The DNA methyltransferase DMAP1 is required for tissue maintenance and planarian regeneration.

The precise regulation of transcription is required for embryonic development, adult tissue turnover, and regeneration. Epigenetic modifications play a crucial role in orchestrating and regulating the transcription of genes. These modifications are important in the transition of pluripotent stem cells and their progeny. Methylation, a key epigenetic modification, influences gene expression through changes in DNA methylation. Work in different organisms has shown that the DNA methyltransferase-1-associated protein (DMAP1) may associate with other molecules to repress transcription through DNA methylation. Thus, DMAP1 is a versatile protein implicated in a myriad of events, including pluripotency maintenance, DNA damage repair, and tumor suppression. While DMAP1 has been extensively studied in vitro, its complex regulation in the context of the adult organism remains unclear. To gain insights into the possible roles of DMAP1 at the organismal level, we used planarian flatworms that possess remarkable regenerative capabilities driven by pluripotent stem cells called neoblast. Our findings demonstrate the evolutionary conservation of DMAP1 in the planarian Schmidtea mediterranea. Functional disruption of DMAP1 through RNA interference revealed its critical role in tissue maintenance, neoblast differentiation, and regeneration in S. mediterranea. Moreover, our analysis unveiled a novel function for DMAP1 in regulating cell death in response to DNA damage and influencing the expression of axial polarity markers. Our findings provide a simplified paradigm for studying DMAP1's function in adult tissues.

Restoration of DNA integrity and the cell cycle by electric stimulation in planarian tissues damaged by ionizing radiation.

Exposure to high levels of ionizing γ radiation leads to irreversible DNA damage and cell death. Here, we establish that exogenous application of electric stimulation enables cellular plasticity and the re-establishment of stem cell activity in tissues damaged by ionizing radiation. We show that subthreshold direct current stimulation (DCS) rapidly restores pluripotent stem cell populations previously eliminated by lethally γ-irradiated tissues of the planarian flatworm Schmidtea mediterranea. Our findings reveal that DCS enhances DNA repair, transcriptional activity, and cell cycle entry in post-mitotic cells. These responses involve rapid increases in cytosolic Ca2+ concentration through the activation of L-type Cav channels and intracellular Ca2+ stores, leading to the activation of immediate early genes and ectopic expression of stem cell markers in post-mitotic cells. Overall, we show the potential of electric current stimulation to reverse the damaging effects of high-dose γ radiation in adult tissues. Furthermore, our results provide mechanistic insights describing how electric stimulation effectively translates into molecular responses capable of regulating fundamental cellular functions without the need for genetic or pharmacological intervention.

Extracellular matrix-regulator MMPA is required for the orderly proliferation of neoblasts and differentiation of ectodermal progenitor cells in the planarian Dugesia japonica.

Matrix metalloproteinases (MMPs) are members of a family of zinc-dependent metallopeptidase proteins that are widely found in plants, animals, and microorganisms. As the regulators of the extracellular matrix and basement membrane, MMPs play an important role in embryogenesis, development, innate immunity, and regeneration. However, the function of MMP family in planarian, a model for regeneration research, is still ambiguous. Here, we cloned 5 MMPs genes from Dugesia japonica and found that DjMMPA was associated with the process of regeneration, neoblasts cell maintenance confusion and destruction. Loss of DjMMPA led to homeostasis confusion and eventually death, owing to neoblasts proliferation disorder. Additionally, DjMMPA RNAi-treated animals had impaired regeneration after amputation. Furthermore, knockdown of DjMMPA had noticeable defects in cell differentiation of ectoderm, especially in eyes and neural progenitor cells, possibly by inhibiting Wnt signaling. Our results suggest that extracellular matrix-regulator MMPA is required for the orderly proliferation of neoblasts and differentiation of ectodermal progenitor cells in the planarian, which provide valuable information for further explorations into the molecular mechanism of MMPS, stem cells, and regeneration.

Establishment of a new method to isolate viable x-ray-sensitive cells from planarian by fluorescence-activated cell sorting.

