regeneration in nature

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How uniform or heterogenous is the planarian blastema?

Planarian regeneration depends on a unique population of pluripotent stem cells called neoblasts. When a planarian is amputated neoblasts close to the wound respond by actively proliferating and migrating towards the wound forming the regenerative blastema in which most of the missing structures will differentiate. Although the pluripotency of the neoblasts as a whole population has been classically suggested and believed, it was in 2011 that the laboratory of Peter Reddien provided strong experimental evidence that at least a part of the neoblast population includes truly pluripotent stem cells. Thus, a single of these pluripotent neoblasts can rescue planarians lethally-irradiated (http://www.ncbi.nlm.nih.gov/pubmed/21566185).

The regenerative blastema has been classically seen as a rather uniform mass of undifferentiated cells, probably multipotent. However, recent data suggest that this could be not entirely true. In a recent hypothesis paper, Reddien discusses this possibility as well as at what stage of regeneration neoblasts and/or neoblast progeny are specified to differentiate into the required cell types (http://www.ncbi.nlm.nih.gov/pubmed/23404104). The author proposes two alternative (although not necessarily mutually exclusive) models: in the “naïve neoblast model” pluripotent neoblasts divide and migrate into the blastema where they would stop dividing and then be specified and differentiated into the proper cell types; alternatively, in the “specialized neoblast model”, there would be specialized neoblasts  that would produce different lineage-committed non-dividing blastema cells. In this last case, therefore, the specification of the blastema cells towards a particular cell fate would occur somehow prior they enter the blastema. The “specialized neoblast model” is supported by the fact that at least for three different cell types: eye-pigment, photoreceptors and protonephridia, specific cell populations of progenitor-like neoblasts express specific transcription factors that specify those neoblasts towards those cell fates (http://www.ncbi.nlm.nih.gov/pubmed/21852957, http://www.ncbi.nlm.nih.gov/pubmed/21937596). Still, they are “neoblasts” according to the definition of neoblast used here: “cells expressing smedwi1 and irradiation sensitive (during the first 24h after irradiation)”.

There is no doubt that those recent findings of progenitor cells for some planarian cell types have re-opened an exciting debate on how heterogeneous are neoblasts in terms of their potentiality. As stated in Reddien’s paper many questions need to be solved, starting with a definition of a neoblast. Also, it would be important to determine up to what extend these specialized neoblasts are not only present in uninjured animals but divide and are maintained in them for regular cell turnover. If specialized neoblasts exist at low numbers in uninjured animals what happens after amputation: do naïve multipotent neoblasts make more specialized neoblasts? Do the few specialized neoblasts divide and make more of them? Or both?

Finally, as both models make an important distinction on whether blastema cells are specified within (naïve model) or outside (specialized model) the blastema, it would be then appropriate to look for a good definition of a blastema. Very often a planarian blastema is defined as the mass of unpigmented tissue that grows below the wound, but body pigmented cells differentiate relatively late so after 5 days of regeneration you have a rather unpigmented mass of cells but in fact inside a new brain, for example, has already differentiated. So, up to what stage during regeneration a blastema is a blastema? And also, how early can we talk about a blastema? Many of the important decisions such as, for example, the establishment of proper polarity are made very early after amputation, and before any sign of new tissue really appearing below the wound. And that polarity decisions are going to affect the cell types you will need; it is not the same regenerating a head with its brain than a tail. So, during regeneration, the signals that generate specialized neoblasts from naïve pluripotent neoblasts where do they come from? From pre-existing cells in the stump? When the blastema is formed, does it have any role in cell specification (in the “specialized neoblast model”)? or does it play a mere organizational role? Anyway the definition of “blastema” would probably deserve another post by itself.

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2 Comments

  1. Jaume Baguñà says:

    The hypothesis paper by Peter W Reddien, timely and well produced, asks whether blastema neoblasts is a uniform population (likely multipotent) of naïve cells or whether it is a heterogeneous one made by specialized neoblasts belonging to different cell lineages. His bets are for the second.

    So is my bet. However, let me introduce three comments. First, it is fair to state that in the past most researchers did not usually think of neoblasts as a uniform population of cells, neither in the intact worm nor within the regenerating blastema. Reasons were twofold: 1) ‘morphological’ neoblasts make from 20 to 35% of total cells according to body length/volume. It is extremely difficult then to consider all of them as “naïve” neoblasts; that is, multipotent, ready to give any cell type on demand; and 2) 50 to 60% of neoblasts never divide, likely being fully determined precursors of specific cell types. This is borne out from experiments using low concentrations of colchicine to stop cell division. Cells steadily accumulate at the metaphase stage up to 4 days of incubation toppleing up to 40-45% of total neoblasts. As for the rest they should be, very likely, fully commited non dividing neoblasts.

