MEK/ERK Signaling Regulates Reconstitution of the Dopaminergic Nerve Circuit in the Planarian Dugesia japonica.

Planarian Dugesia japonica is a flatworm that can autonomously regenerate its own body after an artificial amputation. A recent report showed the role of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway in the head morphogenesis during the planarian regeneration process after amputation; however, neuron-specific regeneration mechanisms have not yet been reported. Here, whether MEK/ERK pathway was involved in the dopaminergic neuronal regeneration in planarians was investigated. Planarians regenerated their body within 14 days after amputation; however, the head region morphogenesis was inhibited by MEK inhibitor U0126 (3 or 10 µM). Furthermore, the number of planarian tyrosine hydroxylase (DjTH)-positive dopaminergic neurons in the regenerated head region was also decreased by U0126. The 6-hydroxydopamine (6-OHDA), a dopaminergic neurotoxin, can decrease the number of dopaminergic neurons; however, planarians can regenerate dopaminergic neurons after injecting 6-OHDA into the intestinal tract. MEK inhibitor PD98059 (30 µM) or U0126 (10 µM) significantly decreased dopaminergic neurons 5 days after the 6-OHDA injection. During the regeneration process of dopaminergic neurons, phosphorylated histone H3 (H3P)-positive stem cells known as "neoblasts" were increased in the head region; however, MEK inhibitors significantly decreased the number of H3P-positive neoblasts. These results suggested that dopaminergic neuronal regeneration in planarian was regulated by the MEK/ERK pathway.

Planarian regeneration between 1960s and 1990s: From skilful baffled ancestors to bold integrative descendants. A personal account.

Scientific fields grow by accretion of knowledge brought up by succesive generations of scientists. With the field of planarian regeneration as a general background, here I give a personal account of it from the late 1960s until the late 1990s when new research groups, namely Americans, broke into it. After a short historical summary, I report how I got into regeneration, why I choose planarians, and most especially the finding and description of the current model organism Schmidtea mediterranea. Next, I concentrate on the quantitative cellular approaches to regeneration, growth, and degrowth undertaken in our lab in Barcelona, and the long struggle to set neoblasts as a totipotent/pluripotent heterogeneous population of mitotic and non-mitotic cells for homeostatic somatic cell renewal, reproduction, and blastema formation during regeneration. Finally, the first attempts by several labs to analyze regeneration in molecular terms (RNAs, proteins, mAbs, gene detection, cloning and expression) are also covered. Along this essay, I am honoured to pay due tribute to our forebears (ancestors) from Morgan and Child to the French School researchers. In spite of being often baffled by planarian 'tricks', they struggled hard to find new techniques, new ways, and new ideas to tackle the main problems of regeneration, paving the way to those (descendants) that followed suit.

A lineage CLOUD for neoblasts.

In planarians, pluripotency can be studied in vivo in the adult animal, making these animals a unique model system where pluripotency-based regeneration (PBR)-and its therapeutic potential-can be investigated. This review focuses on recent findings to build a cloud model of fate restriction likelihood for planarian stem and progenitor cells. Recently, a computational approach based on functional and molecular profiling at the single cell level was proposed for human hematopoietic stem cells. Based on data generated both in vivo and ex vivo, we hypothesized that planarian stem cells could acquire multiple direction lineage biases, following a "badlands" landscape. Instead of a discrete tree-like hierarchy, where the potency of stem/progenitor cells reduces stepwise, we propose a Continuum of LOw-primed UnDifferentiated Planarian Stem/Progenitor Cells (CLOUD-PSPCs). Every subclass of neoblast/progenitor cells is a cloud of likelihood, as the single cell transcriptomics data indicate. The CLOUD-HSPCs concept was substantiated by in vitro data from cell culture; therefore, to confirm the CLOUD-PSPCs model, the planarian community needs to develop new tools, like live cell tracking. Future studies will allow a deeper understanding of PBR in planarian, and the possible implications for regenerative therapies in human.

Post-transcriptional regulation in planarian stem cells.

Planarians are known for their immense regenerative abilities. A pluripotent stem cell population provides the cellular source for this process, as well as for the homeostatic cell turnover of the animals. These stem cells, known as neoblasts, present striking similarities at the morphological and molecular level to germ cells, but however, give rise to somatic tissue. Many RNA binding proteins known to be important for germ cell biology are also required for neoblast function, highlighting the importance of post-transcriptional regulation for stem cell control. Many of its aspects, including alternative splicing, alternative polyadenylation, translational control and mRNA deadenylation, as well as small RNAs such as microRNAs and piRNA are critical for stem cells. Their inhibition often abrogates both regeneration and cell turnover, resulting in lethality. Some of aspects of post-transcriptional regulation are conserved from planarian to mammalian stem cells.

