Rate of regeneration of two arms in the field and its effect on body components in Luidia clathrata (Echinodermata: Asteroidea)

The incidence of arm regeneration, effects of arm regeneration on non-regenerating body components and rate of arm regeneration were examined under field conditions. Approximately 60% of Luidia clathrata found in the upper portion of Tampa Bay, Florida were regenerating one or more arms. Individuals with two arms amputated at the disk edge took 380 days to regenerate completely. Both arms of an individual regenerated at the same rate. Length of regenerating arms initially increased faster than dry mass. No difference was found between non-regenerating and regenerating individuals for the dry mass of the body wall, pyloric caeca, and gonad of a non-regenerating arm. The absolute masses of the gonad and pyloric caeca were low through out the year possibly indicating a low food environment. Based on the rate of regeneration, arm loss just prior to the reproductive season may reduce the reproductive potential of this species.     

Electric currents in Xenopus tadpole tail regeneration

Xenopus laevis tadpoles can regenerate tail, including spinal cord, after partial amputation, but lose this ability during a specific period around stage 45. They regain this ability after stage 45. What happens during this “refractory period” might hold the key to spinal cord regeneration. We hypothesize that electric currents at amputated stumps play significant roles in tail regeneration. We measured electric current at tail stumps following amputation at different developmental stages. Amputation induced large outward currents leaving the stump. In regenerating stumps of stage 40 tadpoles, a remarkable reversal of the current direction occurred around 12-24 h post-amputation, while non-regenerating stumps of stage 45 tadpole maintained outward currents. This reversal of electric current at tail stumps correlates with whether tails regenerate or not (regenerating stage 40—inward current; non-regenerating stage 45—outward current). Reduction of tail stump current using sodium-free solution decreased the rate of regeneration and percentage regeneration. Fin punch wounds healed normally at stages 45 and 48, and in sodium-free solution, suggesting that the absence of tail re-growth at stage 45 is regeneration-specific rather than a general inhibition of wound healing. These data suggest that electric signals might be one of the key players regulating regeneration.     

General characterization of regeneration in Aeolosoma viride (Annelida,Aeolosomatidae)

Regeneration has long been the focus of scientific interest for its potential to restore lost, damaged, or aged tissues and organs. A wide range of regenerative studies have been conducted on different vertebrate and invertebrate model organisms. Annelids are known for their regenerative capacities, and because of their relatively complex organ systems, they are an ideal organism for regeneration study. Our present work focused on the freshwater annelid Aeolosoma viride, an asexually reproducing annelid capable of regenerating both anteriorly and posteriorly. Even though regenerative ability has been documented in this animal in previous studies, detailed characterization of the process is still unavailable. The objective of this study was to evaluate the regenerative ability of A. viride. We described the sequential morphological events during the process of regeneration, such as wound healing and the formation of blastema, mouth, and pygidium. In order to clarify the capacity and type of regeneration, we conducted a series of observations and experiments using a cell proliferation assay. Massive proliferation and the absence of cell migration indicated that the animal regenerates primarily through epimorphosis. Our study of the epimorphic regenerative process of A. viride provides a clearer picture of the evolutionary origin of regeneration in annelids.     

What role do annelid neoblasts play? A comparison of the regeneration patterns in a neoblast-bearing and a neoblast-lacking enchytraeid oligochaete

The term 'neoblast' was originally coined for a particular type of cell that had been observed during annelid regeneration, but is now used to describe the pluripotent/totipotent stem cells that are indispensable for planarian regeneration. Despite having the same name, however, planarian and annelid neoblasts are morphologically and functionally distinct, and many annelid species that lack neoblasts can nonetheless substantially regenerate. To further elucidate the functions of the annelid neoblasts, a comparison was made between the regeneration patterns of two enchytraeid oligochaetes, Enchytraeus japonensis and Enchytraeus buchholzi, which possess and lack neoblasts, respectively. In E. japonensis, which can reproduce asexually by fragmentation and subsequent regeneration, neoblasts are present in all segments except for the eight anterior-most segments including the seven head-specific segments, and all body fragments containing neoblasts can regenerate a complete head and a complete tail, irrespective of the region of the body from which they were originally derived. In E. japonensis, therefore, no antero-posterior gradient of regeneration ability exists in the trunk region. However, when amputation was carried out within the head region, where neoblasts are absent, the number of regenerated segments was found to be dependent on the level of amputation along the body axis. In E. buchholzi, which reproduces only sexually and lacks neoblasts in all segments, complete heads were never regenerated and incomplete (hypomeric) heads could be regenerated only from the anterior region of the body. Such an antero-posterior gradient of regeneration ability was observed for both the anterior and posterior regeneration in the whole body of E. buchholzi. These results indicate that the presence of neoblasts correlates with the absence of an antero-posterior gradient of regeneration ability along the body axis, and suggest that the annelid neoblasts are more essential for efficient asexual reproduction than for the regeneration of missing body parts.     

