The planarian Dugesia japonica can regenerate a complete individual from a head, trunk or tail fragment via activation of somatic pluripotent stem cells. About a century ago, Thomas Hunt Morgan attempted to explain the extraordinary regenerative ability of planarians by positing two opposing morphogenetic gradients of formative "head stuff" and "tail stuff" along the anterior-posterior axis. However, Morgan's hypothesis remains open to debate. Here we show that extracellular signal-related kinase (ERK) and Wnt/β-catenin signalling pathways establish a solid framework for planarian regeneration. Our data suggest that ERK signalling forms a spatial gradient in the anterior region during regeneration. The fibroblast growth factor receptor-like gene nou-darake (which serves as an output of ERK signalling in the differentiating head) and posteriorly biased β-catenin activity negatively regulate ERK signalling along the anterior-posterior axis in distinct manners, and thereby posteriorize regenerating tissues outside the head region to reconstruct a complete head-to-tail axis. On the basis of this knowledge about D. japonica, we proposed that β-catenin signalling is responsible for the lack of head-regenerative ability of tail fragments in the planarian Phagocata kawakatsui, and our confirmation thereof supports the notion that posterior β-catenin signalling negatively modulates the ERK signalling involved in anteriorization across planarian species. These findings suggest that ERK signalling has a pivotal role in triggering globally dynamic differentiation of stem cells in a head-to-tail sequence through a default program that promotes head tissue specification in the absence of posteriorizing signals. Thus, we have confirmed the broad outline of Morgan's hypothesis, and refined it on the basis of our proposed default property of planarian stem cells.

SUMMARYThe robust regenerative ability of planarians depends on a population of somatic stem cells called neoblasts, which are the only mitotic cells in adults and are responsible for blastema formation after amputation. The molecular mechanism underlying neoblast differentiation associated with blastema formation remains unknown. Here, using the planarian Dugesia japonica we found that DjmkpA, a planarian mitogen-activated protein kinase (MAPK) phosphatase-related gene, was specifically expressed in blastema cells in response to increased extracellular signal-related kinase (ERK) activity. Pharmacological and genetic [RNA interference (RNAi)] approaches provided evidence that ERK activity was required for blastema cells to exit the proliferative state and undergo differentiation. By contrast, DjmkpA RNAi induced an increased level of ERK activity and rescued the differentiation defect of blastema cells caused by pharmacological reduction of ERK activity. These observations suggest that ERK signaling plays an instructive role in the cell fate decisions of blastema cells regarding whether to differentiate or not, by inducing DjmkpA as a negative regulator of ERK signaling during planarian regeneration.

The robust regenerative abilities of planarians absolutely depend on a unique population of pluripotent stem cells called neoblasts, which are the only mitotic somatic cells in adult planarians and are responsible for blastema formation after amputation. Little is known about the molecular mechanisms that drive blastema formation during planarian regeneration. Here we found that treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 blocked the entry of neoblasts into the M-phase of the cell cycle, while allowing neoblasts to successfully enter S-phase in the planarian Dugesia japonica. The rapid and efficient blockage of neoblast mitosis by treatment with the JNK inhibitor provided a method to assess whether temporally regulated cell cycle activation drives blastema formation during planarian regeneration. In the early phase of blastema formation, activated JNK was detected prominently in a mitotic region (the ''postblastema'') proximal to the blastema region. Furthermore, we demonstrated that undifferentiated mitotic neoblasts in the postblastema showed highly activated JNK at the single cell level. JNK inhibition by treatment with SP600125 during this period caused a severe defect of blastema formation, which accorded with a drastic decrease of mitotic neoblasts in regenerating animals. By contrast, these animals still retained many undifferentiated neoblasts near the amputation stump. These findings suggest that JNK signaling plays a crucial role in feeding into the blastema neoblasts for differentiation by regulating the G2 ⁄ M transition in the cell cycle during planarian regeneration.

It is difficult to identify mutagen-induced genome-wide somatic mutations using next generation sequencing; hence, mutagenic features of each mutagen and their roles in cancer development require further elucidation. We described Hawk-Seq™, a highly accurate genome sequencing method and the optimal conditions, for using it to construct libraries that would enable the accurate (c.a. 1 error/10 7-10 8 bp) and efficient survey of genome-wide mutations. Genomic mutations in gpt delta mice or Salmonella typhimurium TA100 exposed to methylnitrosourea (MNU), ethylnitrosourea (ENU), diethylnitrosamine (DEN), benzo[a]pyrene (BP), and aristolochic acid (AA) were profiled using Hawk-Seq™ to analyse positions, substitution patterns, or frequencies. The resultant vast mutation data provided high-resolution mutational signatures, including for minor mutational fractions (e.g. G:C>A:T by AA), which enabled the clarification of the mutagenic features of all mutagens. The 96-type mutational signatures of MNU, AA, and BP indicate their partial similarity to signature 11, 22, and 4 or 29, respectively. Meanwhile, signatures attributable to ENU and DEN were highly similar to each other, but not to signature 11, suggesting that the mechanisms of these agents differed from those of typical alkylating agents. Thus, Hawk-Seq™ can clarify genome-wide chemical mutagenicity profiles at extraordinary resolutions, thereby providing insight into mutagen mechanisms and their roles in cancer development.

A unique aspect of planarians is that they can regenerate a brain from somatic pluripotent stem cells called neoblasts, which have the ability to produce themselves (self-renew) and to give rise to all missing cell types during regeneration. Recent molecular studies have revealed that the planarian brain is composed of many distinct neuronal populations, which are evolutionarily and functionally conserved ones, and acts as an information-processing center to elicit distinct behavioral traits depending on a variety of signals arising from the external environment. How can planarians regenerate such a brain? On the basis of our recent findings, here we review the cellular and molecular mechanisms that regulate the stem cell dynamics involved in the brain regeneration of the planarian Dugesia japonica. Our findings suggest the possible value of in vivo planarian studies for guiding regenerative medicine to treat neurodegenerative diseases via interlinking stem cell biology and regeneration biology.