Planarian flatworms as a new model system for understanding the epigenetic regulation of stem cell pluripotency and differentiation.

Planarian flatworms possess pluripotent stem cells (neoblasts) that are able to differentiate into all cell types that constitute the adult body plan. Consequently, planarians possess remarkable regenerative capabilities. Transcriptomic studies have revealed that gene expression is coordinated to maintain neoblast pluripotency, and ensure correct lineage specification during differentiation. But as yet they have not revealed how this regulation of expression is controlled. In this review, we propose that planarians represent a unique and effective system to study the epigenetic regulation of these processes in an in vivo context. We consolidate evidence suggesting that although DNA methylation is likely present in some flatworm lineages, it does not regulate neoblast function in Schmidtea mediterranea. A number of phenotypic studies have documented the role of histone modification and chromatin remodelling complexes in regulating distinct neoblast processes, and we focus on four important examples of planarian epigenetic regulators: Nucleosome Remodeling Deacetylase (NuRD) complex, Polycomb Repressive Complex (PRC), the SET1/MLL methyltransferases, and the nuclear PIWI/piRNA complex. Given the recent advent of ChIP-seq in planarians, we propose future avenues of research that will identify the genomic targets of these complexes allowing for a clearer picture of how neoblast processes are coordinated at the epigenetic level. These insights into neoblast biology may be directly relevant to mammalian stem cells and disease. The unique biology of planarians will also allow us to investigate how extracellular signals feed into epigenetic regulatory networks to govern concerted neoblast responses during regenerative polarity, tissue patterning, and remodelling.

Heat shock protein DNAJA1 stabilizes PIWI proteins to support regeneration and homeostasis of planarian Schmidtea mediterranea.

PIWI proteins are key regulators of germline and somatic stem cells throughout different evolutionary lineages. However, how PIWI proteins themselves are regulated remains largely unknown. To identify candidate proteins that interact with PIWI proteins and regulate their stability, here we established a yeast two-hybrid (Y2H) assay in the planarian species Schmidtea mediterranea We show that DNAJA1, a heat shock protein 40 family member, interacts with the PIWI protein SMEDWI-2, as validated by the Y2H screen and co-immunoprecipitation assays. We found that DNAJA1 is enriched in planarian adult stem cells, the nervous system, and intestinal tissues. DNAJA1-knockdown abolished planarian regeneration and homeostasis, compromised stem cell maintenance and PIWI-interacting RNA (piRNA) biogenesis, and deregulated SMEDWI-1/2 target genes. Mechanistically, we observed that DNAJA1 is required for the stability of SMEDWI-1 and SMEDWI-2 proteins. Furthermore, we noted that human DNAJA1 binds to Piwi-like RNA-mediated gene silencing 1 (PIWIL1) and is required for PIWIL1 stability in human gastric cancer cells. In summary, our results reveal not only an evolutionarily conserved functional link between PIWI and DNAJA1 that is essential for PIWI protein stability and piRNA biogenesis, but also an important role of DNAJA1 in the control of proteins involved in stem cell regulation.

Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian Schmidtea mediterranea.

Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown of smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including zfp-1, are translationally regulated by SMED-PABPC2. Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration.

No head regeneration here: regeneration capacity and stem cell dynamics of Theama mediterranea (Polycladida, Platyhelminthes).

Research on the regeneration potential of flatworms (Platyhelminthes) has been mainly undertaken with planarians (Tricladida), where most species can regenerate a head and no proliferation takes place in the blastema, i.e. the early undifferentiated regenerative tissue. Only few studies are available for an early-branching group within the Platyhelminthes, the Polycladida. Head regeneration in polyclads is not possible, with a single exception from a study performed more than 100 years ago: Cestoplana was reported to be able to regenerate a head if cut a short distance behind the brain. Here, we show that 'Cestoplana' was misdetermined and most likely was the small interstitial polyclad Theama mediterranea. We revisited regeneration capacity and dynamics of T. mediterranea with live observations and stainings of musculature, nervous system, and proliferating and differentiating stem cells. In our experiments, after transversal amputation, only animals retaining more than half of the brain could fully restore the head including the brain. If completely removed, the brain was never found to regenerate to any extent. Different from planarians, but comparable to other free-living flatworms we detected cell proliferation within the posterior regeneration blastema in T. mediterranea. Similar to other free-living flatworms, proliferation did not occur within, but only outside, the differentiating organ primordia. Our results strongly imply that brain regeneration in the absence of the latter is not possible in any polyclad studied so far. Also, it appears that proliferation of stem cells within the regeneration blastema is a plesiomorphy in flatworms and that planarians are derived in this character.