Planarians show outstanding regenerative ability due to the proliferation of neoblasts. Hence the method to isolate planarian neoblasts is important to understand the regeneration process. In our previous study, we reported a method to isolate planarian neoblasts of Dugesia japonica using fluorescence-activated cell sorting (FACS). However, we have not yet succeeded in cultivating these cells even under in vivo conditions after transplantation into x-ray-irradiated planarians. This suggests that dissociated cells might enter apoptotic or necrotic states in the process of fluorescent dye staining and sorting. Here, we developed a new method to isolate viable neoblasts, which can proliferate in the x-ray-irradiated planarians. First, the toxicity of various fluorescence dyes was investigated. All nuclear fluorescent dyes such as Hoechst 33342, DRAQ5, and DyeCycle, showed, more or less, toxicity to mammalian culture cells. In contrast, cytoplasmic fluorescent dye for live cells, calcein AM, was less toxic on these cells. Next, we stained the dissociated planarian cells with only calcein AM, and then collected the x-ray-sensitive fraction. Although the purity of neoblasts was slightly lower than that of the original staining method (ca. 97% → ca. 89%), the sorted cells could actively proliferate when they were injected into x-ray-irradiated planarians. This simple staining and sorting method will provide new opportunities to isolate viable neoblasts and understand regenerating processes.

Sirtuin-1 regulates organismal growth by altering feeding behavior and intestinal morphology in planarians.

Nutrient availability upon feeding leads to an increase in body size in the planarian Schmidtea mediterranea However, it remains unclear how food consumption integrates with cell division at the organismal level. Here, we show that the NAD-dependent protein deacetylases sirtuins are evolutionarily conserved in planarians, and specifically demonstrate that the homolog of human sirtuin-1 (SIRT1) (encoded by Smed-Sirt-1), regulates organismal growth by impairing both feeding behavior and intestinal morphology. Disruption of Smed-Sirt-1 with RNAi or pharmacological inhibition of Sirtuin-1 leads to reduced animal growth. Conversely, enhancement of Sirtuin-1 activity with resveratrol accelerates growth. Differences in growth rates were associated with changes in the amount of time taken to locate food and overall food consumption. Furthermore, Smed-Sirt-1(RNAi) animals displayed reduced cell death and increased stem cell proliferation accompanied by impaired expression of intestinal lineage progenitors and reduced branching of the gut. Taken together, our findings indicate that Sirtuin-1 is a crucial metabolic hub capable of controlling animal behavior, tissue renewal and morphogenesis of the adult intestine.

Planarian stem cell niche, the challenge for understanding tissue regeneration.

Stem cell fate depends on surrounding microenvironment, the so called niche. For this reason, understanding stem cell niche is one of the most challenging target in cell biology field and need to be unraveled with in vivo studies. Planarians offer this unique opportunity, as their stem cells, the neoblasts, are abundant, highly characterized and genetically modifiable by RNA interference in alive animals. However, despite impressive advances have been done in the understanding planarian stem cells and regeneration, only a few information is available in defining signals from differentiated tissues, which affect neoblast stemness and fate. Here, we review on molecular factors that have been found activated in differentiated tissues and directly or indirectly affect neoblast behavior, and we suggest future directions for unravelling this challenge in understanding planarian stem cells.

The true colours of the flatworm: Mechanisms of pigment biosynthesis and pigment cell lineage development in planarians.

Pigment cells serve a variety of important uses across the animal kingdom, and in many species can change and regenerate throughout the lifetime of the organism. The functions of these cells, as well as their origins in both embryonic development and adult regeneration, are not fully understood. Here, we review advances in the study of pigment cells in the freshwater planarian, a model system for stem cell biology and regeneration. Freshwater planarians produce at least three pigment types to generate brown eye and body colouration: melanin, porphyrin, and ommochrome. The body pigments of planarians are produced and contained by a specialized, highly dendritic cell type located in the subepidermal parenchymal space. This cell type is specifically ablated following intense light exposure, a characteristic which has been exploited to discover the gene expression and regeneration of planarian pigment cells. Regenerating pigment cells progress through an immature state marked by upregulation of pigment synthesis genes before differentiating into mature pigment cells; these two states are dynamically regulated in homeostasis to maintain constant body pigmentation. The transcription factors Albino, FoxF-1, and Ets-1, as well as an FGFR-like molecule, are required for proper maintenance of the pigment lineage in both regeneration and homeostasis. These discoveries set the stage for research into external signals that regulate the pigment lineage, as well as possible functions for pigment cells in planarians, including the extra-ocular light response. These insights will address outstanding questions about the evolutionarily-conserved biology of pigment cells.

