regeneration in nature

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Monthly Archives: July 2014

Summer break

Just a short notice to inform you that I am going to take some weeks off for summer break. I will back in September to continue posting about regeneration. The beginning of September will be also an exciting time as there will be the EMBO Conference on Regeneration in Sant Feliu de Guixols (near Barcelona), so I hope we can learn a lot from the data presented there.

Have a nice summer!

Neoblast heterogeneity and planarian regeneration

Compared to other regeneration models, freshwater planarians display a distinctive feature: they regenerate from a unique population of adult pluripotent stem cells, called neoblasts. This makes planarian an excellent model in which to study the behaviour of stem cells in vivo. Although classically seen as a rather homogeneous cell population, several recent studies have suggested that neoblasts could, in fact, constitute a wide heterogeneous population, based on different parameters and features, in which, for instance, several progenitor-like cells for different cell types exist. Now, a beautiful paper from the laboratory of Peter Reddien goes much deeper into their analysis of planarian stem cells and identifies two major and functionally distinct cellular compartments among neoblasts (http://www.ncbi.nlm.nih.gov/pubmed/25017721).

In this study the authors characterized the neoblasts at a single-cell resolution level. Neoblasts are the only proliferative cells in planarians. Fluorescence-activated cell sorting (FACS) can be used to isolate proliferating neoblasts based on their DNA content. The authors then analysed the expression of 96 genes within each of hundreds of individual neoblasts. These genes were selected from a neoblast transcriptome and included, among others, well-known neoblasts and post-mitotic markers, as well as a variety of transcription factors and regulators highly abundant in neoblasts. Hierarchical clustering allowed distinguishing two main classes of equally sized populations of neoblasts: the zeta-class and the sigma-class. The zeta-class neoblasts were characterized by a high expression of zfp-1, g6pd, fgfr-1, p53, soxP-3 and egr-1. On the other hand, the sigma-class neoblast showed low expression levels of the previous genes and high expression of a distinct set of genes: soxP-1, soxP-2, soxB-1, smad6/7, inx-13, pbx-1, fgfr-4 and nlk-1.

In order to discard that these two neoblast populations could be in fact a single population but at different state of the cell cycle the authors isolated by FACS different neoblast populations at different stages of the cell cycle according to their DNA content. Single-cell profiling showed that both classes, zeta- and sigma-neoblasts, were equally present throughout the cell cycle. Also, zeta- and sigma-neoblasts showed a similar broad spatial distribution along the planarian body. Next, the authors checked how these two populations responded to either amputation (anterior or posterior regeneration) or sublethal irradiation. In all cases, after two days of amputation or irradiation the relative abundances of the zeta- and sigma-classes were the same as in control, untreated animals.

Upon amputation, neoblasts display a bimodal proliferating response with a first mitotic peak at 6 hours (all throughout the regenerating fragment) and a second peak at 48 hours (localized at the wound region). Remarkably, the sigma-neoblasts were overrepresented among the mitotic population both at 6 and 48 hours, indicating that these two mitotic peaks derived mainly from the activity of the sigma-neoblasts. Moreover, analyses on the spatial distribution of these neoblasts indicated that the accumulation of neoblasts at the wound region at 48 hours of regeneration depended mainly on the sigma-class neoblasts.

Next, the authors focussed on the functional analysis of zfp-1, a gene specific to the zeta-class. Upon its silencing by RNAi, the animals died in few weeks. Zfp-1 RNAi resulted in the loss of expression of other zeta-class markers without affecting the expression of sigma-class genes. In fact, the silencing of zfp-1 eliminated the zeta-class neoblasts without affecting the sigma-class cellular compartment. In terms of function, the depletion of the zeta-class neoblasts did not interfere with the normal behaviour of the sigma-class as these cells could mount a proper regenerative response generating two mitotic peaks at 6 and 48 hours as in controls. As the animals in which zfp-1 was silenced were still capable of regenerating a blastema the authors checked whether specific cell lineages were affected in those animals. Remarkably, they found that brain, gut, muscle, protonephridia, eyes and pharynx tissues were apparently normal, indicating that the sigma-class neoblasts were capable of differentiating into a broad range of cell types. However, other cells characterized by the expression of previously identified as neoblast early and late progeny markers were depleted. These genes included prog-1 (early progeny) and AGAT-1 (late progeny). These genes label subepidermal cells that undergo rapid cell turnover. However, it was not clear whether those cells defined a particular cell lineage. Here, by doing RNAseq, the authors found out that several transcripts associated with epidermis, cilia and secretory cells were reduced after the silencing of zfp-1. Specifically, they identified nine genes expressed subepidermally (as prog-1) and seven genes expressed at the epidermis, and whose expression was clearly affected after zfp-1 RNAi. Consequently, the epidermal cells were disorganized, thinner and less abundant in those animals. Moreover, after two weeks of BrdU incorporation much fewer epidermal cells were labelled with BrdU, suggesting that zeta-class neoblasts gives rise, at least in part, to an epidermal cell lineage, probably through an intermediate stage defined by the expression of prog-1 and AGAT-1.

Finally, and through some elegant experiments of transplantation of sigma-class neoblasts into previously irradiated (and therefore depleted of any neoblast) planarians, the authors showed how the sigma-class neoblast were capable of generating the zeta-class compartment. Then, and by checking the gene expression profile of individual cells at different stages of the cell cycle, the authors suggest that zeta-class neoblast would derive from sigma-class cells just following their entry into S phase.

