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The planarian Schmidtea mediterranea shows nutrient-dependent whole-body plasticity. Starvation leads to body size reduction, while feeding triggers growth. The balance of cell proliferation and cell death controls cell number, driving organismal body size. Here, we uncovered the role of FoxO in controlling cell death through TUNEL and caspase-3 assays and bak qPCR detection in foxO RNAi planarians., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Tissue regeneration is a complex process involving large changes in cell proliferation, fate determination, and differentiation. Mitochondrial dynamics and metabolism play a crucial role in development and wound repair, but their function in large-scale regeneration remains poorly understood. Planarians offer an excellent model to investigate this process due to their remarkable regenerative abilities. In this study, we examine mitochondrial dynamics during planarian regeneration. We find that knockdown of the mitochondrial fusion gene, opa1, impairs both tissue regeneration and stem cell pluripotency. Interestingly, the regeneration defects caused by opa1 knockdown are rescued by simultaneous knockdown of the mitochondrial fission gene, drp1, which partially restores mitochondrial dynamics. Furthermore, we discover that Mito low stem cells exhibit an enrichment of pluripotency due to their fate choices at earlier stages. Transcriptomic analysis reveals the delicate mitonuclear balance in metabolism and mitochondrial proteins in regeneration, controlled by mitochondrial dynamics. These findings highlight the importance of maintaining mitochondrial dynamics in large-scale tissue regeneration and suggest the potential for manipulating these dynamics to enhance stem cell functionality and regenerative processes., Competing Interests: Competing interests: Z.C. holds the patent of PK Mito dyes (CN111454193). The remaining authors declare no other competing interest. Ethical approval: We support inclusive, diverse, and equitable conduct of research., (© 2024. The Author(s).)

Tissue homeostasis and regeneration involve complex cellular changes. The role of rRNA modification-dependent translational regulation in these processes remains largely unknown. Planarians, renowned for their ability to undergo remarkable tissue regeneration, provide an ideal model for the analysis of differential rRNA regulation in diverse cell types during tissue homeostasis and regeneration. We investigated the role of RNA 2'-O-methyltransferase, Fibrillarin (FBL), in the planarian Schmidtea mediterranea and identified two FBL homologs: Smed-fbl-1 (fbl-1) and Smed-fbl-2 (fbl-2). Both are essential for planarian regeneration, but play distinct roles: fbl-1 is crucial for progenitor cell differentiation, while fbl-2 is important for late-stage epidermal lineage specification. Different 2'-O-methylation patterns were observed upon fbl-1 and fbl-2 knockdown, suggesting their roles in translation of specific mRNA pools during regeneration. Ribo-seq analysis further revealed differing impacts of fbl-1 and fbl-2 knockdown on gene translation. These findings indicate divergent roles of the duplicate fbl genes in specific cell lineage development in planarians and suggest a role of rRNA modifications in translational regulation during tissue maintenance and regeneration., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

Biological evolution has generated a vast array of natural compounds produced by organisms across all domains. Among these, secondary metabolites, selected to enhance an organism's competitiveness in its natural environment, make them a reservoir for discovering new compounds with cytotoxic activity, potentially useful as novel anticancer agents. Slime secretions, the first barrier between epithelial surfaces and the surrounding environment, frequently contain cytotoxic molecules to limit the growth of parasitic organisms. Planarians, freshwater Triclads, continuously secrete a viscous mucus with multiple physiological functions. The chemical composition of planarian mucus has been only partially elucidated, and there are no studies reporting its cytotoxic or cytostatic effects. In this study, we developed a protocol for collecting mucus from Dugesia japonica specimens and we demonstrated that it inhibits the growth of cancer cells by activating cytostatic and ROS-dependent cytotoxic mechanisms inducing lipid droplet accumulation and mitochondrial membrane reorganization. Although further research is needed to identify the specific chemicals responsible for the anticancer activity of planarian mucus, this work opens up numerous research avenues aimed at better understanding the mechanisms of action of this product for potential therapeutic applications.

Planarian flatworms undergo continuous internal turnover, wherein old cells are replaced by the division progeny of adult pluripotent stem cells (neoblasts). How cell turnover is carried out at the organismal level remains an intriguing question in planarians and other systems. While previous studies have predominantly focused on neoblast proliferation, little is known about the processes that mediate cell loss during tissue homeostasis. Here, we use the planarian epidermis as a model to study the mechanisms of cell removal. We established a covalent dye-labeling assay and image analysis pipeline to quantify the cell turnover rate in the planarian epidermis. Our findings indicate that the ventral epidermis is highly dynamic and epidermal cells undergo internalization via basal extrusion, followed by a relocation toward the intestine and ultimately digestion by intestinal phagocytes. Overall, our study reveals a complex homeostatic process of cell clearance that may generally allow planarians to catabolize their own cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Germ cells are regulated by local microenvironments (niches), which secrete instructive cues. Conserved developmental signaling molecules act as niche-derived regulatory factors, yet other types of niche signals remain to be identified. Single-cell RNA-sequencing of sexual planarians revealed niche cells expressing a nonribosomal peptide synthetase ( nrps ). Inhibiting nrps led to loss of female reproductive organs and testis hyperplasia. Mass spectrometry detected the dipeptide β-alanyl-tryptamine (BATT), which is associated with reproductive system development and requires nrps and a monoamine-transmitter-synthetic enzyme Aromatic L-amino acid decarboxylase (AADC) for its production. Exogenous BATT rescued the reproductive defects after nrps or aadc inhibition, restoring fertility. Thus, a nonribosomal, monoamine-derived peptide provided by niche cells acts as a critical signal to trigger planarian reproductive development. These findings reveal an unexpected function for monoamines in niche-germ cell signaling. Furthermore, given the recently reported role for BATT as a male-derived factor required for reproductive maturation of female schistosomes, these results have important implications for the evolution of parasitic flatworms and suggest a potential role for nonribosomal peptides as signaling molecules in other organisms., Competing Interests: Competing interests statement:The authors declare no competing interest.

Successful regeneration of missing tissues requires seamless integration of positional information along the body axes. Planarians, which regenerate from almost any injury, use conserved, developmentally important signaling pathways to pattern the body axes. However, the molecular mechanisms which facilitate cross talk between these signaling pathways to integrate positional information remain poorly understood. Here, we report a p21-activated kinase ( smed-pak1 ) which functionally integrates the anterior-posterior (AP) and the medio-lateral (ML) axes. pak1 inhibits WNT/β-catenin signaling along the AP axis and, functions synergistically with the β-catenin-independent WNT signaling of the ML axis. Furthermore, this functional integration is dependent on warts and merlin -the components of the Hippo/Yorkie (YKI) pathway. Hippo/YKI pathway is a critical regulator of body size in flies and mice, but our data suggest the pathway regulates body axes patterning in planarians. Our study provides a signaling network integrating positional information which can mediate coordinated growth and patterning during planarian regeneration., Competing Interests: Competing interests statement:The authors declare no competing interest.