DNA damage and tissue repair: What we can learn from planaria.

Faithful renewal of aging and damaged tissues is central to organismal lifespan. Stem cells (SCs) generate the cellular progeny that replenish adult tissues across the body but this task becomes increasingly compromised over time. The age related decline in SC-mediated tissue maintenance is a multifactorial event that commonly affects genome integrity. The presence of DNA damage in SCs that are under continuous demand to divide poses a great risk for age-related disorders such as cancer. However, performing analysis of SCs with genomic instability and the DNA damage response during tissue renewal present significant challenges. Here we introduce an alternative experimental system based on the planaria flatworm Schmidtea mediterranea to address at the organismal level studies intersecting SC-mediated tissue renewal in the presence of genomic instability. Planaria have abundant SCs (neoblasts) that maintain high rates of cellular turnover and a variety of molecular tools have been developed to induce DNA damage and dissect how neoblasts respond to this stressor. S. mediterranea displays high evolutionary conservation of DNA repair mechanisms and signaling pathways regulating adult SCs. We describe genetically induced-DNA damage models and highlight body-wide signals affecting cellular decisions such as survival, proliferation, and death in the presence of genomic instability. We also discuss transcriptomic changes in the DNA damage response during injury repair and propose DNA repair as key component of tissue regeneration. Additional studies using planaria will provide insights about mechanisms regulating survival and growth of cells with DNA damage during tissue renewal and regeneration.

Complementary transcriptomic and proteomic analyses reveal the cellular and molecular processes that drive growth and development of Fasciola hepatica in the host liver.

BACKGROUND: The major pathogenesis associated with Fasciola hepatica infection results from the extensive tissue damage caused by the tunnelling and feeding activity of immature flukes during their migration, growth and development in the liver. This is compounded by the pathology caused by host innate and adaptive immune responses that struggle to simultaneously counter infection and repair tissue damage. RESULTS: Complementary transcriptomic and proteomic approaches defined the F. hepatica factors associated with their migration in the liver, and the resulting immune-pathogenesis. Immature liver-stage flukes express ~ 8000 transcripts that are enriched for transcription and translation processes reflective of intensive protein production and signal transduction pathways. Key pathways that regulate neoblast/pluripotent cells, including the PI3K-Akt signalling pathway, are particularly dominant and emphasise the importance of neoblast-like cells for the parasite's rapid development. The liver-stage parasites display different secretome profiles, reflecting their distinct niche within the host, and supports the view that cathepsin peptidases, cathepsin peptidase inhibitors, saposins and leucine aminopeptidases play a central role in the parasite's destructive migration, and digestion of host tissue and blood. Immature flukes are also primed for countering immune attack by secreting immunomodulating fatty acid binding proteins (FABP) and helminth defence molecules (FhHDM). Combined with published host microarray data, our results suggest that considerable immune cell infiltration and subsequent fibrosis of the liver tissue exacerbates oxidative stress within parenchyma that compels the expression of a range of antioxidant molecules within both host and parasite. CONCLUSIONS: The migration of immature F. hepatica parasites within the liver is associated with an increase in protein production, expression of signalling pathways and neoblast proliferation that drive their rapid growth and development. The secretion of a defined set of molecules, particularly cathepsin L peptidases, peptidase-inhibitors, saponins, immune-regulators and antioxidants allow the parasite to negotiate the liver micro-environment, immune attack and increasing levels of oxidative stress. This data contributes to the growing F. hepatica -omics information that can be exploited to understand parasite development more fully and for the design of novel control strategies to prevent host liver tissue destruction and pathology.

