign polarity is an important direction to pursue. In planaria, a single signaling pathway (Wnt/ -catenin) is sufficient for imposing the axial polarity of the blastema and, when mis-expressed, can result in a blastema with the wrong polarity (Gurley et al. 2008). Whether a single pathway, and possibly even the same pathway, is involved in this process in annelids will be an interesting question to address.

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based on histology and gene expression, that germline cells are distributed along the length of the body and that, during anterior regeneration, such cells migrate anteriorly to the wound from several segments away to re-establish the germ line in the regenerated gonad-bearing segments (Tadokoro et al. 2006). By contrast, in Capitella, expression patterns for the same molecular marker used in the Enchytraeus study (a piwi homolog) do not suggest migration of marker-positive cells to the wound (Giani et al. 2011). Whether this difference is because the mechanism of germ-line regeneration differs between these two species or because the markers themselves are expressed differently between them remains to be determined. Additional markers and methods to identify germ-line cells, as well as studies in a broader range of annelids, are needed to address the important question of how the germ line is regenerated in annelids. Finally, new endodermal cells appear to come from old endodermal cells in annelids. Proliferation assays indicate that the cut tip of the gut proliferates and histological studies suggest this tissue gives rise to the new gut (Hill 1970). Importantly, a recent study employing a cell-tracing technique in Lumbriculus has confirmed that old gut tissue does indeed contribute to new gut tissue (Tweeten and Reiner 2012). Cell-tracing studies such as this one are key to validating inferences that previously have been based solely on histological data. In summary, although the question of what cells contribute to regenerated structures in annelids has long been debated (Randolph 1891, 1892; Boilly 1969b; Hill 1970), a working model that appears to be general for annelids is emerging. Specifically, all, or most, of the blastema is thought to arise from cells at, or very near, the site of the wound; only some portion of the mesoderm in one group of annelids (clitellates) is thought possibly to originate from cells that migrate from several segments away (with no evidence for such a long-distance migration from polychaetes). Source-cells appear to dedifferentiate, proliferate at the site of the wound, and then re-differentiate, with each layer of tissue, apparently giving rise to its own layer in the regenerate. Several key questions remain regarding the cellular origins of the blastema, however. First, whether source-cells fully dedifferentiate before proliferating is an important question that needs further investigation; several authors have indicated that cells (specifically of the epidermis and of the muscle layer) may maintain some level of differentiation, and even retain characteristic gene expression, as they proliferate (Fontes et al. 1983; Cornec et al. 1987). Second, the breadth


differentiation program. While results from Owenia are largely consistent with findings in several other annelids (Hill 1970), an alternative mechanism of muscle formation has been described in Hirudo (Grimaldi et al. 2006, 2009). In this annelid, regeneration of muscle has been investigated following the making of lesions in the body wall (since Hirudo does not regenerate segments) and occurs considerably longer after injury (1 month after body lesions in Hirudo versus a few days after transverse amputation in many other annelids). In Hirudo, the mononucleated muscle-cells can proliferate slowly but most of the regenerated muscle-cells are thought to arise from a population of precursor cells, normally associated with the blood vessels in uninjured animals, which migrate to the wound and differentiate into new muscle. Recruitment of these cells to the regenerating areas is thought to be mediated, at least in part, by a growth factor (Grimaldi et al. 2009). Origin of new mesoderm from cells associated with blood vessels has also been suggested for some polychaetes (Hill 1970). Additional studies are needed to determine the generality of either of these processes in the regeneration of muscles. Migration of dissepimentary cells to the wound has been inferred in a range of annelids based on histology of fixed tissue. Many studies have suggested that these cells contribute to the coelomic lining (including septum walls) of new segments and to new muscles (reviewed by Cresp 1964; Hill 1970). Although migration of such cells in ‘‘polychaetes’’ is reported only over short distances (within one segment from the wound) (Cresp 1964; Hill 1970), in a number of clitellates a much more extensive migration is inferred. In clitellates, amputation appears to induce activation of dissepimentary cells over a number of segments and these cells then appear to migrate intersegmentally along the ventral nerve cord to the wound, where they finally proliferate (reviewed by Stephan-Dubois 1954; Cornec et al. 1987). As mentioned above, these migrating cells are sometimes referred to as neoblasts. It remains to be determined whether these cells and this process are characteristic of clitellates or merely most easily seen or more extensive in this group. The fate of these intersegmentally migrating dissepimentary cells is also difficult to ascertain and has been heavily debated (Stephan-Dubois 1954; Hill 1970; Jamieson 1981; Myohara 2012). To help resolve this long-standing issue, studies that directly trace the fates of these migrating cells are critically needed. The source of the germ line in regenerated segments is an important question that also needs greater study. A study in Enchytraeus suggests,

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Conclusions The cellular events of regeneration have been studied in a remarkably broad range of annelid species. Unlike the study of regeneration in many other groups, efforts have not concentrated on just one or two model systems but instead have been distributed over many species. Although this distribution of effort is not without drawbacks (i.e., the picture that has emerged is broad yet still relatively shallow), a benefit is that generalities are apparent for a number of aspects of regeneration across the phylum. Thus, it is possible to compile a broad summary picture of how regeneration typically proceeds in annelids, as has been done here. Whether regeneration in annelids is, in fact, more uniform as a process across the phylum than it is in other phyla remains to be seen. Many important questions remain regarding the early cellular events of regeneration in annelids. Some of these are fundamental, yet understudied or difficult to address. For example, how does the early cellular response to injury differ depending on the type of injury, the severity of injury, and the potential for regeneration? What is the source and nature of cells that contribute to the regenerated structures? Have the major events of regeneration and the cellular origins of regenerated structures evolved across annelid phylogeny, and if so, how? To help address these, and other, important questions, new techniques and approaches should be applied to the study of regeneration in annelids. There is a particular need to develop and apply methods to identify and manipulate specific cell types (e.g., Grimaldi et al. 2011), to image the dynamics of

cells and tissues over long durations in vivo (e.g., Zattara 2012), and to trace the fates of cells (e.g., Weisblat et al. 1978; Meyer et al. 2010; Tweeten and Reiner 2012) during regeneration. Employing such new approaches is likely to yield major advances in our understanding of the cellular basis of regeneration in annelids.

Acknowledgments The author thanks Eduardo Zattara and Duygu ¨ zpolat for extensive and helpful discussions about O regeneration in annelids and Mansi Srivastava, Deirdre Lyons, and Mark Martindale for the opportunity to participate in this symposium.

Funding Participation in the symposium was supported by the Society for Integrative and Comparative Biology.

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