Mining of potentially stem cell-related miRNAs in planarians.

Stem cells and regenerative medicine have recently become important research topics. However, the complex stem cell regulatory networks involved in various microRNA (miRNA)-mediated mechanisms have not yet been fully elucidated. Planarians are ideal animal models for studying stem cells owing to their rich stem cell populations (neoblasts) and extremely strong regeneration capacity. The roles of planarian miRNAs in stem cells and regeneration have long attracted attention. However, previous studies have generally provided simple datasets lacking integrative analysis. Here, we have summarized the miRNA family reported in planarians and highlighted conservation in both sequence and function. Furthermore, we summarized miRNA data related to planarian stem cells and regeneration and screened potential involved candidates. Nevertheless, the roles of these miRNAs in planarian regeneration and stem cells remain unclear. The identification of potential stem cell-related miRNAs offers more precise suggestions and references for future investigations of miRNAs in planarians. Furthermore, it provides potential research avenues for understanding the mechanisms of stem cell regulatory networks. Finally, we compiled a summary of the experimental methods employed for studying planarian miRNAs, with the aim of highlighting special considerations in certain procedures and providing more convenient technical support for future research endeavors.

Planarian Mucus: A Novel Source of Pleiotropic Cytotoxic and Cytostatic Agents against Cancer Cells.

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.

Schild Analysis of the Interaction between Parthenolide and Cocaine Suggests an Allosteric Relationship for Their Effects on Planarian Motility.

The freshwater planarian is an emerging animal model in neuroscience due to its centralized nervous system that closely parallels closely parallels the nervous system of vertebrates. Cocaine, an abused drug, is the 'founding member' of the local anesthetic family. Parthenolide, a sesquiterpene lactone, acts as a behavioral and physiological antagonist of cocaine in planarians and rats, respectively. Previous work from our laboratory showed that both parthenolide and cocaine reduced planarian motility and that parthenolide reversed the cocaine-induced motility decrease at concentrations where parthenolide does not affect the movement of the worms. However, the exact mechanism of the cocaine/parthenolide antagonism is unknown. Here, we report the results of a Schild analysis to explore the parthenolide/cocaine relationship in the planarian Girardia tigrina. The Schild slopes of a family of concentration-response curves of parthenolide ± a single concentration of cocaine and vice versa were -0.55 and -0.36, respectively. These slopes were not statistically different from each other. Interestingly, the slope corresponding to the parthenolide ± cocaine (but not the cocaine ± parthenolide) data set was statistically different from -1. Our data suggest an allosteric relationship between cocaine and parthenolide for their effect on planarian motility. To the best of our knowledge, this is the first study about the mechanism of action of the antagonism between cocaine and parthenolide. Further studies are needed to determine the specific nature of the parthenolide/cocaine target(s) in this organism.

A comparative analysis of planarian genomes reveals regulatory conservation in the face of rapid structural divergence.

The planarian Schmidtea mediterranea is being studied as a model species for regeneration, but the assembly of planarian genomes remains challenging. Here, we report a high-quality haplotype-phased, chromosome-scale genome assembly of the sexual S2 strain of S. mediterranea and high-quality chromosome-scale assemblies of its three close relatives, S. polychroa, S. nova, and S. lugubris. Using hybrid gene annotations and optimized ATAC-seq and ChIP-seq protocols for regulatory element annotation, we provide valuable genome resources for the planarian research community and a first comparative perspective on planarian genome evolution. Our analyses reveal substantial divergence in protein-coding sequences and regulatory regions but considerable conservation within promoter and enhancer annotations. We also find frequent retrotransposon-associated chromosomal inversions and interchromosomal translocations within the genus Schmidtea and, remarkably, independent and nearly complete losses of ancestral metazoan synteny in Schmidtea and two other flatworm groups. Overall, our results suggest that platyhelminth genomes can evolve without syntenic constraints.

Cotinine influences the effect of high and low nicotine concentrations on planarian motility differently.