5-Fluorouracil-treated planarians, a versatile model system for studying stem cell heterogeneity and tissue aging.

BACKGROUND INFORMATION: Planarians are a sound, well-established model system for molecular studies in the field of stem cells, cell differentiation, developmental biology and translational research. Treated stem cell-less planarians produced by X-ray treatment are commonly used to study stem cell transcriptional profile and their role in planarian biological processes. X-ray induces oxidative and DNA damage to differentiated cells, requires expensive radiation machines that are not available in most of the research centres and demand rigorous risk management and dedicated staff. RESULTS: We tested the use of the well-known antimetabolite genotoxic drug 5-fluorouracil which mainly affects proliferating cells in way to demonstrate its use in replacing X-ray treatment. We succeeded in demonstrating ability of high doses of 5-fluorouracil to deplete Dugesia japonica stem cells and in identifying a 5-fluorouracil transiently resistant population of lineage committed stem cells. CONCLUSIONS AND SIGNIFICANCE: Our results encourage the use of 5-fluorouracil-treated planarians as a model system for studying mechanisms of resistance to genotoxicants, planarian stem cell heterogeneity and molecular cascades of tissue aging.

An Emerging System to Study Photosymbiosis, Brain Regeneration, Chronobiology, and Behavior: The Marine Acoel Symsagittifera roscoffensis.

The acoel worm Symsagittifera roscoffensis, an early offshoot of the Bilateria and the only well-studied marine acoel that lives in a photosymbiotic relationship, exhibits a centralized nervous system, brain regeneration, and a wide repertoire of complex behaviors such as circatidal rhythmicity, photo/geotaxis, and social interactions. While this animal can be collected by the thousands and is studied historically, significant progress is made over the last decade to develop it as an emerging marine model. The authors here present the feasibility of culturing it in the laboratory and describe the progress made on different areas, including genomic and tissue architectures, highlighting the associated challenges. In light of these developments, and on the ability to access abundant synchronized embryos, the authors put forward S. roscoffensis as a marine system to revisit questions in the areas of photosymbiosis, regeneration, chronobiology, and the study of complex behaviors from a molecular and evolutionary perspective.

Poly(ADP-Ribose) Polymerase-3 Regulates Regeneration in Planarians.

Protein ADP-ribosylation is a reversible post-translational modification (PTM) process that plays fundamental roles in cell signaling. The covalent attachment of ADP ribose polymers is executed by PAR polymerases (PARP) and it is essential for chromatin organization, DNA repair, cell cycle, transcription, and replication, among other critical cellular events. The process of PARylation or polyADP-ribosylation is dynamic and takes place across many tissues undergoing renewal and repair, but the molecular mechanisms regulating this PTM remain mostly unknown. Here, we introduce the use of the planarian Schmidtea mediterranea as a tractable model to study PARylation in the complexity of the adult body that is under constant renewal and is capable of regenerating damaged tissues. We identified the evolutionary conservation of PARP signaling that is expressed in planarian stem cells and differentiated tissues. We also demonstrate that Smed-PARP-3 homolog is required for proper regeneration of tissues in the anterior region of the animal. Furthermore, our results demonstrate, Smed-PARP-3(RNAi) disrupts the timely location of injury-induced cell death near the anterior facing wounds and also affects the regeneration of the central nervous system. Our work reveals novel roles for PARylation in large-scale regeneration and provides a simplified platform to investigate PARP signaling in the complexity of the adult body.

Secrets from immortal worms: What can we learn about biological ageing from the planarian model system?

Understanding how some animals are immortal and avoid the ageing process is important. We currently know very little about how they achieve this. Research with genetic model systems has revealed the existence of conserved genetic pathways and molecular processes that affect longevity. Most of these established model organisms have relatively short lifespans. Here we consider the use of planarians, with an immortal life-history that is able to entirely avoid the ageing process. These animals are capable of profound feats of regeneration fueled by a population of adult stem cells called neoblasts. These cells are capable of indefinite self-renewal that has underpinned the evolution of animals that reproduce only by fission, having disposed of the germline, and must therefore be somatically immortal and avoid the ageing process. How they do this is only now starting to be understood. Here we suggest that the evidence so far supports the hypothesis that the lack of ageing is an emergent property of both being highly regenerative and the evolution of highly effective mechanisms for ensuring genome stability in the neoblast stem cell population. The details of these mechanisms could prove to be very informative in understanding how the causes of ageing can be avoided, slowed or even reversed.