Whole-body regeneration requires the ability to produce the full repertoire of adult cell types. The planarian Schmidtea mediterranea contains over 125 cell types, which can be regenerated from a stem cell population called neoblasts. Neoblast fate choice can be regulated by the expression of fate-specific transcription factors (FSTFs). How fate choices are made and distributed across neoblasts versus their post-mitotic progeny remains unclear. We used single-cell RNA sequencing to systematically map fate choices made in S/G 2 /M neoblasts and, separately, in their post-mitotic progeny that serve as progenitors for all adult cell types. We defined transcription factor expression signatures associated with all detected fates, identifying numerous new progenitor classes and FSTFs that regulate them. Our work generates an atlas of stem cell fates with associated transcription factor signatures for most cell types in a complete adult organism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neoblasts are the only cells capable of proliferation in planarians. The traditional flow cytometry protocol using Hoechst inhibits the cell cycle. Here, we present a protocol for culturing and functionally manipulating planarian neoblasts using SiR-DNA-based flow cytometry. We describe steps for cell dissociation and staining, flow cytometry, and cell collection and culture. We then detail procedures for Nanoluciferase mRNA transfection. This protocol facilitates further investigations into the pluripotency and regeneration mechanisms within neoblasts. For complete details on the use and execution of this protocol, please refer to Lei et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cell corpse removal is a critical component of both development and homeostasis throughout the animal kingdom. Extensive research has revealed many of the mechanisms involved in corpse removal, typically involving engulfment and digestion by another cell; however, the dynamics of cell corpse clearance in adult tissues remain unclear. Here, we track cell death in the adult planarian Schmidtea mediterranea and find that, following light-induced cell death, pigment cell corpses transit to the gut and are excreted from the animal. Gut phagocytes, previously only known to phagocytose food, are required for pigment cells to enter the gut lumen. Finally, we show that the planarian ortholog of ced-12/engulfment and cell motility (ELMO) is required for corpse phagocytosis and removal through the gut. In total, we present a mechanism of cell clearance in an adult organism involving transit of dead cells to the gut, transport into the gut by phagocytes, and physical excretion of debris., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The freshwater planarian Dugesia japonica maintains an abundant heterogeneous cell population called neoblasts, which include adult pluripotent stem cells. Thus, it is an excellent model organism for stem cell and regeneration research. Recently, many single-cell RNA sequencing (scRNA-seq) databases of several model organisms, including other planarian species, have become publicly available; these are powerful and useful resources to search for gene expression in various tissues and cells. However, the only scRNA-seq dataset for D. japonica has been limited by the number of genes detected. Herein, we collected D. japonica cells, and conducted an scRNA-seq analysis. A novel, automatic, iterative cell clustering strategy produced a dataset of 3,404 cells, which could be classified into 63 cell types based on gene expression profiles. We introduced two examples for utilizing the scRNA-seq dataset in this study using D. japonica. First, the dataset provided results consistent with previous studies as well as novel functionally relevant insights, that is, the expression of DjMTA and DjP2X-A genes in neoblasts that give rise to differentiated cells. Second, we conducted an integrative analysis of the scRNA-seq dataset and time-course bulk RNA-seq of irradiated animals, demonstrating that the dataset can help interpret differentially expressed genes captured via bulk RNA-seq. Using the R package "Seurat" and GSE223927, researchers can easily access and utilize this dataset., (© 2023 Japanese Society of Developmental Biologists.)

Adult stem cells (ASCs) are pluripotent cells with the capacity to self-renew and constantly replace lost cells due to physiological turnover or injury. Understanding the molecular mechanisms of the precise coordination of stem cell proliferation and proper cell fate decision is important to regeneration and organismal homeostasis. The planarian epidermis provides a highly tractable model to study ASC complex dynamic due to the distinct spatiotemporal differentiation stages during lineage development. Here, we identified the myosin regulatory light chain (MRLC) homologue in the Dugesia japonica transcriptome. We found high expression levels of MRLC in wound region during regeneration and also expressed in late epidermal progenitors as an essential regulator of the lineage from neoblasts to mature epidermal cells. We investigated the function of MRLC using in situ hybridization, real-time polymerase chain reaction and double fluorescent and uncovered the potential mechanism. Knockdown of MRLC leads to a remarkable increase in cell death, causes severe abnormalities during regeneration and homeostasis and eventually leads to animal death. The global decrease in epidermal cell in MRLC RNAi animals induces accelerated epidermal proliferation and differentiation. Additionally, we find that MRLC is co-expressed with cdc42 and acts cooperatively to control the epidermal lineage development by affecting cell death. Our results uncover an important role of MRLC, as an inhibitor of apoptosis, involves in epidermal development., (© 2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Regenerative abilities vary dramatically across animals. Even amongst planarian flatworms, well-known for complete regeneration from tiny body fragments, some species have restricted regeneration abilities while others are almost entirely regeneration incompetent. Here, we assemble a diverse live collection of 40 planarian species to probe the evolution of head regeneration in the group. Combining quantification of species-specific head-regeneration abilities with a comprehensive transcriptome-based phylogeny reconstruction, we show multiple independent transitions between robust whole-body regeneration and restricted regeneration in freshwater species. RNA-mediated genetic interference inhibition of canonical Wnt signalling in RNA-mediated genetic interference-sensitive species bypassed all head-regeneration defects, suggesting that the Wnt pathway is linked to the emergence of planarian regeneration defects. Our finding that Wnt signalling has multiple roles in the reproductive system of the model species Schmidtea mediterranea raises the possibility that a trade-off between egg-laying, asexual reproduction by fission/regeneration and Wnt signalling drives regenerative trait evolution. Although quantitative comparisons of Wnt signalling levels, yolk content and reproductive strategy across our species collection remained inconclusive, they revealed divergent Wnt signalling roles in the reproductive system of planarians. Altogether, our study establishes planarians as a model taxon for comparative regeneration research and presents a framework for the mechanistic evolution of regenerative abilities., (© 2023. The Author(s).)

Regeneration requires mechanisms for producing a wide array of cell types. Neoblasts are stem cells in the planarian Schmidtea mediterranea that undergo fate specification to produce over 125 adult cell types. Fate specification in neoblasts can be regulated through expression of fate-specific transcription factors. We utilize multiplexed error-robust fluorescence in situ hybridization (MERFISH) and whole-mount FISH to characterize fate choice distribution of stem cells within planarians. Fate choices are often made distant from target tissues and in a highly intermingled manner, with neighboring neoblasts frequently making divergent fate choices for tissues of different location and function. We propose that pattern formation is driven primarily by the migratory assortment of progenitors from mixed and spatially distributed fate-specified stem cells and that fate choice involves stem-cell intrinsic processes., (© 2023. The Author(s).)