Previous work from our laboratory showed that cotinine, a nicotine metabolite, reverses three nicotine-induced behavioral effects in freshwater planarians: motility decrease, seizure-like movements, and withdrawal-like behaviors. The present work explored whether cotinine, a nicotine metabolite, antagonized the nicotine-induced effects on planarian motility in a concentration-dependent manner. We found that nicotine decreased planarian motility at nicotine concentrations above 60 µM but increased planarian velocity at concentrations equal to or below 50 µM, in agreement with previous data. Cotinine did not affect planarian motility at a concentration range between 250 and 2750 µM. Furthermore, we found that cotinine alleviated the 100 µM nicotine-induced motility decrease in a concentration-dependent manner and reversed the low nicotine concentration motility increase, albeit in a concentration-independent manner. The apparent concentration-dependent alleviation of >60 µM nicotine-induced motility decrease by cotinine suggests an orthosteric relationship between nicotine and cotinine. On the other hand, the evident concentration-independent cotinine alleviation of the increase in motility induced by 50 µM nicotine suggests an allosteric relationship. Our data is consistent with the existing literature about the relationship between nicotine and cotinine in various models, reinforcing the case for the usefulness of the planarian model in pharmacological studies.

Adaptive robustness through incoherent signaling mechanisms in a regenerative brain.

Animal behavior emerges from collective dynamics of neurons, making it vulnerable to damage. Paradoxically, many organisms exhibit a remarkable ability to maintain significant behavior even after large-scale neural injury. Molecular underpinnings of this extreme robustness remain largely unknown. Here, we develop a quantitative pipeline to measure long-lasting latent states in planarian flatworm behaviors during whole-brain regeneration. By combining >20,000 animal trials with neural network modeling, we show that long-range volumetric peptidergic signals allow the planarian to rapidly restore coarse behavior output after large perturbations to the nervous system, while slow restoration of small-molecule neuromodulator functions refines precision. This relies on the different time and length scales of neuropeptide and small-molecule transmission to generate incoherent patterns of neural activity that competitively regulate behavior. Controlling behavior through opposing communication mechanisms creates a more robust system than either alone and may serve as a generalizable approach for constructing robust neural networks.

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

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

Morphometric analysis and functional insights into the serotonergic system of Girardia tigrina (Tricladida, Platyhelminthes).

Using immunocytochemistry, serotonergic nerve elements were documented in the nervous system of the planarian Girardia tigrina. Serotonin-immunopositive components were observed in the brain, ventral, dorsal and longitudinal nerve cords, transverse nerve commissures connecting the nerve cords, and in the nerve plexus. Whole-mount preparations of G. tigrina were analyzed by fluorescent and confocal laser scanning microscopy. An essential quantitative morphometric measurement of serotonin-immunopositive structures was conducted in three body regions (anterior, middle, and posterior) of the planarian. The number of serotonin neurons was maximal in the head region. The ventral nerve cords gradually decreased in thickness from anterior to posterior body ends. Physiological action of exogenously applied serotonin was studied in G. tigrina for the first time. It was found that serotonin (0.1 and 1 µmol L-1) accelerated eye regeneration. The transcriptome sequencing performed for the first time for the planarian G. tigrina revealed the transcripts of the tryptophan hydroxylase (trph), amino acid decarboxylase (aadc) and serotonin transporter (sert) genes. The data obtained indicate the presence of the components of serotonin pathway in G. tigrina. The identified transcripts can take part in serotonin turnover and participate in the realization of biological effects of serotonin in planarians, associated with eyes regeneration and differentiation.

Planarian LDB and SSDP proteins scaffold transcriptional complexes for regeneration and patterning.

Sequence-specific transcription factors often function as components of large regulatory complexes. LIM-domain binding protein (LDB) and single-stranded DNA-binding protein (SSDP) function as core scaffolds of transcriptional complexes in animals and plants. Little is known about potential partners and functions for LDB/SSDP complexes in the context of tissue regeneration. In this work, we find that planarian LDB1 and SSDP2 promote tissue regeneration, with a particular function in anterior regeneration and mediolateral polarity reestablishment. We find that LDB1 and SSDP2 interact with one another and with characterized planarian LIM-HD proteins Arrowhead, Islet1, and Lhx1/5-1. We also show that SSDP2 and LDB1 function with islet1 in polarity reestablishment and with lhx1/5-1 in serotonergic neuron maturation. Finally, we find new roles for LDB1 and SSDP2 in regulating gene expression in the planarian intestine and parenchyma; these functions are likely LIM-HD-independent. Together, our work provides insight into LDB/SSDP complexes in a highly regenerative organism. Further, our work provides a strong starting point for identifying and characterizing potential binding partners of LDB1 and SSDP2 and for exploring roles for these proteins in diverse aspects of planarian physiology.