EvoRegen in animals: Time to uncover deep conservation or convergence of adult stem cell evolution and regenerative processes.

How do animals regenerate specialised tissues or their entire body after a traumatic injury, how has this ability evolved and what are the genetic and cellular components underpinning this remarkable feat? While some progress has been made in understanding mechanisms, relatively little is known about the evolution of regenerative ability. Which elements of regeneration are due to lineage specific evolutionary novelties or have deeply conserved roots within the Metazoa remains an open question. The renaissance in regeneration research, fuelled by the development of modern functional and comparative genomics, now enable us to gain a detailed understanding of both the mechanisms and evolutionary forces underpinning regeneration in diverse animal phyla. Here we review existing and emerging model systems, with the focus on invertebrates, for studying regeneration. We summarize findings across these taxa that tell us something about the evolution of adult stem cell types that fuel regeneration and the growing evidence that many highly regenerative animals harbor adult stem cells with a gene expression profile that overlaps with germline stem cells. We propose a framework in which regenerative ability broadly evolves through changes in the extent to which stem cells generated through embryogenesis are maintained into the adult life history.

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.

teashirt is required for head-versus-tail regeneration polarity in planarians.

Regeneration requires that the identities of new cells are properly specified to replace missing tissues. The Wnt signaling pathway serves a central role in specifying posterior cell fates during planarian regeneration. We identified a gene encoding a homolog of the Teashirt family of zinc-finger proteins in the planarian Schmidtea mediterranea to be a target of Wnt signaling in intact animals and at posterior-facing wounds. Inhibition of Smed-teashirt (teashirt) by RNA interference (RNAi) resulted in the regeneration of heads in place of tails, a phenotype previously observed with RNAi of the Wnt pathway genes ß-catenin-1, wnt1, Dvl-1/2 or wntless. teashirt was required for ß-catenin-1-dependent activation of posterior genes during regeneration. These findings identify teashirt as a transcriptional target of Wnt signaling required for Wnt-mediated specification of posterior blastemas.

PIWI homologs mediate histone H4 mRNA localization to planarian chromatoid bodies.

The well-known regenerative abilities of planarian flatworms are attributed to a population of adult stem cells called neoblasts that proliferate and differentiate to produce all cell types. A characteristic feature of neoblasts is the presence of large cytoplasmic ribonucleoprotein granules named chromatoid bodies, the function of which has remained largely elusive. This study shows that histone mRNAs are a common component of chromatoid bodies. Our experiments also demonstrate that accumulation of histone mRNAs, which is typically restricted to the S phase of eukaryotic cells, is extended during the cell cycle of neoblasts. The planarian PIWI homologs SMEDWI-1 and SMEDWI-3 are required for proper localization of germinal histone H4 (gH4) mRNA to chromatoid bodies. The association between histone mRNA and chromatoid body components extends beyond gH4 mRNA, since transcripts of other core histone genes were also found in these structures. Additionally, piRNAs corresponding to loci of every core histone type have been identified. Altogether, this work provides evidence that links PIWI proteins and chromatoid bodies to histone mRNA regulation in planarian stem cells. The molecular similarities between neoblasts and undifferentiated cells of other organisms raise the possibility that PIWI proteins might also regulate histone mRNAs in stem cells and germ cells of other metazoans.

Genome-wide analysis of the bHLH gene family in planarians identifies factors required for adult neurogenesis and neuronal regeneration.