The origin of the sterility observed in ex-fissiparous freshwater planarians with hyperplasic ovaries has yet to be explained. To improve our understanding of this enigmatic phenomenon, immunofluorescence staining and confocal microscopy examination were used the assess autophagy, apoptosis, cytoskeleton, and epigenetics markers in the hyperplasic ovaries of ex-fissiparous individuals and the normal ovaries of sexual individuals. Immunofluorescence positivity for the autophagic marker microtubule-associated protein 1 light chain 3 (LC3) was significantly lower in the hyperplasic ovary than in the normal ovary. Compared with the normal ovary, the hyperplasic ovary exhibited significantly higher immunofluorescence positivity for the apoptotic marker caspase 3, suggesting that autophagy and apoptosis are closely associated in this pathogenicity. Furthermore, the level of global DNA (cytosine-5)-methyltransferase 3A (DNMT3) protein expression was significantly higher in the normal ovary than in the hyperplasic ovary, suggesting that DNA methylation is involved in the infertility phenomenon. The cytoskeleton marker actin also exhibited relatively higher immunofluorescence intensity in the normal ovary than in the hyperplasic ovary, consistent with previous findings on the role of cytoskeleton architecture in oocyte maturation. These results help improve our understanding of the causes of infertility in ex-fissiparous planarians with hyperplasic ovaries and provide new insights that will facilitate future studies on this mysterious pathogenicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

As stem cells divide, they acquire mutations that can be passed on to daughter cells. To mitigate potentially deleterious outcomes, cells activate the DNA damage response (DDR) network, which governs several cellular outcomes following DNA damage, including repairing DNA or undergoing apoptosis. At the helm of the DDR are three PI3-like kinases including Ataxia-Telangiectasia Mutated (ATM). We report here that knockdown of ATM in planarian flatworms enables stem cells to withstand lethal doses of radiation which would otherwise induce cell death. In this context, stem cells circumvent apoptosis, replicate their DNA, and recover function using homologous recombination-mediated DNA repair. Despite radiation exposure, atm knockdown animals survive long-term and regenerate new tissues. These effects occur independently of ATM's canonical downstream effector p53. Together, our results demonstrate that in planarians, ATM promotes radiation-induced apoptosis. This acute, ATM-dependent apoptosis is a key determinant of long-term animal survival. Our results suggest that inhibition of ATM in these organisms could, therefore, potentially favor cell survival after radiation without obvious effects on stem cell behavior., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Regeneration is the regrowth of damaged tissues or organs, a vital process in response to damages from primitive organisms to higher mammals. Planarian possesses active whole-body regenerative capability owing to its vast reservoir of adult stem cells, neoblasts, providing an ideal model to delineate the underlying mechanisms for regeneration. RNA N 6 -methyladenosine (m 6 A) modification participates in many biological processes, including stem cell self-renewal and differentiation, in particular the regeneration of haematopoietic stem cells and axons. However, how m 6 A controls regeneration at the whole-organism level remains largely unknown. Here, we demonstrate that the depletion of m 6 A methyltransferase regulatory subunit wtap abolishes planarian regeneration, potentially through regulating genes related to cell-cell communication and cell cycle. Single-cell RNA-seq (scRNA-seq) analysis unveils that the wtap knockdown induces a unique type of neural progenitor-like cells (NP-like cells), characterized by specific expression of the cell-cell communication ligand grn. Intriguingly, the depletion of m 6 A-modified transcripts grn, cdk9 or cdk7 partially rescues the defective regeneration of planarian caused by wtap knockdown. Overall, our study reveals an indispensable role of m 6 A modification in regulating whole-organism regeneration., (© 2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

The serine hydrolase acetylcholinesterase (AChE) is an important neuronal enzyme which catalyzes the hydrolysis of the neurotransmitter acetylcholine and other choline esters. The breakdown of acetylcholine by AChE terminates synaptic transmission and regulates neuromuscular communication. AChE inhibition is a common mode of action of various insecticides, such as carbamates and organophosphorus pesticides. Freshwater planarians, especially the species Dugesia japonica, have been shown to possess AChE activity and to be a suitable alternative model for studying the effects of pesticides in vivo. AChE activity can be quantified in homogenates using the Ellman assay. However, this biochemical assay requires specialized equipment and large numbers of planarians. Here, we present a protocol for visualizing AChE activity in individual planarians. Activity staining can be completed in several hours and can be executed using standard laboratory equipment (a fume hood, nutator, and light microscope with imaging capability). We describe the steps for preparing the reagents, and the staining and imaging of the planarians. Planarians are treated with 10% acetic acid and fixed with 4% paraformaldehyde and then incubated in a staining solution containing the substrate acetylthiocholine. After incubation in the staining solution for 3.5 hr on a nutator at 4°C, or stationary on ice, planarians are washed and mounted for imaging. Using exposure to an organophosphorus pesticide as an example, we show how AChE inhibition leads to a loss of staining. Thus, this simple method can be used to qualitatively evaluate AChE inhibition due to chemical exposure or RNA interference, providing a new tool for mechanistic studies of effects on the cholinergic system. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparing the staining solution Basic Protocol 2: Fixing, staining, and imaging whole-mount planarian specimens for visualization of acetylcholinesterase activity., (© 2023 Wiley Periodicals LLC.)

CK1α (Casein kinase 1α) is a member of the casein kinase 1(CK1) family that is involved in diverse cellular processes, but its functions remain unclear in stem cell development. Freshwater planarians are capable of whole-body regeneration, making it a classic model for the study of regeneration, tissue homeostasis, and polarity in vivo. To investigate the roles of CK1α in regeneration and homeostasis progress, we characterize a homolog of CK1α from planarian Dugesia japonica . We find that Djck1α , which shows an enriched expression pattern in the nascent tissues, is widely expressed especially in the medial regions of planarians. Knockdown of CK1α by RNAi presents a thicker body due to dorsal hyperplasia, along with defects in the medial tissues including nerve proliferation, missing epidermis, intestine disturbance, and hyper-proliferation during the progression of regeneration and homeostasis. Moreover, we find that the ck1α RNAi animals exhibit expansion of the midline marker slit . The eye deficiency induced by slit RNAi can be rescued by ck1α and slit double RNAi. These results suggest that ck1α is required for the medial tissue regeneration and maintenance in planarian Dugesia japonica by regulating the expression of slit , which helps to further investigate the regulation of planarian mediolateral axis.