Neurophysiological measurements of planarian brain activity: a unique model for neuroscience research.

Planarians are well-known model organisms for regeneration and developmental biology research due to their remarkable regenerative capacity. Here, we aim to advocate for the use of planaria as a valuable model for neurobiology, as well. Planarians have most of the major qualities of more developed organisms, including a primal brain. These traits combined with their exceptional regeneration capabilities, allow neurobiological experiments not possible in any other model organism, as we demonstrate by electrophysiological recording from planaria with two heads that control a shared body. To facilitate planarian neuroscience research, we developed an extracellular multi-unit recording procedure for the planarians fragile brain (Dugesia japonica). We created a semi-intact preparation restrained with fine dissection pins, enabling hours of reliable recording, via a suction electrode. Here, we demonstrate the feasibility and potential of planarian neurophysiological research by characterizing the neuronal activity during simple learning processes and responses to various stimuli. In addition, we examined the use of linalool as anesthetic agent to allows recordings from an intact, large worm and for fine electrophysiological approaches such as intracellular recording. The demonstrated ability for neurophysiological measurements, along with the inherent advantages of planarians, promotes this exceptional model organism for neuroscience research.

Purification of Planarian Stem Cells Using a Draq5-Based FACS Approach.

Planarians are flatworms that have the remarkable ability to regenerate entirely new animals. This regenerative ability requires abundant adult stem cells called neoblasts, which are relatively small in size, sensitive to irradiation and the only proliferative cells in the animal. Despite the lack of cell surface markers, fluorescence-activated cell sorting (FACS) protocols have been developed to discriminate and isolate neoblasts, based on DNA content. Here, we describe a protocol that combines staining of far-red DNA dye Draq5, Calcein-AM and DAPI, along with a shortened processing time. This profiling strategy can be used to functionally characterize the neoblast population in pharmacologically-treated or gene knockdown animals. Highly purified neoblasts can be analyzed with downstream assays, such as in situ hybridization and RNA sequencing.

Behavioral and physiological responses of Girardia tigrina exposed to polyethylene microplastics.

Microplastic particles appear in great abundance and variety in freshwater ecosystems across the globe, spanning lakes and rivers, with increasingly frequent exposure of aquatic organisms. Studies on the mechanisms of any effects of plastic particles are still scarce, particularly in relation to the regenerative capacity of biota, for which there is no established model organism; however, planaria have shown sensitivity for assessing these risks to the aquatic environment. Thus, the present study aimed to investigate the behavioral and regeneration responses of the freshwater planaria Girardia tigrina exposed to polyethylene (PE) microplastics (MPs) incorporated into their food source. The greatest effect was observed on planarian regeneration, which was manifested at 10 µg/mg liver. Planaria reproduction and fertility were affected at 50 µg/mg liver; however, planaria locomotion was not affected at the concentrations evaluated. Mid-infrared absorption spectroscopy (FT-IR) was used to identify the constituent polymers, and ingestion of the polyethylene microplastic by the planaria was confirmed by infrared spectroscopy. The results highlight the potential adverse effects of exposure to polyethylene microplastic and show that the reproductive behavior and regeneration of a freshwater organism can be indicators of toxicity resulting from environmental pollution.

Single-cell genomic profiling to study regeneration.

Regenerative capacities and strategies vary dramatically across animals, as well as between cell types, organs, and with age. In recent years, high-throughput single-cell transcriptomics and other single-cell profiling technologies have been applied to many animal models to gain an understanding of the cellular and molecular mechanisms underlying regeneration. Here, we review recent single-cell studies of regeneration in diverse contexts and summarize key concepts that have emerged. The immense regenerative capacity of some invertebrates, exemplified by planarians, is driven mainly by the differentiation of abundant adult pluripotent stem cells, whereas in many other cases, regeneration involves the reactivation of embryonic or developmental gene-regulatory networks in differentiated cell types. However, regeneration also differs from development in many ways, including the use of regeneration-specific cell types and gene regulatory networks.

Epidermal turnover in the planarian Schmidtea mediterranea involves basal cell extrusion and intestinal digestion.

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.

Ecotoxicological evaluation and regeneration impairment of planarians by dibutyl phthalate.