Regeneration of posterior segments and terminal structures in the bearded fireworm,Hermodice carunculata (Annelida: Amphinomidae)

Like many other annelids, bearded fireworms, Hermodice carunculata, are capable of regenerating posterior body segments and terminal structures lost to amputation. Although previous research has examined anterior regeneration in other fireworm species, posterior regenerative ability in fireworms remains poorly studied. As the morphology of the anal lobe (a small, fleshy terminal structure of unknown function) has been used to distinguish East and West Atlantic H. carunculata populations, there is a more imminent need to understand the morphology and organization of tissues in specimens undergoing posterior regeneration, and the timeframe in which significant developmental changes occur. To further investigate this phenomenon, we amputated the posterior segments of living H. carunculata specimens collected from the Gulf of Mexico and monitored posterior regeneration over a 6‐month study period. Although many aspects of posterior regeneration in H. carunculata are consistent with the findings of other annelid regeneration studies, histological analysis revealed that once formed, anal lobe morphology remains relatively unchanged at all stages of posterior regeneration; East Atlantic morphotypes were not observed in the West Atlantic specimens studied here. Additionally, we found that the ventral nerve chord, which is partially responsible for the regeneration of lost body parts in polychaete annelids, terminates within the anal lobe, suggesting that this structure may play a role in the formation of new segments. J. Morphol. 275:1103–1112, 2014. © 2014 Wiley Periodicals, Inc.     

Evidence of regenerative ability in Myxicola infundibulum (Annelida,Sabellida): evolutionary and systematic implications

Members of only a few species of annelids are reported as being incapable of regeneration; of these, Myxicola infundibulum is the only example in the family Sabellidae. Interestingly, its congener Myxicola aesthetica exhibits noteworthy regenerative ability. Unambiguously identifying non‐regenerating species is critical to reconstructing how regenerative abilities evolved within the phylum. However, studies designed specifically to assess the regenerative potential of M. infundibulum have never been performed. In this study, we aimed to confirm the lack of regeneration ability of M. infundibulum, reported previously for Atlantic specimens, or to determine the extent to which regeneration occurs. Our results showed that individuals from the Mediterranean Sea (Adriatic Sea) do undergo regeneration of lost body parts, although to a lesser extent than do other sabellids. Therefore, M. infundibulum should no longer be considered a non‐regenerating species. At present, uncertainties regarding phylogenetic relationships of Sabellidae prevent inferences about the polarity of change in M. infundibulum. Since our findings are counter to those of previous studies which describe Atlantic specimens as non‐regenerating, more extensive analysis is required to ascertain if they could actually belong to a different species than Mediterranean M. infundibulum, accounting for these differences in reported regenerative capacity.     

Dynamic gene expression profiles during arm regeneration in the brittle star Amphiura filiformis

Echinoderms and in particular brittle stars display a remarkable ability to regenerate lost or damaged tissues. They offer an excellent model in which to study regeneration displaying extensive regenerative ability and close relationship to vertebrates providing the opportunity for comparative studies. Previous studies of gene expression during arm regeneration in brittle stars have focused on single genes commonly associated with the regenerative process. In this study we present the first microarray investigation of gene expression during arm regeneration in the brittle star Amphiura filiformis. We show the large-scale gene expression changes associated with the complex process of regeneration with over 50% of the clones measured showing a significant change at some point during the process when compared to non-regenerating arms. Particular attention is paid to genes associated with Hox gene expression regulation, neuronal development and the bone morphogenic protein BMP-1. Our data give an insight into the molecular control required during the various stages of regeneration from the stem cell rich blastema stage through to the highly differentiated regenerate. This work also forms an important basis for future gene expression investigations in this emerging model of limb regeneration.     