Planarians have become a powerful model system for stem cell research and regeneration. While the tool kit for mechanistic investigations has been steadily expanding over the last decade, robust genetic tools for transgene expression are still lacking. We describe here methods for in vivo and in vitro mRNA transfection of the planarian species Schmidtea mediterranea. These methods utilize the commercially available TransIT-mRNA transfection reagent to efficiently deliver mRNA encoding a synthetic nanoluciferase reporter. Using a luminescent reporter overcomes the bright autofluorescent background of planarian tissues and allows quantitative measurements of protein expression levels. Collectively, our methods provide the means for heterologous reporter expression in planarian cells and the basis for future development of transgenic techniques., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Polybrominated diphenyl ethers (PBDEs) are widely used as brominated flame retardants in the manufacturing industry, belonging to persistent organic pollutants in the environment. Planarians are the freshwater worms, with strong regenerative ability and extreme sensitivity to environmental toxicants. This study aimed to evaluate the potential acute comprehensive effects of PBDE-47/-209 on freshwater planarians. Methods to detect the effects include: detection of oxidative stress, observation of morphology and histology, detection of DNA fragmentation, and detection of cell proliferation and apoptosis. In the PBDE-47 treatment group, planarians showed increased oxidative stress intensity, severe tissue damage, increased DNA fragmentation level, and increased cell proliferation and apoptosis. In the PBDE-209 treatment group, planarians showed decreased oxidative stress intensity, slight tissue damage, almost unchanged DNA fragmentation level and apoptosis, proliferation increased only on the first day after treatment. In conclusion, both PBDE-47 and PBDE-209 are dangerous environmental hazardous material that can disrupt planarians homeostasis, while the toxicity of PBDE-47 is sever than PBDE-209 that PBDE-47 can lead to the death of planarians., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Single-cell transcriptomics has revolutionised biology allowing the quantification of gene expression in individual cells. Since each single cell contains cell type specific mRNAs, these techniques enable the classification of cell identities. Therefore, single cell methods have been used to explore the repertoire of cell types (the single cell atlas) of different organisms, including freshwater planarians. Nowadays, planarians are one of the most prominent animal models in single cell biology. They have been studied at the single cell level for over a decade using most of the available single cell methodological approaches. These include plate-based methods, such as qPCR, nanodroplet methods and in situ barcoding methods. Because of these studies, we now have a very good picture of planarian cell types and their differentiation trajectories. Planarian regenerative properties and other characteristics, such as their developmental plasticity and their capacity to reproduce asexually, ensure that another decade of single cell biology in planarians is yet to come. Here, we review these characteristics, the new biological insights that have been obtained by single-cell transcriptomics and outline the perspectives for the future., (© 2022 The Author(s).)

Extracellular matrix (ECM) is an important component of stem cell niche. Remodeling of ECM mediated by ECM regulators, such as matrix metalloproteinases (MMPs) plays a vital role in stem cell function. However, the mechanisms that modulate the function of ECM regulators in the stem cell niche are understudied. Here, we explored the role of the transcription factor (TF) ETS-1, which is expressed in the cathepsin-positive cell population, in regulating the expression of the ECM regulator, mt-mmpA, thereby modulating basement membrane thickness. In planarians, the basement membrane around the gut/inner parenchyma is thought to act as a niche for pluripotent stem cells. It has been shown that the early epidermal progenitors migrate outwards from this region and progressively differentiate to maintain the terminal epidermis. Our data shows that thickening of the basement membrane in the absence of ets-1 results in defective migration of stem cell progeny. Furthermore, the absence of ets-1 leads to a defective epidermal progenitor landscape, despite its lack of expression in those cell types. Together, our results demonstrate the active role of ECM remodeling in regulating tissue homeostasis and regeneration in the planarian Schmidtea mediterranea. This article has an associated First Person interview with one of the co-first authors of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Planarian is an ideal model system of studying regeneration. Stem cell system and positional control genes (PCGs) are two important factors for perfect regeneration of planarians and they combine to promote their regeneration. Even so, how wounds regulate proliferation and neoblast fate is still important areas to address. Ptpn11 (Protein tyrosine phosphatase non-receptor type 11), one of PTP (Protein tyrosine phosphatase) family members, plays an important role in cellular processes including cell survival, proliferation, differentiation and apoptosis. Nevertheless, the role of ptpn11 in the planarian regeneration has not been fully studied. In this study, we identify the Djptpn11 gene to observe its function in planarian regeneration. The results reveal that the regeneration is severely inhibited and cause the disorder homeostasis in planarians. Furthermore, the stem cells proliferation and differentiation decreases while the apoptosis increases following Djptpn11 RNAi. At the same time, Djptpn11 affects the expression levels of early wound response genes (Djegr2, Dj1-jun, Djrunt1, Djwnt1 and Djnotum). Djwnt1 and Djnotum are two key Wnt signaling pathway genes and Djptpn11 affects the expression levels of Djwnt1 and Djnotum in the early and late stages of planarian regeneration. In general, Djptpn11 is indispensable for the homeostasis and regeneration of planarian by affecting the stem cells, early wound response genes and the Wnt pathway., Competing Interests: Declaration of competing interest This paper is not under simultaneous consideration by any other journal. And all the authors listed have seen the manuscript and approved to submit to your journal. The authors have declared that no competing interests exist., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

P53 is a widely studied tumor suppressor that plays important roles in cell-cycle regulation, cell death, and DNA damage repair. P53 is found throughout metazoans, even in invertebrates that do not develop malignancies. The prevailing theory for why these invertebrates possess a tumor suppressor is that P53 originally evolved to protect the germline of early metazoans from genotoxic stress such as ultraviolet radiation. This theory is largely based upon functional data from only three invertebrates, omitting important groups of animals including flatworms. Previous studies in the freshwater planarian flatworm Schmidtea mediterranea suggested that flatworm P53 plays an important role in stem cell maintenance and skin production, but these studies did not directly test for any tumor suppressor functions. To better understand the function of P53 homologs across diverse flatworms, we examined the function of two different P53 homologs in the parasitic flatworm Schistosoma mansoni . The first P53 homolog ( p53-1 ) is orthologous to S. mediterranea P53( Smed-p53 ) and human TP53 and regulates flatworm stem cell maintenance and skin production. The second P53 homolog ( p53-2 ) is a parasite-specific paralog that is conserved across parasitic flatworms and is required for the normal response to genotoxic stress in S. mansoni . We then found that Smed-p53 does not seem to play any role in the planarian response to genotoxic stress. The existence of this parasite-specific paralog that bears a tumor suppressor-like function in parasitic flatworms implies that the ability to respond to genotoxic stress in parasitic flatworms may have arisen from convergent evolution.

Background: Planarian has attracted increasing attentions in the regeneration field for its usefulness as an important biological model organism attributing to its strong regeneration ability. Both the complexity of multiple regulatory networks and their coordinate functions contribute to the maintenance of normal cellular homeostasis and the process of regeneration in planarian. The polarity, size, location and number of regeneration tissues are regulated by diverse mechanisms. In this review we summarize the recent advances about the importance genetic and molecular mechanisms for regeneration control on various tissues in planarian., Methods: A comprehensive literature search of original articles published in recent years was performed in regards to the molecular mechanism of each cell types during the planarian regeneration, including neoblast, nerve system, eye spot, excretory system and epidermal., Results: Available molecular mechanisms gave us an overview of regeneration process in every tissue. The sense of injuries and initiation of regeneration is regulated by diverse genes like follistatin and ERK signaling. The Neoblasts differentiate into tissue progenitors under the regulation of genes such as egfr-3. The regeneration polarity is controlled by Wnt pathway, BMP pathway and bioelectric signals. The neoblast within the blastema differentiate into desired cell types and regenerate the missing tissues. Those tissue specific genes regulate the tissue progenitor cells to differentiate into desired cell types to complete the regeneration process., Conclusion: All tissue types in planarian participate in the regeneration process regulated by distinct molecular factors and cellular signaling pathways. The neoblasts play vital roles in tissue regeneration and morphology maintenance. These studies provide new insights into the molecular mechanisms for regulating planarian regeneration., (© 2022 The Authors. Cell Proliferation published by European Cell Proliferation Society and John Wiley & Sons Ltd.)

