regeneration in nature

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Arm regeneration in cuttlefish

Many animals are able to regenerate different types of appendages (understood as body wall outgrowths). Among vertebrates, limbs and tails from salamanders and lizards are well-known examples. On the other side, some invertebrates also regenerate appendages such us parapodia, tentacles, opercula, palps and gills. Molluscs comprise a very diverse phylum with an extreme morphological diversity. Up to date, no known representative has been shown to be able to regenerate the whole body. However, several mollusc species can regenerate a variety of structures such as: foot, tentacles, siphon, shell and mantle, and even the head. Cephalopod molluscs are well known for their capacity to regenerate their arms. However, very little quantitative data is available about this process and even less about the cellular and molecular mechanisms involved. In a paper from the laboratory of Jedediah Tressler Nathan J. Tublitz the authors provide a detailed description and quantification of the regeneration process in two species of cuttlefish (http://www.ncbi.nlm.nih.gov/pubmed/23982859). Studying arm regeneration in cephalopods is also important as these structures are as complex as many vertebrates appendages.

It is also obvious that an initial detailed description and quantitative data on the regeneration process in terms of growth rate, behavioural changes, timing of regeneration and functional recovery is necessary prior to tackle the cellular and molecular mechanisms that regulate it, especially in those non-model animals.

First, the authors present the data on Sepia officinalis in which the right 3rd arm was amputated from nine juveniles.   In all cases, the amputated arms were regenerated by 39 days, after which the newly formed arms were indistinguishable from the contralateral control arms. They divided the regeneration process in 5 stages: at stage I (days 0-3) the leading edge of the arm appeared smooth with little bleeding. Two days after the amputation the regenerating arm appeared frayed and covered with a mucus-like substance. At this stage only a few new suction cups and no new chromatophores were observed. As a consequence of the amputation the behaviour of the animals was significantly altered as they showed: unbalanced swimming, impaired prey manipulation for ingestion, altered normal body posturing behaviours and lack of any colour change behaviour. Stage II (days 4-15) was characterized by the smooth, slightly hemispherical appearance displayed by the leading edge of the regenerating arm. Growth across the entire width of the arm was symmetrical. New suction cups and chromatophores were seen at this stage. Also, a normal balanced swimming was seen and from day 9 normal food manipulation reappeared. Stage III (days 16-20) began with the appearance of a growth bud on the lateral side of the leading edge, resulting in an asymmetric shape of the regenerating arm. The new arm kept growing at a higher rate that the contralateral control and new suction cups and chromatophores were added at the distal tip. By the end of this stage the regenerating arm was also used for normal body postures. Stage IV (days 21-24) was defined by the emergence of an elongated tip from the growth bud. Suction cup regeneration appeared to be completed by the end of this stage. In the final stage V (days 25-39) the elongated tip took on a tapered appearance. New chromatophores were added until their density in the tip resembled that from control arms. Here, all the checked behaviours, including the brown tip behaviour, were recovered.

In the other species, Sepia pharaonis, arms were also regenerated in 39 days and followed the same 5 stages as in S. officinalis. Based on the location of the new suction cups, chromatophores and the presence of the growth bud and elongated tip, it seemed that new tissue was added directly to the tip of the regenerating arm. Whereas the growth rate of the regenerating arm was quite constant throughout regeneration in S. pharaonis, it varied depending on the stage in S. officinalis.

In summary, in this paper the authors report on the fine description of arm regeneration in two species of cuttlefish at the morphological and behavioural recovery levels. Next step should be to get insights into the cellular and molecular processes governing this regenerative process.

Evolution of regeneration in spiralians

One of the on-going debates in the regeneration field concerns how the regenerative capabilities shown by different animal groups have evolved. When considering the animal phylogeny we can see how most phyla contain species capable of regenerating. However, a huge variability exists in: 1) the regeneration power shown by closely related species and 2) the biological level of regeneration as depending on the model they can regenerate only specific cell types, or some tissues and/or organs, or structures and complex parts (for example, a limb) or, finally, the real champions capable of regenerating the whole body.

A recent paper by Alexandra Bely and colleagues discusses about the evolution of regeneration, especially within the spiralians (http://www.ijdb.ehu.es/web/paper/140142ab/regeneration-in-spiralians-evolutionary-patterns-and-developmental-processes). As the authors raise, an important question is whether regeneration variation among bilaterians is the results of regeneration losses, independent gains or a combination of both. That is, was regeneration a feature that was already present in the last common ancestor of all bilaterians and has been lost in some taxonomic groups? or, alternatively, is something that has independently appeared in some groups and not others?