Regional signals in the planarian body guide stem cell fate in the presence of genomic instability.

Cellular fate decisions are influenced by their topographical location in the adult body. For instance, tissue repair and neoplastic growth are greater in anterior than in posterior regions of adult animals. However, the molecular underpinnings of these regional differences are unknown. We identified a regional switch in the adult planarian body upon systemic disruption of homologous recombination with RNA-interference of Rad51 Rad51 knockdown increases DNA double-strand breaks (DSBs) throughout the body, but stem cells react differently depending on their location along the anteroposterior axis. In the presence of extensive DSBs, cells in the anterior part of the body resist death, whereas cells in the posterior region undergo apoptosis. Furthermore, we found that proliferation of cells with DNA damage is induced in the presence of brain tissue and that the retinoblastoma pathway enables overproliferation of cells with DSBs while attending to the demands of tissue growth and repair. Our results implicate both autonomous and non-autonomous mechanisms as key mediators of regional cell behavior and cellular transformation in the adult body.

Planarian Stem Cell Heterogeneity.

Planarian (Platyhelminthes, Triclads) are free-living flatworms endowed with extraordinary regenerative capabilities, i.e., the ability to rebuild any missing body parts also from small fragments. Planarian regenerative capabilities fascinated scientific community since early 1800, including high-standing scientists such as J.T. Morgan and C. M. Child. Today, it is known that planarian regeneration is due to the presence of a wide population of stem cells, the so-called neoblasts. However, the understanding of the nature of cells orchestrating planarian regeneration was a long journey, and several questions still remain unanswered. In this chapter, beginning from the definition of the classical concept of neoblast, we review progressive discoveries that have brought to the modern view of these cells as a highly heterogeneous population of stem cells including pluripotent stem cells and undifferentiated populations of committed progenies.

Searching for non-transposable targets of planarian nuclear PIWI in pluripotent stem cells and differentiated cells.

Nuclear PIWIs together with their guide RNAs (piRNAs) epigenetically silence various genes including transposons in many organisms. In planarians, the nuclear piwi family gene, DjpiwiB is specifically transcribed in adult pluripotent stem cells (adult PSC, neoblast), but not in differentiated cells. However, the protein accumulates in the nuclei of both neoblasts and their descendant differentiated cells. Interestingly, PIWI(DjPiwiB)-piRNA complexes are indispensable for the repression of transposable genes at the onset of differentiation from neoblasts. Here, we conducted a comparative transcriptome analysis between control and DjpiwiB(RNAi) animals to identify non-transposable target genes of the DjPiwiB-piRNA complexes. Using bioinformatic analyses and RNAi we demonstrate that DjPiwiB-piRNA complexes are required for the proper expression of Djmcm2 and Djhistone h4 in neoblasts and that DjPiwiB-piRNA complexes regulate the transient expression of Djcalu during neoblast differentiation. Thus, DjPiwiB-piRNA complexes regulate the correct expression patterns during neoblast self-renewal and differentiation.

Genome and transcriptome of the regeneration-competent flatworm, Macrostomum lignano.

The free-living flatworm, Macrostomum lignano has an impressive regenerative capacity. Following injury, it can regenerate almost an entirely new organism because of the presence of an abundant somatic stem cell population, the neoblasts. This set of unique properties makes many flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell-fate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of M. lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ∼75% of its sequence being comprised of simple repeats and transposon sequences. This has made high-quality assembly from Illumina reads alone impossible (N50=222 bp). We therefore generated 130× coverage by long sequencing reads from the Pacific Biosciences platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene-expression patterns during regeneration, examining pathways important to stem cell function.

Planarians as models of cadmium-induced neoplasia provide measurable benchmarks for mechanistic studies.

Bioassays of planarian neoplasia highlight the potential of these organisms as useful standards to assess whether environmental toxins such as cadmium promote tumorigenesis. These studies complement other investigations into the exceptional healing and regeneration of planarians - processes that are driven by a population of active stem cells, or neoblasts, which are likely transformed during planarian tumor growth. Our goal was to determine if planarian tumorigenesis assays are amenable to mechanistic studies of cadmium carcinogenesis. To that end we demonstrate, by examining both counts of cell populations by size, and instances of mitosis, that the activity of the stem cell population can be monitored. We also provide evidence that specific biomodulators can affect the potential of planarian neoplastic growth, in that an inhibitor of metalloproteinases effectively blocked the development of the lesions. From these results, we infer that neoblast activity does respond to cadmium-induced tumor growth, and that metalloproteinases are required for the progression of cancer in the planarian.