Planarians have become an established model system to study regeneration and stem cells, but the regulatory elements in the genome remain almost entirely undescribed. Here, by integrating epigenetic and expression data we use multiple sources of evidence to predict enhancer elements active in the adult stem cell populations that drive regeneration. We have used ChIP-seq data to identify genomic regions with histone modifications consistent with enhancer activity, and ATAC-seq data to identify accessible chromatin. Overlapping these signals allowed for the identification of a set of high-confidence candidate enhancers predicted to be active in planarian adult stem cells. These enhancers are enriched for predicted transcription factor (TF) binding sites for TFs and TF families expressed in planarian adult stem cells. Footprinting analyses provided further evidence that these potential TF binding sites are likely to be occupied in adult stem cells. We integrated these analyses to build testable hypotheses for the regulatory function of TFs in stem cells, both with respect to how pluripotency might be regulated, and to how lineage differentiation programs are controlled. We found that our predicted GRNs were independently supported by existing TF RNAi/RNA-seq datasets, providing further evidence that our work predicts active enhancers that regulate adult stem cells and regenerative mechanisms., Competing Interests: JN, DS, AD, AA No competing interests declared, (© 2022, Neiro et al.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Heat shock protein 70 family (HSP70s) is one of the most conserved and important group of HSPs as molecular chaperones, which plays an important role in cytoprotection, anti-apoptosis and so on. However, the molecular mechanism of HSP70s in animal regeneration remains to be delineated. In this study, we investigate the roles of HSP70s in regeneration of planarian. The four genes, Djhsp70a, Djhsp70b, Djhsp70c, and Djhsp70d of the HSP70s, are selected from the transcriptome database, because of their high expression levels in planarians. We then study the biological roles of each gene by conducting various experimental techniques, including RNAi, RT-PCR, WISH, Whole-mount immunostaining and TUNEL. The results show: (1) External stressors, such as temperature, tissue damage and ionic liquid upregulate the expression of Djhsp70s significantly. (2) The gene expression of Djhsp70s in planarians exhibits dynamic patterns. According to the result of WISH, the Djhsp70s are mainly expressed in parenchymal tissues on both sides of the body as well as blastema. It is consistent with the data of qRT-PCR. (3) After RNA interference of Djhsp70s, the worms experience cephalic regression and lysis, body curling, stagnant regeneration and death. (4) Knockdown of Djhsp70s affect the cell proliferation and apoptosis. These results suggest that Djhsp70s are not only conserved in cytoprotection, but involved in homeostasis maintenance and regeneration process by regulating coordination of cell proliferation and apoptosis in planarians., (Copyright © 2022. Published by Elsevier B.V.)

Planaria are known for their ability to completely regenerate upon fissioning or experimental amputation. Yet, metabolic costs of regeneration have not been directly measured in planaria. Our goal was to establish the relationships between oxygen consumption (V̇O2), regeneration, and reproductive mode for asexual and sexual strains of Schmidtea mediterranea. We hypothesized that V̇O 2 would vary by regeneration day for both sexual and asexual S. mediterranea, reflecting different costs of tissue reconstruction, but with an additional cost for regenerating sexual organs. Testes regeneration and body mass, as indicators of regeneration progress, and routine mass-specific V̇O 2 as a function of maturity, regeneration, and reproductive mode, were measured over a 22-day regeneration period. Testes growth was highest in sexually mature adults, ~1/2 that in 14-day post-amputation sexual adults, and not detectable in juveniles and hatchlings. Mass-specific routine V̇O 2 in sexuals was highest in mature controls at ~23 μl O 2 /g/h, but only half that in juveniles, hatchlings, and 14 day post-amputation adults. Both intact and 14-day post-amputation asexuals had a mass-specific routine V̇O 2 of ~10-12 μl O 2 /g/h. The sum of V̇O 2 of all amputated sections was ~100% higher than pre-amputation levels in the first 6 days of regeneration in asexuals, but not sexuals. There was no significant difference in V̇O 2 of head, middle, and tail sections during regeneration. Overall, the highest metabolic costs associated with regeneration occurred during the initial 1-6 days of regeneration in both strains, but regeneration costs for sexual structures were not reflected in major V̇O 2 differences between sexual and asexual strains., (Copyright © 2021 Elsevier Inc. All rights reserved.)

O-Glycans on cell surfaces play important roles in cell-cell, cell-matrix and receptor-ligand interaction. Therefore, glycan-based interactions are important for tissue regeneration and homeostasis. Free-living flatworm Schmidtea mediterranea, because of its robust regenerative potential, is of great interest in the field of stem cell biology and tissue regeneration. Nevertheless, information on the composition and structure of O-glycans in planaria is unknown. Using mass spectrometry and in silico approaches, we characterized the glycome and the related transcriptome of mucin-type O-glycans of planarian S. mediterranea. Mucin-type O-glycans were composed of multiple isomeric, methylated, and unusually extended mono- and disubstituted O-N-acetylgalactosamine structures. Extensions made of hexoses and 3-O-methyl hexoses were the glycoforms observed. From glycotranscriptomic analysis, 60 genes belonging to five distinct enzyme classes were identified to be involved in mucin-type O-glycan biosynthesis. These genes shared homology with those in other invertebrate systems. Although a majority of the genes involved in mucin-type O-glycan biosynthesis were highly expressed during organogenesis and in differentiated cells, a few select genes in each enzyme class were specifically enriched during early embryogenesis. Our results indicate a unique temporal and spatial role for mucin-type O-glycans during embryogenesis and organogenesis and in adulthood. In summary, this is the first report on O-glycans in planaria. This study expands the structural and biosynthetic possibilities in cellular glycosylation in the invertebrate glycome and provides a framework towards understanding the biological role of mucin-type O-glycans in tissue regeneration using planarians., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Planarian flatworms are popular invertebrate models for basic research on stem cell biology and regeneration. These animals are commonly maintained on a diet of homogenized calf liver or boiled egg yolk in the laboratory, introducing a source of uncontrolled experimental variability., Results: Here, we report the development of defined diets, prepared entirely from standardized, commercially sourced ingredients, for the freshwater species Schmidtea mediterranea, Dugesia japonica, and Girardia dorotocephala. These food sources provide an opportunity to test the effects of specific nutritional variables on biological phenomena of interest. Defined diet consumption was not sufficient for growth and only partially induced the increase in stem cell division that normally accompanies feeding, suggesting these responses are not solely determined by caloric intake. Our defined diet formulations enable delivery of double-stranded RNA for gene knockdown in a manner that provides unique advantages in some experimental contexts. We also present a new approach for preserving tissue integrity during hydrogen peroxide bleaching of liver-fed animals., Conclusions: These tools will empower research on the connections between diet, metabolism, and stem cell biology in the experimentally tractable planarian system., (© 2021 American Association for Anatomy.)