Commonly utilized as a plasticizer in the food and chemical sectors, Dibutyl phthalate (DBP) poses threats to the environment and human well-being as it seeps or moves into the surroundings. Nevertheless, research on the harmfulness of DBP to aquatic organisms is limited, and its impact on stem cells and tissue regeneration remains unidentified. Planarians, recognized for their robust regenerative capabilities and sensitivity to aquatic pollutants, are emerging animal models in toxicology. This study investigated the comprehensive toxicity effects of environmentally relevant levels of DBP on planarians. It revealed potential toxicity mechanisms through the use of immunofluorescence, chromatin dispersion assay, Western blot, quantitative real-time fluorescence quantitative PCR (qRT-PCR), chromatin behavioral and histological analyses, immunofluorescence, and terminal dUTP nickel-end labeling (TUNEL). Findings illustrated that DBP caused morphological and motor abnormalities, tissue damage, regenerative inhibition, and developmental neurotoxicity. Further research revealed increased apoptosis and suppressed stem cell proliferation and differentiation, disrupting a balance of cell proliferation and death, ultimately leading to morphological defects and functional abnormalities. This was attributed to oxidative stress and DNA damage caused by excessive release of reactive oxygen species (ROS). This exploration furnishes fresh perspectives on evaluating the toxicity peril posed by DBP in aquatic organisms.

Theoretical insights, degradation, and sub-lethal toxicity of thiamethoxam to the planarian Girardia tigrina.

Advanced oxidative processes, such as Photo-Fenton, transform organic contaminants due to the attack by radicals. In this context, the lethal and sub-lethal effects of the Cruiser® 350FS (CRZ) with the active ingredient thiamethoxam (TMX) were investigated using the planarian Girardia tigrina. Degradation of thiamethoxam by the Fenton process was also assessed by using theoretical studies and the efficiency of Solar-Fenton versus Fenton. The 48 h LC50 value of CRZ for planarians was 478.6 mg L-1. The regeneration of planarians was significantly affected for concentrations ≥ 17 mg·L-1 of TMX (24 h). The Solar-Fenton showed a high degradation percentage reaching ~70%. The theoretical model showed the atoms of the TMX molecule that will suffer attacks from the formed radicals. Current results open new perspectives concerning the treatment of TMX in the aquatic environment because the 70% degradation seems to be sufficient to reach concentrations that do not induce sub-lethal effects in planarians. Further studies should determine if the by-products generated might be toxic for planaria or other organisms.

A niche-derived nonribosomal peptide triggers planarian sexual development.

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 ß-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.

A PAK family kinase and the Hippo/Yorkie pathway modulate WNT signaling to functionally integrate body axes during regeneration.

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/ß-catenin signaling along the AP axis and, functions synergistically with the ß-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.

SoxB family genes delay regeneration and cause abnormal movement in Dugesia japonica.

SoxB subfamily is an important branch of Sox family and plays a key role in animal physiological process, but little is known about their function in planarian regeneration. This study aims to evaluate the function of DjSoxB family genes in intact and regenerating planarians Dugesia japonica. Here, we amplify the full-length cDNA of DjSoxB1 and DjSoxB2 in D. japonica by rapid amplification of the cDNA ends (RACE), detect the expression of DjSoxB family genes in planarian. The results show that DjSoxBs are expressed in parenchymal tissue and the hybridization signals partially disappear after irradiation indicates DjSoxB family genes are expressed in neoblasts. After the RNA interference (RNAi) of DjSoxB1, DjSoxB2 and DjSoxB3 separately, the numbers of proliferative cells are all reduced that causes planarians show slower growth of blastema in the early stage of regeneration, and nerves of planarians are affected that the movement speed of planarians decreases in varying degrees. Specially, planarians in the DjSoxB3 RNAi group show shrinkage and twisting. Overall, this study reveals that DjSoxB family genes play a role in cell proliferation during regeneration. They also play an important role in the maintenance of normal nerve function and nerve regeneration. These results provide directions for the functional study of SoxB family genes and provide an important foundation for planarian regeneration.

The people behind the papers - Jason Ko and Daniel Lobo.

Planarians grow when they are fed and shrink during periods of starvation. However, it is unclear how they maintain appropriate body proportions as their size changes. A new paper in Development investigates the differences between growth and shrinkage dynamics and builds a mathematical model to explore the mechanisms underpinning these two processes. To learn more about the story behind the paper, we caught up with first author, Jason Ko, and corresponding author, Daniel Lobo, Associate Professor at the University of Maryland.