Evolutionary loss of animal regeneration: pattern and process

The ability to regenerate lost or damaged body parts is widespread among animals and provides obvious potential benefits. It is therefore perplexing that this ability has become greatly restricted or completely lost in many lineages. Despite growing interest in the cellular and molecular basis of regeneration, our understanding of how and why regenerative abilities are lost remains rudimentary. In an effort to develop a framework for studying losses of regeneration, here I outline an approach for rigorously identifying such losses, review broad patterns of regenerative ability across animals, describe some of the clearest examples of regeneration loss, discuss some possible scenarios by which regeneration may be lost, and review recent work in annelids that is providing new insights into loss of regenerative ability.     

Activity of antioxidant enzymes and secondary metabolites during in vitro regeneration of Sterculia urens

The changes in the activities of antioxidant enzymes and amounts of proteins, phenols, and flavonoids in regenerating and non-regenerating calli during organogenesis of Sterculia urens were monitored. Maximum growth of calli and the most efficient regeneration of shoots occurred on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^?3 benzylaminopurine (BAP) + 2 or 4 mg dm^?3 naphtalene acetic acid (NAA). Peroxidase (POD), catalase, and superoxide dismutase activities increased in the regenerating calli but decreased in the non-regenerating calli. Six POD isoenzymes were detected. Protein content decreased in the non-regenerating calli and increased significantly during regeneration of shoots from callus. Total phenols and flavonoids increased in the non regenerating calli. SDS-PAGE analysis revealed a role of many proteins in organogenesis.     

Hyaluronidase activity and glycosaminoglycan synthesis in the amputated newt limb: comparison of denervated, nonregenerating limbs with regenerates.

Amputated, regenerating forelimbs have been compared with the contralateral, denervated non-regenerating limb stumps in the adult newt Notophthalmus viridescens, with respect to hyaluronidase activity and the incorporation of ³H-acetate into glycosaminoglycans (GAG). At 10 days after amputation, which is the time of maximum hyaluronate production in the early growing regenerate, incorporation of ³H-acetate into GAG (cpm/mg protein) in the denervated, nonregenerating limb stump was approximately 50% of that in the contralateral regenerating limbs. At this stage, hyaluronate was the major GAG being produced, but the ratio of incorporation into hyaluronate relative to chondroitin sulfate was reduced in the denervated limbs. In intact, nonamputated limbs, the incorporation into GAG was 5% of that in the regenerating limb 10 days after amputation, and 10% of that in the denervated stumps.At 25 days, cartilage is forming and chondroitin sulfate synthesis predominates in the normal regenerate whilst the contralateral, denervated limb stumps are forming scars. GAG synthesis in the latter was less than one-quarter the level seen in the regenerating limbs, mostly due to low incorporation into chondroitin sulfate.Hyaluronidase activity, which appears in the regenerating limb during differentiation of skeletal elements (20–45 days), was not detectable in limbs denervated early enough to prevent regeneration. However, limbs denervated after formation of the blastema will regenerate without nerve, and hyaluronidase activity in such limbs was normal. Thus, hyaluronidase activity appears when regeneration reaches the cartilage deposition stage, with or without nerve.     

Early segregation of germ and somatic lineages during gonadal regeneration in the annelid Enchytraeus japonensis

Although regeneration studies are useful for understanding how organs renew, little information is available about regeneration of reproductive organs and germ cells. We here describe the behavior of germ-cell precursors during regeneration of the oligochaete annelid worm Enchytraeus japonensis, which has the remarkable feature of undergoing asexual (by fission) and sexual reproduction . We first found that the gonad can regenerate from any body fragment yielded by fission during asexual reproduction. We then examined behavior of germ-cell lineage during this regenerative process, by using a homolog of the Piwi gene (Ej-piwi) as a marker. We found that in asexually growing animals, specialized cells expressing Ej-piwi are distributed widely in the body as single cells. These cells seem to serve as a reservoir of germ-cell precursors because during asexual propagation these cells migrate into the regenerating tissue, where they ultimately settle in the prospective gonads, and give rise to germ cells upon sexualization. These cells are distinct from the neoblasts, thought to be stem cells in other animals. This is the first report to directly show that the germ and somatic lineages are segregated in asexually growing animals and behave differently during regeneration.     