The freshwater planarian Dugesia japonica is a good in vivo model for studying the function of piwi genes in adult pluripotent stem cell (aPSC) due to their abundant aPSCs. Generally, PIWI family proteins encoded by piwi genes bind to small noncoding RNAs called piRNAs (PIWI-interacting piRNAs). The analysis of PIWI-piRNA complexes in the planarian is useful for revealing the functions of piwi genes in the aPSC system. In this chapter, we present an immunoprecipitation protocol for PIWI-piRNA complexes from whole planarians., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Gravity alterations elicit complex and mostly detrimental effects on biological systems. Among these, a prominent role is occupied by oxidative stress, with consequences for tissue homeostasis and development. Studies in altered gravity are relevant for both Earth and space biomedicine, but their implementation using whole organisms is often troublesome. Here we utilize planarians, simple worm model for stem cell and regeneration biology, to characterize the pathogenic mechanisms brought by artificial gravity alterations. In particular, we provide a comprehensive evaluation of molecular responses in intact and regenerating specimens, and demonstrate a protective action from the space-apt for nanotechnological antioxidant cerium oxide nanoparticles., (© 2021 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

All animals, other than Platyhelminthes, produce eggs containing yolk, referred to as "entolecithal" eggs. However, only Neoophora, in the phylum Platyhelminthes, produce "ectolecithal" eggs (egg capsules), in which yolk is stored in the vitelline cells surrounding oocytes. Vitelline cells are derived from vitellaria (yolk glands). Vitellaria are important reproductive organs that may be studied to elucidate unique mechanisms that have been evolutionarily conserved within Platyhelminthes. Currently, only limited molecular level information is available on vitellaria. The current study identified major vitellaria-specific proteins in a freshwater planarian, Dugesia ryukyuensis, using peptide mass fingerprinting (PMF) and expression analyses. Amino acid sequence analysis and orthology analysis via OrthoFinder ver.2.3.8 indicated that the identified major vitellaria-specific novel yolk ferritins were conserved in planarians (Tricladida). Because ferritins play an important role in Fe (iron) storage, we examined the metal elements contained in vitellaria and ectolecithal eggs, using non-heme iron histochemistry, elemental analysis based on inductively coupled plasma mass spectrometry and transmission electron microscopy- energy-dispersive X-ray spectroscopy analysis. Interestingly, vitellaria and egg capsules contained large amounts of aluminum (Al), but not Fe. The knockdown of the yolk ferritin genes caused a decrease in the volume of egg capsules, abnormality in juveniles, and increase in Al content in vitellaria. Yolk ferritins of D. ryukyuensis may regulate Al concentration in vitellaria via their pooling function of Al and protect the egg capsule production and normal embryogenesis from Al toxicity., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

P-Element-induced wimpy testis (Piwi) subfamily proteins form complexes that bind to Piwi-interacting RNA. This interaction is crucial for stem cell regulation and formation, maintenance of germline stem cells, and gametogenesis in several metazoans. Planarians are effective models for studying stem cells. In the planarian Dugesia ryukyuensis, DrPiwi-1 is essential for the development of germ cells, but not somatic cells and sexual organs. DrPiwi-2 is indispensable for regeneration. In this study, we aimed to investigate the effects of Piwi on the differentiation of germ cells using monoclonal antibodies against DrPiwi-1 and DrPiwi-2. DrPiwi-1 and DrPiwi-2 co-localized more in immature germ cells than in mature germ cells in the ovary. DrPiwi-1 was found in the cytoplasm of early oogonia as undifferentiated germ cells, whereas DrPiwi-2 was found to localize not only in the nuclei but also in the cytoplasm of early oogonia. In descendant germ cells (oocytes), DrPiwi-2 was not present in the cytoplasm, but was strongly detected in the nucleolus. Moreover, we found that DrPiwi-1 forms a complex with DrPiwi-2. The cause of DrPiwi-1 depletion may be the severe reduction in the DrPiwi-2 level in the cytoplasm of oogonia. These results suggest that the formation of the DrPiwi-1 and DrPiwi-2 complex in the cytoplasm of oogonia is essential for oocyte differentiation. Our findings support the conclusion that DrPiwi-1 forms a complex with DrPiwi-2 in the cytoplasm of undifferentiated germ cells, and it signifies the start of gametogenesis. In contrast, in the testes, Drpiwi-1 was found in undifferentiated germ cells (spermatogonia), whereas DrPiwi-2 was found in descendant germ cells (spermatocytes). The process of germ cell differentiation from adult stem cells in planarians may be regulated in different ways in female and male germ lines by the Piwi family., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

In developmental biology, the regulation of stem cell plasticity and differentiation remains an open question. CBP(CREB-binding protein)/p300 is a conserved gene family that functions as a transcriptional co-activator and plays important roles in a wide range of cellular processes, including cell death, the DNA damage response, and tumorigenesis. The acetyl transferase activity of CBPs is particularly important, as histone and non-histone acetylation results in changes in chromatin architecture and protein activity that affect gene expression. Many studies have described the conserved functions of CBP/p300 in stem cell proliferation and differentiation. The planarian Schmidtea mediterranea is an excellent model for the in vivo study of the molecular mechanisms underlying stem cell differentiation during regeneration. However, how this process is regulated genetically and epigenetically is not well-understood yet. We identified 5 distinct Smed-cbp genes in S. mediterranea that show different expression patterns. Functional analyses revealed that Smed-cbp-2 appears to be essential for stem cell maintenance. On the other hand, the silencing of Smed-cbp-3 resulted in the growth of blastemas that were apparently normal, but remained largely unpigmented and undifferentiated. Smed-cbp-3 silencing also affected the differentiation of several cell lineages including neural, epidermal, digestive, and excretory cell types. Finally, we analysed the predicted interactomes of CBP-2 and CBP-3 as an initial step to better understand their functions in planarian stem cell biology. Our results indicate that planarian cbp genes play key roles in stem cell maintenance and differentiation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Under physiological conditions, the complex planarian neoblast system is a composite of hierarchically organized stem cell sub-populations with sigma-class neoblasts, including clonogenic neoblasts, endowed with larger self-renewal and differentiation capabilities, thus generating all the other sub-populations and dominating the regenerative process. This complex system responds to differentiated tissue demands, ensuring a continuous cell turnover in a way to replace aged specialized cells and maintain tissue functionality. Potency of the neoblast system can be appreciated under challenging conditions in which these stem cells are massively depleted and the few remaining repopulate the entire body, ensuring animal resilience. These challenging conditions offer the possibility to deepen the relationships among different neoblast sub-populations, allowing to expose uncanonical properties that are negligible under physiological conditions. In this paper, we employ short, sub-lethal 5-fluorouracil treatment to specifically affect proliferating cells passing through the S phase and demonstrate that S-phase slowdown triggers a shift in the transcriptional profile of sigma neoblasts, which reduces the expression of their hallmark sox-P1 . Later, some cells reactivate sox-P1 expression, suggesting that some neoblasts in the earlier steps of commitment could modulate their expression profile, reacquiring a wider differentiative potential.