Muscle-derived matrix metalloproteinase regulates stem cell proliferation in planarians.

BACKGROUND: Matrix metalloproteinases (MMPs) are a large family of regulatory enzymes that function in extracellular matrix degradation and facilitate a diverse range of cellular processes. Despite the significant focus on the activities of MMPs in human disease, there is a lack of substantial knowledge regarding their normal physiological roles and their role in regulating aspects of stem cell biology. The freshwater planarian Schmidtea mediterranea (S. mediterranea) is an excellent system in which to study robust and nearly unlimited regeneration, guided by a population of mitotically active stem cells, termed neoblasts. RESULTS: We characterized MMPs in the context of planarian stem cells, specifically exploring the role of S. mediterranea MT-MMPB. Using in situ hybridization and available functional genomic tools, we observed that mt-mmpB is expressed in the dorsoventral muscle cells, and its loss results in a reduction in animal size accompanied by a decrease in mitotic cells, suggesting that it plays a unique role in regulating stem cell proliferation. CONCLUSIONS: The novel findings of this study bring to light the unique and critical roles that muscles play in regulating neoblast function, and more broadly, highlight the importance of MMPs in stem cell biology. Developmental Dynamics 245:963-970, 2016. © 2016 Wiley Periodicals, Inc.

Long-term time-lapse live imaging reveals extensive cell migration during annelid regeneration.

BACKGROUND: Time-lapse imaging has proven highly valuable for studying development, yielding data of much finer resolution than traditional "still-shot" studies and allowing direct examination of tissue and cell dynamics. A major challenge for time-lapse imaging of animals is keeping specimens immobile yet healthy for extended periods of time. Although this is often feasible for embryos, the difficulty of immobilizing typically motile juvenile and adult stages remains a persistent obstacle to time-lapse imaging of post-embryonic development. RESULTS: Here we describe a new method for long-duration time-lapse imaging of adults of the small freshwater annelid Pristina leidyi and use this method to investigate its regenerative processes. Specimens are immobilized with tetrodotoxin, resulting in irreversible paralysis yet apparently normal regeneration, and mounted in agarose surrounded by culture water or halocarbon oil, to prevent dehydration but allowing gas exchange. Using this method, worms can be imaged continuously and at high spatial-temporal resolution for up to 5 days, spanning the entire regeneration process. We performed a fine-scale analysis of regeneration growth rate and characterized cell migration dynamics during early regeneration. Our studies reveal the migration of several putative cell types, including one strongly resembling published descriptions of annelid neoblasts, a cell type suggested to be migratory based on "still-shot" studies and long hypothesized to be linked to regenerative success in annelids. CONCLUSIONS: Combining neurotoxin-based paralysis, live mounting techniques and a starvation-tolerant study system has allowed us to obtain the most extensive high-resolution longitudinal recordings of full anterior and posterior regeneration in an invertebrate, and to detect and characterize several cell types undergoing extensive migration during this process. We expect the tetrodotoxin paralysis and time-lapse imaging methods presented here to be broadly useful in studying other animals and of particular value for studying post-embryonic development.

Identification of neoblast- and regeneration-specific miRNAs in the planarian Schmidtea mediterranea.

In recent years, the planarian Schmidtea mediterranea has emerged as a tractable model system to study stem cell biology and regeneration. MicroRNAs are small RNA species that control gene expression by modulating translational repression and mRNA stability and have been implicated in the regulation of various cellular processes. Though recent studies have identified several miRNAs in S. mediterranea, their expression in neoblast subpopulations and during regeneration has not been examined. Here, we identify several miRNAs whose expression is enriched in different neoblast subpopulations and in regenerating tissue at different time points in S. mediterranea. Some of these miRNAs were enriched within 3 h post-amputation and may, therefore, play a role in wound healing and/or neoblast migration. Our results also revealed miRNAs, such as sme-miR-2d-3p and the sme-miR-124 family, whose expression is enriched in the cephalic ganglia, are also expressed in the brain primordium during CNS regeneration. These results provide new insight into the potential biological functions of miRNAs in neoblasts and regeneration in planarians.

Extracellular vesicles promote proliferation in an animal model of regeneration.

