Freshwater planarians are truly amazing animals as they can regenerate the whole body from a tiny piece of them based on the presence of a unique population of adult pluripotent somatic stem cells. These cells, named neoblasts, are the only proliferative cells and the source of all regenerative cells. In recent years, several genes have been reported to be necessary for proper neoblast function and properties such as proliferation and self-renewal. However, we are still far to fully understand how gene expression is regulated in these stem cells. Now a recent paper from the laboratories of Aziz Aboobaker and Nikolaus Rajewsky reports on a post-transcriptional regulatory mechanism that would control neoblast differentiation during planarian regeneration and homeostasis (http://www.ncbi.nlm.nih.gov/pubmed/24367277).
mRNA degradation is an important process to control gene expression at a post-transcriptional level. A first step during mRNA degradation is deadenylation (shortening of the poly(A) tail) mediated by the CCR4-NOT complex. Little is know, however, about the function of this complex and deadenylation itself in stem cell regulation. Therefore, the authors analysed the function of Smed-not1, a planarian homologue of Not1, a central protein of the CCR4-NOT complex. Smed-not1 is expressed in neoblasts, their postmitotic progeny as well as in differentiated cells of the central nervous system (CNS). Upon the silencing of Smed-not1 the animals were able to start the regenerative process and made small blastemas; however, after few days these blastemas stopped growing, did not differentiate, regressed and finally all the planarians died. A similar observation was found in intact non-regenerating animals: Smed-not1 RNAi lead to head regression starting after 15-20 days and, eventually, all treated planarians died. Because this phenotype of head regression and lethality had been previously associated to depletion of the neoblast population the authors checked first cell proliferation after silencing Smed-not1 in intact animals. Remarkably, cell proliferation was not significantly affected in these animals, suggesting that the primary function of Smed-not1 could be related to neoblast differentiation more than to neoblast maintenance and proliferation.
Next, the authors analysed in more detail the neoblasts and their progeny after silencing Smed-not1. Normal numbers of Smedwi-1 positive cells (neoblasts) were found up to 15 days after RNAi, whereas a significant decrease was apparent by day 20. Smed-nb21.11e cells (early progeny) were normally present by day 10 but started to decline in number after day 15. Finally, Smed-agat-1 cells (late progeny) increased by day 10 and from that point started to decline in number. By day 20 a significant reduction in the presence of these cells was observed. Overall, these results somehow support the idea that the silencing of Smed-not1 primarily affected cell differentiation rather than neoblast maintenance, as changes in the proper number of neoblast progeny and a visible head regression preceded the decrease of neoblasts. An interesting and puzzling result was obtained when the authors quantified the levels of the transcripts of these genes after Smed-not1 RNAi. Thus, for Smed-nb21.11e the levels of mRNA were the same as controls after 15 days, despite the reduced number of positive cells observed by in situ hybridizations. On the other hand, the levels of Smed-agat-1 mRNA in Smed-not-1(RNAi) animals were almost two-fold higher than in controls after 15 days, in huge contrast with the observation that these animals had similar numbers of Smed-agat-1 cells than controls. The authors concluded that Smed-agat-1 transcripts were accumulated in a decreasing number of Smed-agat-1 cells.
As the CCR4-NOT complex regulates deadenylation previous to mRNA degradation one possibility could be that the increase in the levels of mRNA observed after Smed-not1 RNAi could be due to an impaired deadenylation of such transcripts. If this is true then, an increased frequency of poly(A) tail lengths should be observed. And this is exactly what the authors found for Smed-agat-1 and Smed-nb21-11e after Smed-not1 RNAi. Then, they performed similar experiments for the neoblast markers Smedwi-1, Smedtud-1, Smed-vasa-1 and Smed-pcna and in all cases they found an increased in the average length of their poly(A) tails together with increased transcript levels. Importantly, these changes were not observed for genes expressed specifically in differentiated cells.
As Smedtud-1 and Smed-vasa-1 are expressed also in the CNS the authors wondered whether the increased in their mRNAs levels after Smed-not1 was found in all the cells expressing these genes or only in the neoblasts or the CNS. To solve this question they combined RNAi with irradiation, as it is well known that 24 hours after a certain dose of irradiation all neoblasts are effectively eliminated. The rationale here was that if Smed-not1 action on deadenylation was limited to the transcripts present in the neoblasts, then after irradiation no differences should be observed between control and Smed-not1 RNAi animals in terms of both transcript levels and poly(A) length. And this is precisely what they observed, confirming that the effects of Smed-not1 on the mRNAs of these genes were confined to neoblasts.
In summary, the results presented here offer novel insights into the post-transcriptional regulation of neoblasts. As the authors propose, after Smed-not1 RNAi the transcripts of several genes important for neoblast function would accumulate without being degraded and subsequently preventing somehow neoblast differentiation. Further studies will help to better characterize the function of the CCR4-NOT complex in regulating stem cells not only in planarian but also in other animal models.