In summary, this study identifies two clearly distinct classes of neoblasts based on their gene expression profiles, response to wounding and cell differentiation potential. The data presented here also indicates that these two classes can be also rather heterogeneous themselves. Thus, for instance, within the sigma-class the authors suggest the existence of a subclass, the gamma-neoblast, characterized by the expression of some genes that had been previously related to the planarian gut, indicating that those gamma-class cells could be related to the gut lineage.

JNK balances cell death and proliferation during planarian regeneration and remodeling

In any developmental system cell proliferation and cell death need to be tightly regulated to ensure proper growth, morphogenesis and patterning. During regeneration these cellular processes must be also coordinated in order to achieve a well-proportioned animal de novo upon regeneration completion. A recent paper from the laboratory of Emili Saló and Teresa Adell (http://www.ncbi.nlm.nih.gov/pubmed/24922054) reports on the key function of the JNK pathway in regulating these events in regenerating and degrowing planarians. JNK is a stress-activated protein kinase belonging to the MAPK family that, in other systems, has been implicated in the regulation of cell cycle, wound healing, neurodegenerative disorders and cancer.

Planarian JNK is expressed in the central nervous system as well as in the neoblasts (planarian adult pluripotent stem cells). Upon silencing of this gene by RNAi, regeneration was severely inhibited as the treated animals regenerated very small blastemas with aberrant differentiation of the new structures within them. JNK RNAi did not affect the early expression of the polarity determinants notum and wnt1, at those stages in which polarity is re-established. However, the latter expression of these genes was significantly attenuated indicating that JNK is somehow required for the maintenance of the expression of such polarity genes. Whereas in other systems such as Drosophila the JNK pathway is required for wound closure, this was not affected after JNK silencing in planarians. However, JNK RNAi resulted in the failure to activate the expression of several wound-induced genes.

Upon amputation neoblast proliferation dynamics displays a bimodal response. There is a first proliferative peak at 6 h after amputation that is systemic throughout all the regenerating pieces, and that it has been associated to wounding. A second mitotic peak is seen at 48 h of regeneration concentrated around the wound region; this second peak is associated to tissue loss and the regenerative response that leads to blastema formation. JNK RNAi does not affect the number of neoblasts or the proportion of actively cycling cells. However, the authors observed that the first mitotic peak was elevated and the second peak occurred about 10 h earlier than in controls. CldU labeling combined with piwi1 (a neoblast-specific marker) and phosphohistone H3 (a marker of the entering to the M phase of the cell cycle) suggests that JNK RNAi induces the shortening of the G2 phase of the cell cycle and, therefore, neoblast enter faster into mitosis. This shortening of the G2 did not affect the capacity of those neoblasts to give rise to normal numbers of post-mitotic progeny.

In addition to neoblast proliferation and blastema formation, the pre-existing tissues must go through a remodeling process so the regenerated animal achieves proper body proportions. This remodeling is largely dependent on cell death. After amputation apoptotic cell death, in planarians, follows also a bimodal response with a first apoptotic peak at 4 h post amputation concentrated at the wound region, and a second apoptotic peak at 3 days of regeneration systemically found throughout the entire regenerating fragment. This second apoptotic peak has been associated to the remodeling of the pre-existing tissues. JNK RNAi inhibited these two apoptotic peaks. Remarkably, the inhibition of apoptosis was accompanied by an increased proliferation in those pre-existing tissues that need to go through remodeling. As a consequence, these treated animals were incapable of readjusting the position of pre-existing organs such as the pharynx to restore proper body proportions. Overall, these results indicate that JNK is necessary to trigger a proper apoptotic response.

During planarian regeneration the second mitotic and apoptotic peaks are related to tissue loss whereas the first peaks are related to a general systemic response to wounding. Thus, after a wound that does not imply tissue loss only the first mitotic and apoptotic peaks are observed. Interestingly, JNK RNAi did not affect neither the wound-associated proliferative and apoptotic responses or the normal expression of wound-induced genes. In contrast, the proliferative and apoptotic response after small injuries that imply loss of small amounts of tissues depended on the function of JNK, as it happens upon amputation of large regions. Therefore, JNK is required for regeneration in those contexts in which tissue has been lost.

In addition to their amazing regenerative capabilities, planarians are very plastic animals as they constantly grow and degrow depending on food availability. Planarian growth and degrowth depend upon the balance of cell proliferation and cell death. Again these two cellular processes must be tightly regulated as animals keep proper body proportions at any time. In starved animals that consequently will degrow, the silencing of JNK inhibits apoptosis without affecting the proliferation rates. This inhibition of apoptosis is accompanied by the impairment of proper body re-scaling during degrowth. Remarkably, JNK RNAi did not affect the apoptotic response in growing animals and they underwent through proper body re-scaling during their growth. Therefore, the authors conclude that JNK is required for the apoptotic-driven remodeling that takes place in degrowing animals to maintain proper body proportions.

In summary, JNK is required to trigger a proper regenerative response after any wound that results in tissue loss. There, JNK is required to induce apoptosis, regulate the onset of mitosis in neoblasts and trigger the expression of wound-induced genes. Moreover, in intact starved animals that are degrowing JNK is necessary to induce the apoptotic driven tissue remodeling and rescaling of proper body proportions.

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