One of the many fascinating questions about regeneration is why some animals can regenerate and others not. In this sense, several times in this blog I have pointed out the importance of comparative studies between closely related species with different regenerative capabilities. Last week three independent studies from the laboratories of Phil Newmark (http://www.ncbi.nlm.nih.gov/pubmed/23883929), Jochen Rink (http://www.ncbi.nlm.nih.gov/pubmed/23883932) and Kiyokazu Agata (http://www.ncbi.nlm.nih.gov/pubmed/23883928) published in Nature, reported the recovery of head regrowth in regeneration-deficient planarians after silencing a particular signalling pathway.
Freshwater planarians are among the champions of regeneration as they can regenerate a whole animal (including a complete, functional central nervous system) from a tiny piece of their bodies. However, not all planarian species show the same regenerative competence. One striking observation is that several species can regrow a new head when amputated pre-pharyngeally (in planarians, the pharynx is located in the mid-body region), but those same animals cannot regenerate a head when amputated post-pharyngeally. So, what makes that those tail pieces cannot regenerate a new head even if bearing comparable numbers of stem cells as tail pieces from regeneration-competence species?
In order to address this question the laboratory of Phil Newmark used RNAseq to characterize those transcripts that were differentially expressed between pre-pharyngeal regeneration-proficient and post-pharyngeal regeneration-deficient tissues after amputation, in the species Procotyla fluviatilis. When analyzing the data they realized that many of the transcripts over-represented in the regeneration-deficient tissues corresponded to genes of the Wnt signaling pathway. Previous studies have shown that the Wnt/b-catenin pathway plays a pivotal role in specifying anterior versus posterior identity during planarian regeneration. Thus, the silencing of b-catenin transforms any tail blastema into a head fate. Conversely, the knockdown of an inhibitor of b-catenin (such as APC or axins) transforms any head blastema into tails. These results have been interpreted as b-catenin activity being required to specify posterior identity whereas in the absence of b-catenin anterior identity is specified. Because many Wnt ligands were over-expressed in regeneration-deficient tissues the authors hypothesized that an active Wnt/b-catenin pathway could be the responsible of blocking anterior regeneration in those post-pharyngeal tissues. To test it they simply silenced a b-catenin homologue and, amazingly, a new head regenerated from those regeneration-deficient tail pieces. These results indicate that those regeneration-deficient tissues are competent to express and unfold a head regeneration program but fail at the initial stage of re-establishment of axial polarity. This early step would be necessary to proceed with the subsequent regenerative events (blastema growth and differentiation into a new head). Conversely, an over-activation of the Wnt/b-catenin pathway (through the silencing of the pathway inhibitor APC), lead to the growth of ectopic tails from those regenerating tail pieces.
In the study by Jochen Rink’s lab they worked with the species Dendrocoelum lacteum. Similarly to what it is described for P. fluviatilis, after amputation at the post-pharyngeal level, the resulting tail pieces normally heal the wound and stem cells within it respond to amputation by increasing their proliferation. So, a blastema is initially formed but never grows and differentiates into a new head. In the case of D. lacteum the authors deep sequenced the transcriptomes at 8 eight time points of regeneration (0, 4, 12, 16, 24, 48, 72 and 120 hours) from pre-pharyngeal regeneration-competent tissues and post-pharyngeal regeneration-deficient tissues. In both cases, amputation lead to the normal upregulation of a collection of previously reported wound-response genes as well as a similar stem cell activation response. However, the authors saw how tail pieces failed to activate a set of head-specific genes while at the same time maintained the expression of tail markers. This was in sharp contrast to what happens in pre-pharyngeal regenerating tissues in which head markers were activated and the expression of tail markers was downregulated. Because what it is known about the Wnt/b-catenin pathway the authors speculated that the differences between pre-pharyngeal and post-pharyngeal regenerating pieces could be due either from inappropriate high levels of Wnt signaling and/or from insufficient Wnt inhibitory capacity. Therefore, they silenced b-catenin in tail pieces and, as a result, these were able then to regenerate a new head. Conversely, the silencing of APC gave rise to the regeneration of ectopic tails.
Finally, the work by Kiyokazu Agata and Yoshihiko Umesono followed a complete different strategy in the planarian Phagocata kawakatsui but also showed how the silencing of b-catenin rescues head regeneration from regeneration-deficient tail pieces.
In summary, the three studies show how just by simply modulating the Wnt/b-catenin signalling pathway you can transform a regeneration-deficient tissue into a regeneration-competent tissue capable then of re-growing a new head with a fully functional and complex brain. Further studies should determine why in these species the post-pharyngeal pieces, upon amputation, fail to modulate the Wnt/b-catenin pathway to re-specify proper polarity and subsequently trigger the head-regeneration program. These terribly exciting results may help us to understand the mechanisms responsible for the loss of regeneration capacities through evolution as well as provide insights into how to promote regrowth in regeneration-deficient models.