    A second argument against the ‘naïve’ neoblast model are the beautiful and under-quoted experiments by Gremigni and co-workers made in the late 70s-early 80s. Briefly, they showed that during regeneration some blastema cells already ‘determined’ either to male (as seen by being diploid) and female (as seen by being hexaploid) germ line could go back and change their lineage to any sort of somatic cell lineage. This meant that prior to cutting at least some neoblasts were at the early stages of determination (oogonia and spermatogonia) to the germ line. Common sense, however unproved, suggests that intact worms should have scores and scores of neoblasts at different stages towards specific cell lineages.

    My final comment hinges on a cell number exercise dealing with the likely number of clonogenic neoblasts (or cNeoblasts according to Wagner et al, 2011). A 10mm long planarian is made by 1 million cells and, accordingly, it bears from 200.000 to 250.000 neoblasts. A square mm of a 10mm long planarian body has close to 30.000 total cells (close to the minimum fragment able to regenerate, bearing 10.000 cells according to Montgomery and Coward, 1974, though never substantiated. By the way, anyone knows how they counted the number of cells?). This tiny fragment should have from 6 to 8.000 neoblasts. If we assume that 1% of total neoblasts are cNeoblasts, then 60 to 80 of them are cNeoblasts, enough to repopulate and make to regenerate the tiny fragment. And even assuming they represent a mere 0,1% of total neoblasts (close to the estimated percentage of mouse haemopoietic stem cells) that will make from 6 to 8 cNeoblasts; again, able to repopulate, according to the injection experiment of a single cNeoblast, reported in Wagner et al (2011), this tiny fragment and make it to regenerate. In summary, cNeoblasts could easily represent a mere 0,1 to 1% of total neoblasts. Being so low, the inescapable conclusion is that the rest should be neoblasts at different stages of determination/early differentiation, still able to proliferate.

    All in all, this points to the need to shift our ‘regeneration-centric’ view of planarian biology. Most planarians NEVER regenerate, either because they can’t or because even if they can they never use it (being sexual and avoiding predation). BUT, ALL PLANARIANS, able to regenerate or not, are continuously replacing dying cells (mostly differentiated cells) by new cells differentiating from neoblasts. If regeneration is called upon (predation, asexual reproduction, scalpels,…), lots of determined neoblasts are already there ready to give, if needed, different cell types. Moreover, cNeoblasts and neoblasts at the very early stages of determination, and likely able to go back to multipotent neoblasts, could start proliferation and differentiation to the new cell types needed according to axial position. We are in need of more basic planarian biology!!!

    Besides the main questions posed, Reddien paper is important because it points at the need to develop additional cell-marking tools (transgenics, mAbs, specific single cell labelling, etc,..) to characterize described and additional lineages. This would benefit the analysis of daily cell renewal, growth and degrowth, regeneration, ageing, and the why and how of different reproduction strategies.

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    • I really appreciate this comment by Jaume Baguñà because points out several previous experiments and pieces of data that appear to argue against the possibility that the neoblasts, as a whole population, could be considered as “naïve neoblasts”. In this sense I think that it is convenient to put together those comments with the most current data mainly derived from the laboratory of Peter Reddien (and discussed in the original post), in which distinct populations of specialized neoblasts (progenitors) have been characterized through their labeling with specific transcription factors. So, in the emerging view of neoblasts as being form by a small percentage of truly “naïve” multipotent cells and a major population on “progenitor” cells it will become necessary to identify additional markers for the distinct cell lineages.

      Another aspect discussed in the hypothesis paper by Peter Reddien was about the neoblasts that form the blastema. Here, again, in addition of the distinction between “naïve” and “specialized” neoblasts it will be important to agree with a consensus definition of blastema, as i pointed out in my original post. One piece of data to take in account is that neoblasts have been mainly defined based upon the expression of the gene smedwi1. But it has been also described that during regeneration and from day 2 most of the “neoblasts” that accumulate below the wound forming the blastema stop expressing smedwi1 although they keep being positive for the protein SMEDWI1 as they exit the cell cycle and start differentiating. Thus, based on those definitions, the “blastema” would be then formed by “specialized” non-dividing progenitors. What remains to be determined, among others, is WHEN and WHERE “naïve” neoblasts become “specialized” progenitors during the regeneration of the several cell types.

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Francesc Cebrià

Francesc Cebrià

Francesc Cebrià

I am a Biologist and Professor at the University of Barcelona. I do my research on a fascinating animal: freshwater planarians. You can cut them in as many pieces as you want and each piece will regenerate a complete new flatworm in very few days. In this blog I will keep you updated on the latest news on the field of animal regeneration. You will be able to follow the latest research on how planarians, axolotls, newts, cnidarians and other animals are able to regenerate parts of their bodies

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