The ability to respond to light has profoundly shaped life. Animals with eyes overwhelmingly rely on their visual circuits for mediating light-induced coordinated movements. Building on previously reported behaviors, we report the discovery of an organized, eye-independent (extraocular), body-wide photosensory framework that allows even a head-removed animal to move like an intact animal. Despite possessing sensitive cerebral eyes and a centralized brain that controls most behaviors, head-removed planarians show acute, coordinated ultraviolet-A (UV-A) aversive phototaxis. We find this eye-brain-independent phototaxis is mediated by two noncanonical rhabdomeric opsins, the first known function for this newly classified opsin-clade. We uncover a unique array of dual-opsin-expressing photoreceptor cells that line the periphery of animal body, are proximal to a body-wide nerve net, and mediate UV-A phototaxis by engaging multiple modes of locomotion. Unlike embryonically developing cerebral eyes that are functional when animals hatch, the body-wide photosensory array matures postembryonically in "adult-like animals." Notably, apart from head-removed phototaxis, the body-wide, extraocular sensory organization also impacts physiology of intact animals. Low-dose UV-A, but not visible light (ocular-stimulus), is able to arouse intact worms that have naturally cycled to an inactive/rest-like state. This wavelength selective, low-light arousal of resting animals is noncanonical-opsin dependent but eye independent. Our discovery of an autonomous, multifunctional, late-maturing, organized body-wide photosensory system establishes a paradigm in sensory biology and evolution of light sensing., Competing Interests: The authors declare no competing interest.

A strict coordination between pro- and antioxidative molecules is needed for normal animal physiology, although their exact function and dynamics during regeneration and development remains largely unknown. Via in vivo imaging, we were able to locate and discriminate between reactive oxygen species (ROS) in real-time during different physiological stages of the highly regenerative planarian Schmidtea mediterranea . All ROS signals were strong enough to overcome the detected autofluorescence. Combined with an in situ characterisation and quantification of the transcription of several antioxidant genes, our data showed that the planarian gut and epidermis have a well-equipped redox system. Pharmacological inhibition or RNA interference of either side of the redox balance resulted in alterations in the regeneration process, characterised by decreased blastema sizes and delayed neurodevelopment, thereby affecting tails more than heads. Focusing on glutathione, a central component in the redox balance, we found that it is highly present in planarians and that a significant reduction in glutathione content led to regenerative failure with tissue lesions, characterised by underlying stem cell alterations. This exploratory study indicates that ROS and antioxidants are tightly intertwined and should be studied as a whole to fully comprehend the function of the redox balance in animal physiology.

Mitochondrial state changes were shown to be critical for stem cell function. However, variation in the mitochondrial content in stem cells and the implication, if any, on differentiation is poorly understood. Here, using cellular and molecular studies, we show that the planarian pluripotent stem cells (PSCs) have low mitochondrial mass compared with their progenitors. Transplantation experiments provided functional validation that neoblasts with low mitochondrial mass are the true PSCs. Further, the mitochondrial mass correlated with OxPhos and inhibiting the transition to OxPhos dependent metabolism in cultured cells resulted in higher PSCs. In summary, we show that low mitochondrial mass is a hallmark of PSCs in planaria and provide a mechanism to isolate live, functionally active, PSCs from different cell cycle stages (G0/G1 and S, G2/M). Our study demonstrates that the change in mitochondrial metabolism, a feature of PSCs is conserved in planaria and highlights its role in organismal regeneration., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Chromatin modifications function as critical regulators of gene expression and cellular identity, especially in the regulation and maintenance of the pluripotent state. However, many studies of chromatin modification in stem cells-and pluripotent stem cells in particular-are performed in mammalian stem cell culture, an in vitro condition mimicking a very transient state during mammalian development. Thus, new models for studying pluripotent stem cells in vivo could be helpful for understanding the roles of chromatin modification, for confirming prior in vitro studies, and for exploring evolution of the pluripotent state. The freshwater flatworm, Schmidtea mediterranea, is an excellent model for studying adult pluripotent stem cells, particularly in the context of robust, whole-body regeneration. To identify chromatin modifying and remodeling enzymes critical for planarian regeneration and stem cell maintenance, we took a candidate approach and screened planarian homologs of 25 genes known to regulate chromatin biology in other organisms. Through our study, we identified six genes with novel functions in planarian homeostasis, regeneration, and behavior. Of the list of genes characterized, we identified five planarian homologs of the mammalian CREB-Binding Protein (CBP) and p300 family of histone acetyltransferases, representing an expansion of this family in planarians. We find that two planarian CBP family members are required for planarian survival, with knockdown of Smed-CBP2 and Smed-CBP3 causing distinct defects in stem cell maintenance or function. Loss of CBP2 causes a quick, dramatic loss of stem cells, while knockdown of CBP3 affects stem cells more narrowly, influencing differentiation of several cell types that include neuronal subtypes and cells of the eye. Further, we find that Smed-CBP1 is required for planarian fissioning behavior. We propose that the division of labor among a diversified CBP family in planarians presents an opportunity to dissect specific functions of a broadly important histone acetyltransferase family., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mechanical stress during cell migration may be a previously unappreciated source of genome instability, but the extent to which this happens in any animal in vivo remains unknown. We consider an in vivo system where the adult stem cells of planarian flatworms are required to migrate to a distal wound site. We observe a relationship between adult stem cell migration and ongoing DNA damage and repair during tissue regeneration. Migrating planarian stem cells undergo changes in nuclear shape and exhibit increased levels of DNA damage. Increased DNA damage levels reduce once stem cells reach the wound site. Stem cells in which DNA damage is induced prior to wounding take longer to initiate migration and migrating stem cell populations are more sensitive to further DNA damage than stationary stem cells. RNAi-mediated knockdown of DNA repair pathway components blocks normal stem cell migration, confirming that active DNA repair pathways are required to allow successful migration to a distal wound site. Together these findings provide evidence that levels of migration-coupled-DNA-damage are significant in adult stem cells and that ongoing migration requires DNA repair mechanisms. Our findings reveal that migration of normal stem cells in vivo represents an unappreciated source of damage, which could be a significant source of mutations in animals during development or during long-term tissue homeostasis., Competing Interests: SS, DS, PA, NK, AD, JT, MH, AA No competing interests declared, (© 2021, Sahu et al.)