In contrast to most well-studied model organisms, planarians have a remarkable ability to completely regenerate a functional nervous system from a pluripotent stem cell population. Thus, planarians provide a powerful model to identify genes required for adult neurogenesis in vivo. We analyzed the basic helix-loop-helix (bHLH) family of transcription factors, many of which are crucial for nervous system development and have been implicated in human diseases. However, their potential roles in adult neurogenesis or central nervous system (CNS) function are not well understood. We identified 44 planarian bHLH homologs, determined their patterns of expression in the animal and assessed their functions using RNAi. We found nine bHLHs expressed in stem cells and neurons that are required for CNS regeneration. Our analyses revealed that homologs of coe, hes (hesl-3) and sim label progenitors in intact planarians, and following amputation we observed an enrichment of coe(+) and sim(+) progenitors near the wound site. RNAi knockdown of coe, hesl-3 or sim led to defects in CNS regeneration, including failure of the cephalic ganglia to properly pattern and a loss of expression of distinct neuronal subtype markers. Together, these data indicate that coe, hesl-3 and sim label neural progenitor cells, which serve to generate new neurons in uninjured or regenerating animals. Our study demonstrates that this model will be useful to investigate how stem cells interpret and respond to genetic and environmental cues in the CNS and to examine the role of bHLH transcription factors in adult tissue regeneration.

Light and electron microscopic studies of the intestinal epithelium in Notoplana humilis (Platyhelminthes, Polycladida): the contribution of mesodermal/gastrodermal neoblasts to intestinal regeneration.

Some free-living flatworms in the phylum Platyhelminthes possess strong regenerative capability that depends on putative pluripotent stem cells known as neoblasts. These neoblasts are defined based on several criteria, including their proliferative capacity and the presence of cellular components known as chromatoid bodies. Polyclads, which are marine flatworms, have the potential to be a good model system for stem cell research, yet little information is available regarding neoblasts and regeneration. In this study, transmission electron microscopy and immunostaining analyses, using antibodies against phospho-histone H3 and BrdU, were used to identify two populations of neoblasts in the polyclad Notoplana humilis: mesodermal neoblasts (located in the mesenchymal space) and gastrodermal neoblasts (located within the intestine, where granular club cells and phagocytic cells are also located). Light and electron microscopic analyses also suggested that phagocytic cells and mesodermal/gastrodermal neoblasts, but not granular club cells, migrated into blastemas and remodeled the intestine during regeneration. Therefore, we suggest that, in polyclads, intestinal regeneration is accomplished by mechanisms underlying both morphallaxis (remodeling of pre-existing tissues) and epimorphosis (de novo tissue formation derived from mesodermal/gastrodermal neoblasts). Based on the assumption that gastrodermal neoblasts, which are derived from mesodermal neoblasts, are intestinal stem cells, we propose a model to study intestinal regeneration.

The DNA Methyltransferase DMAP1 is Required for Tissue Maintenance and Planarian Regeneration.

The precise regulation of transcription is required for embryonic development, adult tissue turnover, and regeneration. Epigenetic modifications play a crucial role in orchestrating and regulating the transcription of genes. These modifications are important in the transition of pluripotent stem cells and their progeny. Methylation, a key epigenetic modification, influences gene expression through changes in histone tails and direct DNA methylation. Work in different organisms has shown that the DNA methyltransferase-1-associated protein (DMAP1) may associate with other molecules to repress transcription through DNA methylation. Thus, DMAP1 is a versatile protein implicated in a myriad of events, including pluripotency maintenance, DNA damage repair, and tumor suppression. While DMAP1 has been extensively studied in vitro, its complex regulation in the context of the adult organism remains unclear. To gain insights into the possible roles of DMAP1 at the organismal level, we used planarian flatworms that possess remarkable regenerative capabilities driven by pluripotent stem cells called neoblast. Our findings demonstrate the evolutionary conservation of DMAP1 in the planarian Schmidtea mediterranea. Functional disruption of DMAP1 through RNA interference revealed its critical role in tissue maintenance, neoblast differentiation, and regeneration in S. mediterranea. Moreover, our analysis unveiled a novel function for DMAP1 in regulating cell death in response to DNA damage and influencing the expression of axial polarity markers. Our findings provide a simplified paradigm for studying DMAP1's epigenetic regulation in adult tissues.