Evidence for the local evolution of mechanisms underlying limb regeneration in salamanders

The most extensive regenerative ability in adult vertebrates is found in the salamanders. Although it is often suggested that regeneration is an ancestral property for vertebrates, our studies on the cell-surface three-finger-protein Prod 1 provide clear evidence for the importance of local evolution of limb regeneration in salamanders. Prod 1 is implicated in both patterning and growth in the regeneration of limbs. It interacts with well-conserved proteins such as the epidermal growth-factor receptor and the anterior gradient protein that are widely expressed in phylogeny. A detailed analysis of the structure and sequence of Prod 1 in relation to other vertebrate three-finger proteins in mammals and zebra fish supports the view that it is a salamander-specific protein. This is the first example of a taxon-specific protein that is clearly implicated in the mechanisms of regeneration. We propose the hypothesis that regeneration depends on the activity of taxon-specific components in orchestrating a cellular machinery that is extensively conserved between regenerating and non-regenerating taxa. This hypothesis has significant implications for our outlook on regeneration in vertebrates, as well as for the strategies employed in extending regenerative ability in mammals.     

Structure and anterior regeneration of musculature and nervous system in Cirratulus cf. cirratus (Cirratulidae,Annelida)

Annelids provide suitable models for studying regeneration. By now, comprehensive information is restricted to only a few taxa. For many other annelids, comparative data are scarce or even missing. Here, we describe the regeneration of a member of the Cirratulus cirratus species complex. Using phalloidin‐labeling and antibody‐stainings combined with subsequent confocal laser scanning microscopy, we provide data about the organization of body wall musculature and nervous system of intact specimens, as well as about anteriorly regenerating specimens. Our analyses show that C. cf. cirratus exhibits a prominent longitudinal muscle layer forming a dorsal muscle plate, two ventral muscle strands and a ventral‐median muscle fiber. The circular musculature forms closed rings which are interrupted in the area of parapodia. The nervous system of C. cf. cirratus shows a typical rope‐ladder like arrangement and the circumesophageal connectives exhibit two separate roots leading to the brain. During regeneration, the nervous system redevelops remarkably earlier than the musculature, first constituting a tripartite loop‐like structure which later become the circumesophageal connectives. Regeneration of longitudinal musculature starts with diffuse ingrowth and subsequent structuring into the blastema. In contrast, circular musculature develops independently inside the blastema. Our findings constitute the first analysis of regeneration for a member of the Cirratuliformia on a structural level. Summarizing the regeneration process in C. cf. cirratus, five main phases can be subdivided: 1) wound closure, 2) blastema formation, 3) blastema differentiation, 4) resegmentation, and 5) growth, respectively elongation. Additionally, the described tripartite loop‐like structure of the regenerating nervous system has not been reported for any other annelid taxon. In contrast, the regeneration of circular and longitudinal musculature originating from different groups of cells seems to be a general pattern in annelid regeneration. J. Morphol. 275:1418–1430, 2014. © 2014 Wiley Periodicals, Inc.     

Morphological,cellular and molecular characterization of posterior regeneration in the marine annelid Platynereis dumerilii

Regeneration, the ability to restore body parts after an injury or an amputation, is a widespread but highly variable and complex phenomenon in animals. While having fascinated scientists for centuries, fundamental questions about the cellular basis of animal regeneration as well as its evolutionary history remain largely unanswered. Here, we present a study of regeneration of the marine annelid Platynereis dumerilii, an emerging comparative developmental biology model, which, like many other annelids, displays important regenerative abilities. When P. dumerilii worms are amputated, they are able to regenerate the posteriormost differentiated part of their body and a stem cell-rich growth zone that allows the production of new segments replacing the amputated ones. We show that posterior regeneration is a rapid process that follows a well reproducible path and timeline, going through specific stages that we thoroughly defined. Wound healing is achieved one day after amputation and a regeneration blastema forms one day later. At this time point, some tissue specification already occurs, and a functional posterior growth zone is re-established as early as three days after amputation. Regeneration timing is only influenced, in a minor manner, by worm size. Comparable regenerative abilities are found for amputations performed at different positions along the antero-posterior axis of the worm, except when amputation planes are very close to the pharynx. Regenerative abilities persist upon repeated amputations without important alterations of the process. We also show that intense cell proliferation occurs during regeneration and that cell divisions are required for regeneration to proceed normally. Finally, 5-ethynyl-2’-deoxyuridine (EdU) pulse and chase experiments suggest that blastemal cells mostly derive from the segment immediately abutting the amputation plane. The detailed characterization of P. dumerilii posterior body regeneration presented in this article provides the foundation for future mechanistic and comparative studies of regeneration in this species.     