The extracellular matrix (ECM) provides a precise physical and molecular environment for cell maintenance, self-renewal, and differentiation in the stem cell niche. However, the nature and organization of the ECM niche is not well understood. The adult freshwater planarian Schmidtea mediterranea maintains a large population of multipotent stem cells (neoblasts), presenting an ideal model to study the role of the ECM niche in stem cell regulation. Here we tested the function of 165 planarian homologs of ECM and ECM-related genes in neoblast regulation. We identified the collagen gene family as one with differential effects in promoting or suppressing proliferation of neoblasts. col4-1, encoding a type IV collagen α-chain, had the strongest effect. RNA interference (RNAi) of col4-1 impaired tissue maintenance and regeneration, causing tissue regression. Finally, we provide evidence for an interaction between type IV collagen, the discoidin domain receptor, and neuregulin-7 (NRG-7), which constitutes a mechanism to regulate the balance of symmetric and asymmetric division of neoblasts via the NRG-7/EGFR pathway., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

tRNA-derived fragments (tRFs) have recently gained a lot of scientific interest due to their diverse regulatory roles in several cellular processes. However, their function in dynamic biological processes such as development and regeneration remains unexplored. Here, we show that tRFs are dynamically expressed during planarian regeneration, suggesting a possible role for these small RNAs in the regulation of regeneration. In order to characterize planarian tRFs, we first annotated 457 tRNAs in S. mediterranea combining two tRNA prediction algorithms. Annotation of tRNAs facilitated the identification of three main species of tRFs in planarians-the shorter tRF-5s and itRFs, and the abundantly expressed 5'-tsRNAs. Spatial profiling of tRFs in sequential transverse sections of planarians revealed diverse expression patterns of these small RNAs, including those that are enriched in the head and pharyngeal regions. Expression analysis of these tRF species revealed dynamic expression of these small RNAs over the course of regeneration suggesting an important role in planarian anterior and posterior regeneration. Finally, we show that 5'-tsRNA in planaria interact with all three SMEDWI proteins and an involvement of AGO1 in the processing of itRFs. In summary, our findings implicate a novel role for tRFs in planarian regeneration, highlighting their importance in regulating complex systemic processes. Our study adds to the catalog of posttranscriptional regulatory systems in planaria, providing valuable insights on the biogenesis and the function of tRFs in neoblasts and planarian regeneration., (© 2021 Lakshmanan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Across the animal kingdom, adult tissue homeostasis is regulated by adult stem cell activity, which is commonly dysregulated in human cancers. However, identifying key regulators of stem cells in the milieu of thousands of genes dysregulated in a given cancer is challenging. Here, using a comparative genomics approach between planarian adult stem cells and patient-derived glioblastoma stem cells (GSCs), we identify and demonstrate the role of DEAD-box helicase DDX56 in regulating aspects of stemness in four stem cell systems: planarians, mouse neural stem cells, human GSCs, and a fly model of glioblastoma. In a human GSC line, DDX56 localizes to the nucleolus, and using planarians, when DDX56 is lost, stem cells dysregulate expression of ribosomal RNAs and lose nucleolar integrity prior to stem cell death. Together, a comparative genomic approach can be used to uncover conserved stemness regulators that are functional in both normal and cancer stem cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the planarian field, two techniques are mostly used for protein detection: immunohistochemistry (IHC) and western blotting. While IHC is great for visualizing the spatial distribution of proteins in whole organisms, it has limitations in antibody availability and issues related to nonspecific expression. The use of western blotting can circumvent nonspecific expression, providing a dependable way to quantify proteins of interest. Here, we present a standardized, easily reproducible protocol with details on protein extractions of whole planarians and western blotting. For complete details on the use and execution of this protocol, please refer to Ziman et al. (2020a)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Despite extensive research on molecular pathways controlling the process of regeneration in model organisms, little is known about the actual initiation signals necessary to induce regeneration. Recently, the activation of ERK signaling has been shown to be required to initiate regeneration in planarians. However, how ERK signaling is activated remains unknown. Reactive Oxygen Species (ROS) are well-known early signals necessary for regeneration in several models, including planarians. Still, the probable interplay between ROS and MAPK/ERK has not yet been described. Here, by interfering with major mediators (ROS, EGFR and MAPK/ERK), we were able to identify wound-induced ROS, and specifically H 2 O 2 , as upstream cues in the activation of regeneration. Our data demonstrate new relationships between regeneration-related ROS production and MAPK/ERK activation at the earliest regeneration stages, as well as the involvement of the EGFR-signaling pathway. Our results suggest that (1) ROS and/or H 2 O 2 have the potential to rescue regeneration after MEK-inhibition, either by H 2 O 2 -treatment or light therapy, (2) ROS and/or H 2 O 2 are required for the activation of MAPK/ERK signaling pathway, (3) the EGFR pathway can mediate ROS production and the activation of MAPK/ERK during planarian regeneration.

The extracellular matrix (ECM) is a three-dimensional network of macromolecules that provides a microenvironment capable of supporting and regulating cell functions. However, only a few research organisms are available for the systematic dissection of the composition and functions of the ECM, particularly during regeneration. We utilized the free-living flatworm Schmidtea mediterranea to develop an integrative approach consisting of decellularization, proteomics, and RNAi to characterize and investigate ECM functions during tissue homeostasis and regeneration. ECM-enriched samples were isolated from planarians, and their proteomes were characterized by LC-MS/MS. The functions of identified ECM components were interrogated using RNA interference. Using this approach, we found that heparan sulfate proteoglycan is essential for tissue regeneration. Our strategy provides an experimental approach for identifying both known and novel ECM components involved in regeneration., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background Information: Dense multicilia in protozoa and metazoa generate a strong force important for locomotion and extracellular fluid flow. During ciliogenesis, multiciliated cells produce hundreds of centrioles to serve as basal bodies through various pathways including deuterosome-dependent (DD), hyper-activated mother centriole-dependent (MCD) and basal bodydependent (BBD) pathways. The centrosome-free planarian Schmidtea mediterranea is widely used for regeneration studies because its neoblasts are capable of regenerating any body part after injury. However, it is currently unclear how the flatworms generate massive centrioles for multiciliated cells in the pharynx and body epidermis when their cells are initially centriole-free., Results: In this study, we investigate the progress of centriole amplification during the pharynx regeneration. We observe that the planarian pharyngeal epithelial cells generate their centrioles asynchronously through a de novo pathway. Most of the de novo centrioles are formed individually, whereas the remaining ones are assembled in pairs, possibly by sharing a cartwheel, or in small clusters lacking a nucleation center. Further RNAi experiments show that the known key factors of centriole duplication, including Cep152, Plk4 and Sas6, are crucial for the centriole amplification., Conclusions and Significance: Our study demonstrates the distinct process of massive centriole biogenesis in S. mediterranea and helps to understand the diversity of centriole biogenesis during evolution., (© 2020 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)