A transcription factor atlas of stem cell fate in planarians.

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/G2/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.

Protocol for culturing and functionally manipulating planarian neoblasts using SiR-DNA-based flow cytometry.

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.

Planarians require ced-12/elmo-1 to clear dead cells by excretion through the gut.

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.

A resource of single-cell gene expression profiles in a planarian Dugesia japonica.

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.

MRLC controls apoptotic cell death and functions to regulate epidermal development during planarian regeneration and homeostasis.

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.

Fate specification is spatially intermingled across planarian stem cells.

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.

Evolutionary dynamics of whole-body regeneration across planarian flatworms.

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.

Genome-Wide Analysis of Planarian piRNAs.

In planarian flatworms, the piRNA pathway is operated by three PIWI proteins, termed SMEDWI-1, SMEDWI-2, and SMEDWI-3 (SMEDWI = Schmidtea mediterranea PIWI). The interplay between these three PIWI proteins and their associated small noncoding RNAs, termed piRNAs, fuels the outstanding regenerative abilities of planarians, enables tissue homeostasis, and, ultimately, ensures animal survival. As the molecular targets of PIWI proteins are determined by the sequences of their co-bound piRNAs, it is imperative to identify these sequences by next-generation sequencing applications. Following sequencing, the genomic targets and the regulatory potential of the isolated piRNA populations need to be uncovered. To that end, here we present a bioinformatics analysis pipeline for processing and systematic characterization of planarian piRNAs. The pipeline includes steps for the removal of PCR duplicates based on unique molecular identifier (UMI) sequences, and it accounts for piRNA multimapping to different loci in the genome. Importantly, our protocol also includes a fully automated pipeline that is freely available at GitHub. Together with the piRNA isolation and library preparation protocol (see accompanying chapter), the presented computational pipeline enables researchers to explore the functional role of the piRNA pathway in flatworm biology.

mRNA Transfection of S. mediterranea for Luminescence Analysis.

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.

Using autophagy, apoptosis, cytoskeleton, and epigenetics markers to investigate the origin of infertility in ex-fissiparous freshwater planarian individuals (nomen nudum species) with hyperplasic ovaries.

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.

m6 A promotes planarian regeneration.

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 N6 -methyladenosine (m6 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 m6 A controls regeneration at the whole-organism level remains largely unknown. Here, we demonstrate that the depletion of m6 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 m6 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 m6 A modification in regulating whole-organism regeneration.

Inhibition of ATM kinase rescues planarian regeneration after lethal radiation.

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.

Acetylcholinesterase Activity Staining in Freshwater Planarians.

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.

Djck1α Is Required for Proper Regeneration and Maintenance of the Medial Tissues in Planarians.

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.

PBDEs disrupt homeostasis maintenance and regeneration of planarians due to DNA damage, proliferation and apoptosis anomaly.

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.

Single-cell transcriptomics in planaria: new tools allow new insights into cellular and evolutionary features.

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.

S. mediterranea ETS-1 regulates the function of cathepsin-positive cells and the epidermal lineage landscape via basement membrane remodeling.

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.

Convergent evolution of a genotoxic stress response in a parasite-specific p53 homolog.

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.

Identification of putative enhancer-like elements predicts regulatory networks active in planarian adult stem cells.

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.

Djptpn11 is indispensable for planarian regeneration by affecting early wound response genes expression and the Wnt pathway.

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.