In order to address these questions it is absolutely necessary to gather as much information as possible about the regeneration capabilities of as many animal groups as possible and, more importantly, characterize the cellular and molecular processes that guide regeneration in those animals. Also, it is important to understand why very closely related species can differ significantly in their regenerative capabilities. In this review, the authors focussed on the Spiralia, a large and diverse protostome clade composed of 13 phyla including annelids, molluscs, nemerteans, platyhelminthes and rotifers. Importantly, there is an important variability of regeneration not only between different spiralian phyla but also within them.

Thus, annelids include species that can regenerate every part of the body, including some that can regenerate a whole animal from a single body segment, as well as species totally incapable of regenerating a single segment lost. In general, the capacity to regenerate posterior segments is very broadly distributed within the phylum. In contrast, the ability to regenerate anterior segments is much more variable and, in fact, the failure to regenerate anterior segments has been shown in over a third of the families from which data are available. Nemerteans can undergo growth and degrowth indicating processes of remodelling. Some animals can be maintained starved for over a year shrinking in size but otherwise apparently happy. Among them, regeneration of the proboscis (used for prey capture), tail and head occurs in some groups, and some species can regenerate the whole animal from a tiny body piece. Posterior regeneration is not in general very well documented because the lack of easily scorable structures. Anterior regeneration appears to be very limited within this group although some species of one particular family can regenerate a complete head. However, this seems to be an exception within this phylum, which could imply that it might be a regeneration gain of this particular family.

Among Platyhelminthes, the triclads (planarians) are the best known in terms of their regenerative capabilities. However, it is also true that a number of groups of this phylum have much more limited regenerative capacities. Also, within this phylum posterior regeneration appears to be more widespread than anterior regeneration. Finally, within molluscs we do not have any representative capable of regenerating the whole body. However, different species can regenerate specific structures such as the foot, anterior neural elements, tentacles and even the entire head in some gastropods.

Next, the authors review what is known about the cellular and molecular basis of regeneration within these different phyla. Thus, in annelids after amputation there is a rapid muscle contraction to seal the wound. During the very first stages of wound healing and regeneration, proliferation throughout most of the body seems to be shut down. At the same time there is a large cell migration response towards the wound. After wound healing, cells near the wound start proliferating forming a regenerative blastema. The origin of the regenerative cells within the blastema seems to come from the proliferation of the three tissue layers close to the wound. The role of annelid neoblasts (undifferentiated cells) in regeneration is still under debate. Also, several genes have been shown to be expressed within the blastema including markers of stem cells and germline as well as Hox genes. Interestingly, all these genes expressed within the blastema are also detected during normal growth in the posterior growth zone. This suggests a shared molecular mechanism between regeneration and growth. Finally, regeneration does not uniquely imply the formation of new tissues and structures but also a remodelling of the pre-existing tissues.

In nemerteans, amputation is followed by muscle contraction and wound healing followed by a phase of cell proliferation and the formation of a regenerative blastema, much more evident during anterior regeneration than in posterior regenerates. Unfortunately, the origin of the regenerative cells of the blastema is obscure, and although some old studies pointed to the role of some putative undifferentiated and totipotent cells scattered in the extracellular matrix, more recent studies does not seem to support the existence of such undifferentiated cells. Also, very little is known about the genetic program triggered during regeneration within the blastema cells, except some studies reporting the expression of pax6 and otx in the regenerating central nervous system. Within platyhelminthes, most of the cellular and molecular data of the regenerative process comes from planarians, macrostomids are providing also some interesting data. In planarians, wound healing is followed by the local proliferation of totipotent stem cells (known as neoblasts) closed to the wound that originate a regenerative blastema in which the new structures differentiate. Remodelling of the pre-existing tissues is also necessary to achieve normal body proportions of the regenerated animal. Recently, many papers have reported on different genes and signalling pathways that regulate proper regeneration in planarians. However, much more data should be provided from those taxonomic groups that have either poor regenerative capabilities or for which the cellular and molecular basis of their regenerative capacities are currently unknown. Finally, very little is known about the cellular and molecular processes involved in regeneration in molluscs. A recent report on octopus arm regeneration suggests that a mass of mesenchymal undifferentiated cells would accumulate below the wound forming a highly proliferative blastema.

From all these data and comparative analysis in these spiralian phyla the authors draw four main conclusions: 1) the ability to regenerate the whole body seems to be present in only a subset of representatives of each of these groups. From a phylogenetic perspective numerous increases and/or decreases in regeneration ability have occurred across these phyla. This raises that the possibility that regeneration may not be homologous across them needs to be considered; 2) posterior regeneration appears to be more widespread than anterior regeneration; 3) all phyla include a blastema stage, although the origin of the regenerative cells that form it may be different, and 4) in all these phyla the capacity for continuous growth and degrowth is well documented, suggesting a mechanistic relationship or common set of elements and features between these processes and regeneration.

In summary, how the capacity of regeneration has evolved is also a fascinating field of study that requires much more sampling and data collection for the required comparative analyses.

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