Extracellular vesicles (EVs) are secreted nanoparticles composed of a lipid bilayer that carry lipid, protein, and nucleic acid cargo between cells as a mode of intercellular communication. Although EVs can promote tissue repair in mammals, their roles in animals with greater regenerative capacity are not well understood. Planarian flatworms are capable of whole body regeneration due to pluripotent somatic stem cells called neoblasts that proliferate in response to injury. Here, using transmission electron microscopy, nanoparticle tracking analysis, and protein content examination, we showed that EVs enriched from the tissues of the planarian Schmidtea mediterranea had similar morphology and size as other eukaryotic EVs, and that these EVs carried orthologs of the conserved EV biogenesis regulators ALIX and TSG101. PKH67-labeled EVs were taken up more quickly by S/G2 neoblasts than G1 neoblasts/early progeny and differentiated cells. When injected into living planarians, EVs from regenerating tissue fragments enhanced upregulation of neoblast-associated transcripts. In addition, EV injection increased the number of F-ara-EdU-labelled cells by 49% as compared to buffer injection only. Our findings demonstrate that regenerating planarians produce EVs that promote stem cell proliferation, and suggest the planarian as an amenable in vivo model for the study of EV function during regeneration.

microRNAs in parasites and parasite infection.

miRNAs, a subclass of small regulatory RNAs, are present from ancient unicellular protozoans to parasitic helminths and parasitic arthropods. The miRNA-silencing mechanism appears, however, to be absent in a number of protozoan parasites. Protozoan miRNAs and components of their silencing machinery possess features different from other eukaryotes, providing some clues on the evolution of the RNA-induced silencing machinery. miRNA functions possibly associate with neoblast biology, development, physiology, infection and immunity of parasites. Parasite infection can alter host miRNA expression that can favor both parasite clearance and infection. miRNA pathways are, thus, a potential target for the therapeutic control of parasitic diseases.

Persistence of Nocturnality in Decapitated and Bisected Flatworms.

The ability of flatworms to regenerate entire brain structures, and indeed much of their body from mere fragments of the whole animal, presents the unique opportunity to observe the development of day-night rhythms in adult animals. In many animals, young are arrhythmic, and their species-specific timing of activity develops as the animal matures. In this study, we created two flatworm cohorts, housed in isolation, that were regenerating either (1) the brain in a decapitated animal, or (2) major body structures in a bisected, tailless animal. In this way, we observed how bisection influenced the level of activity and diel rhythmicity, and how these developed as each flatworm regenerated. Here, we demonstrate that intact flatworms were predominantly active at night, with peaks in activity seen in the hours after lights-off and before lights-on. While decapitated and tailless flatworms could still move, both were less active than the original animal, and both segments retained a nocturnal lifestyle. Furthermore, decapitated flatworms, once regenerated, again showed a U-shaped pattern of nocturnal activity reminiscent of the two night-time peaks seen in the original animal. These results could be used to further investigate how regeneration may affect motor control and motor output, or to further investigate the presence of a clock in the flatworm brain.

Stem cell proliferation and differentiation during larval metamorphosis of the model tapeworm Hymenolepis microstoma.

Background: Tapeworm larvae cause important diseases in humans and domestic animals. During infection, the first larval stage undergoes a metamorphosis where tissues are formed de novo from a population of stem cells called germinative cells. This process is difficult to study for human pathogens, as these larvae are infectious and difficult to obtain in the laboratory. Methods: In this work, we analyzed cell proliferation and differentiation during larval metamorphosis in the model tapeworm Hymenolepis microstoma, by in vivo labelling of proliferating cells with the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU), tracing their differentiation with a suite of specific molecular markers for different cell types. Results: Proliferating cells are very abundant and fast-cycling during early metamorphosis: the total number of cells duplicates every ten hours, and the length of G2 is only 75 minutes. New tegumental, muscle and nerve cells differentiate from this pool of proliferating germinative cells, and these processes are very fast, as differentiation markers for neurons and muscle cells appear within 24 hours after exiting the cell cycle, and fusion of new cells to the tegumental syncytium can be detected after only 4 hours. Tegumental and muscle cells appear from early stages of metamorphosis (24 to 48 hours post-infection); in contrast, most markers for differentiating neurons appear later, and the detection of synapsin and neuropeptides correlates with scolex retraction. Finally, we identified populations of proliferating cells that express conserved genes associated with neuronal progenitors and precursors, suggesting the existence of tissue-specific lineages among germinative cells. Discussion: These results provide for the first time a comprehensive view of the development of new tissues during tapeworm larval metamorphosis, providing a framework for similar studies in human and veterinary pathogens.