Regeneration: Rewarding,but potentially risky

Some bilaterally symmetric animals, such as flatworms, annelids, and nemerteans, are renowned for their outstanding regeneration capacity—even a fraction of the body can give rise to a complete new animal. However, not all species of these taxa can regenerate equally well—some cannot regenerate at all. If regeneration was purely beneficial, why cannot all of members of the flat, round, and ribbon worms regenerate? At that, why cannot all other bilaterians, including humans, regenerate as well? Regeneration capacity is an obvious advantage in accidental, predatory, and parasitic loss of body parts and is also closely intertwined with asexual reproduction strategies. Regeneration is suspected to play a role in life span extension or even rejuvenation. An answer for reduced or missing regeneration capacity in many species may be found in limitations of the body plan, high costs, and inherent dangers of regeneration. Defects in adults and juveniles are shown, and similarities between development and regeneration are pointed out. With a focus on some worms, but also highlighting comparisons with other animal taxa, putative reasons for a limited and an advanced regeneration capacity are discussed in this article. Birth Defects Research (Part C) 84:257–264, 2008. © 2008 Wiley‐Liss, Inc.     

In vitro culture of Cucumis sativus L.

Summary The ability to regenerate plants from leaf explants has been tested for three highly inbred cucumber lines (B, G, S), their reciprocal hybrids, F₂ and BC₁ generations. The lines differed from each other in their regenerating ability, which was expressed by the percentage of explants regenerating embryoidal callus and mean number of plantlets per plant. Thus, the lines could be classified as frequently (B), intermediately (G) or occasionally regenerating ones (S). There were no reciprocal cross differences in the regeneration. It was found that the intermediately and intensively regenerating lines contain two pairs of dominant genes responsible for plant regeneration, characterized by complementary and probably additive interaction. The frequently regenerating line differed from the intermediately regenerating in the effect of one gene. It is supposed that the above-mentioned genes belong to three different loci. The ability to regenerate plants from leaf expiants had high heritability.     

Trochophora larvae: cell-lineages, ciliary bands, and body regions. 1. Annelida and Mollusca

The trochophora concept and the literature on cleavage patterns and differentiation of ectodermal structures in annelids ("polychaetes") and molluscs are reviewed. The early development shows some variation within both phyla, and the cephalopods have a highly modified development. Nevertheless, there are conspicuous similarities between the early development of the two phyla, related to the highly conserved spiral cleavage pattern. Apical and cerebral ganglia have almost identical origin in the two phyla, and the cell-lineage of the prototroch is identical, except for minor variations between species. The cell-lineage of the metatrochs is almost unknown, but the telotroch of annelids and the "telotroch" of the gastropod Patella originate from the 2d-cell, as does the gastrotroch in the few species which have been studied. The segmented annelid body, i.e. the region behind the peristome, develops through addition of new ectoderm from a ring of 2d-cells just in front of the telotroch. This whole region is thus derived from 2d-cells. Conversely, the mollusc body is covered by descendants of cells from both the C and D quadrants and a growth zone is not apparent. This supports the notion that the molluscs are not segmented like the annelids, and that the repeated structures seen in polyplacophorans and monoplacophorans do not represent a segmentation homologous to that of the annelids.     

Mammalian regeneration and regenerative medicine

Mammals are generally considered to be poor regenerators, yet there are a handful of mammalian models that display a robust ability to regenerate. One such system is the regenerating tips of digits in both humans and mice. In vitro studies of regenerating fetal human and mouse digit tips display both anatomical and molecular similarities, indicating that the mouse digit is a clinically relevant model. At the same time, genetic studies on mouse digit tip regeneration have identified signaling pathways required for the regeneration response that parallel those known to be important for regeneration in lower vertebrates. In addition, recent studies establish that digit tip regeneration involves the formation of a blastema that shares similarities with the amphibian blastema, thus establishing a conceptual bridge between clinical application and basic research in regeneration. In this review we discuss how the study of endogenous regenerating mammalian systems is enhancing our understanding of regenerative mechanisms and helping to shed light on the development of therapeutic strategies in regenerative medicine. Birth Defects Research (Part C) 84:265–280, 2008. © 2008 Wiley‐Liss, Inc.