SummaryDjPum, a homologue of Drosophila Pumilio, is essential to planarian stem cell maintenance

Profiling neoblast gene expression

Comparison of the gene-expression profiles of planarians in which all adult pluripotent stem cells (neoblasts) were eliminated and wildtype worms identified a putative neoblast-restricted gene set. This included many genes involved in chromatin modeling and RNA metabolism, suggesting that epigenetic modifications and post-transcriptional regulation are important for neoblast regulation.

Planarian regeneration, based upon totipotent stem cells, the neoblasts, provides a unique opportunity to study in vivo the molecular program that defines a stem cell. In this study, we report the identification of DjPiwi-1, a planarian homologue of Drosophila Piwi. Expression analysis showed that DjPiwi-1 transcripts are preferentially accumulated in small cells distributed along the midline of the dorsal parenchyma. DjPiwi-1 transcripts were not detectable after X-ray irradiation by whole mount in situ hybridization. Real time reverse transcriptase polymerase chain reaction analysis confirmed the significant reduction of DjPiwi-1 expression after X-ray treatment. However, the presence of residual DjPiwi-1 transcription suggests that, although the majority of DjPiwi-1-positive cells can be neoblasts, this gene is also expressed in differentiating/differentiated cells. During regeneration DjPiwi-1-positive cells reorganize along the midline of the stump and no accumulation of hybridization signal was observed either in the blastema area or in the parenchymal region beneath the blastema. DjPiwi-1-positive cells, as well as the DjMCM2-expressing neoblasts located along the midline and those spread all over the parenchyma, showed a lower tolerance to X-ray with respect to the DjMCM2-expressing neoblasts distributed along the lateral lines of the parenchyma. Taken together, these findings suggest the presence of different neoblast subpopulations in planarians.

Planarians are a model system for studying adult stem cells, as they possess the neoblasts, a population of pluripotent adult stem cells able to give rise to both somatic and germ cells. Although over the last years several efforts have been made to shed light on neoblast biology, only recent evidence indicate that this population of cells is heterogeneous. In this study we irradiated planarians with different non-lethal X-ray doses (1-5 Gy) and we identified subpopulations of neoblasts with diverse levels of tolerance to X-rays. We demonstrated that a dramatic reduction of neoblasts occurred soon after non-lethal irradiations and that de-novo proliferation of some radioresistant cells re-established the primary neoblast number. In particular, a strong proliferation activity occurred at the ventral side of irradiated animals close to the nervous system. The produced cells migrated towards the dorsal parenchyma and, together with some dorsal radioresistant cells, reconstituted the entire neoblast population demonstrating the extreme plasticity of this adult stem cell system.

A conserved network of nuclear proteins is crucial to eye formation in both vertebrates and invertebrates. The finding that freshwater planarians can regenerate eyes without the contribution of Pax6 suggests that alternative combinations of regulatory elements may control the morphogenesis of the prototypic planarian eye. To further dissect the molecular events controlling eye regeneration in planarians, we investigated the role of eyes absent (Djeya) and six-1 (Djsix-1) genes in Dugesia japonica. These genes are expressed in both regenerating eyes and in differentiated photoreceptors of intact adults. Through RNAi studies, we show that Djsix-1 and Djeya are both critical for the regeneration of normal eyes in planarians and genetically cooperate in vivo to establish correct eye cell differentiation. We further demonstrate that the genetic interaction is mediated by physical interaction between the evolutionarily conserved domains of these two proteins. These data indicate that planarians use cooperatively Djsix-1 and Djeya for the proper specification of photoreceptors, implicating that the mechanism involving their evolutionarily conserved domains can be very ancient. Finally, both Djsix-1 and Djeya double-stranded RNA are substantially more effective at producing no-eye phenotypes in the second round of regeneration. This is probably due to the significant plasticity of the planarian model system, based on the presence of a stable population of totipotent stem cells, which ensure the rapid cell turnover of all differentiated cell types.