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.

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.

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 controlling 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.

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.

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.

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.

Conserved Genes in Highly Regenerative Metazoans Are Associated with Planarian Regeneration.

Metazoan species depict a wide spectrum of regeneration ability which calls into question the evolutionary origins of the underlying processes. Since species with high regeneration ability are widely distributed throughout metazoans, there is a possibility that the metazoan ancestor had an underlying common molecular mechanism. Early metazoans like sponges possess high regenerative ability, but, due to the large differences they have with Cnidaria and Bilateria regarding symmetry and neuronal systems, it can be inferred that this regenerative ability is different. We hypothesized that the last common ancestor of Cnidaria and Bilateria possessed remarkable regenerative ability which was lost during evolution. We separated Cnidaria and Bilateria into three classes possessing whole-body regenerating, high regenerative ability, and low regenerative ability. Using a multiway BLAST and gene phylogeny approach, we identified genes conserved in whole-body regenerating species and lost in low regenerative ability species and labeled them Cnidaria and Bilaterian regeneration genes. Through transcription factor analysis, we identified that Cnidaria and Bilaterian regeneration genes were associated with an overabundance of homeodomain regulatory elements. RNA interference of Cnidaria and Bilaterian regeneration genes resulted in loss of regeneration phenotype for HRJDa, HRJDb, DUF21, DISP3, and ARMR genes. We observed that DUF21 knockdown was highly lethal in the early stages of regeneration indicating a potential role in wound response. Also, HRJDa, HRJDb, DISP3, and ARMR knockdown showed loss of regeneration phenotype after second amputation. The results strongly correlate with their respective RNA-seq profiles. We propose that Cnidaria and Bilaterian regeneration genes play a major role in regeneration across highly regenerative Cnidaria and Bilateria.

Exposure to polystyrene microplastics and perfluorooctane sulfonate disrupt the homeostasis of intact planarians and the growth of regenerating planarians.

Microplastics (MPs) and perfluorinated compounds (PFAS) are widespread in the global ecosystem. MPs have the ability to adsorb organic contaminants such as perfluorooctane sulfonate (PFOS), leading to combined effects. The current work aims to explore the individual and combined toxicological effects of polystyrene (PS) and PFOS on the growth and nerves of the freshwater planarian (Dugesia japonica). The results showed that PS particles could adsorb PFOS. PS and PFOS impeded the regeneration of decapitated planarians eyespots, whereas the combined treatment increased the locomotor speed of intact planarians. PS and PFOS caused significant DNA damage, while co-treatment with different PS concentrations aggravated and attenuated DNA damage, respectively. Further studies at the molecular level have shown that PS and PFOS affect the proliferation and differentiation of neoblasts in both intact and regenerating planarians, alter the expression levels of neuronal genes, and impede the development of the nervous system. PS and PFOS not only disrupted the homeostasis of intact planarians, but also inhibited the regeneration of decapitated planarians. This study is the first to assess the multiple toxicity of PS and PFOS to planarians after combined exposure. It provides a basis for the environmental and human health risks of MPs and PFAS.

Identification and characterization of TatD DNase in planarian Dugesia japonica and its antibiofilm effect.

TatD DNase, a key enzyme in vertebrates and invertebrates, plays a pivotal role in various physiological processes. Dugesia japonica (D. japonica), a flatworm species, has remarkable regenerative capabilities and possesses a simplified immune system. However, the existence and biological functions of TatD DNase in D. japonica require further investigation. Here, we obtained the open reading frame (ORF) of DjTatD and demonstrated its conservation. The three-dimensional structure of DjTatD revealed its active site and binding mechanism. To investigate its enzymological properties, we overexpressed, purified, and characterized recombinant DjTatD (rDjTatD). We observed that DjTatD was primarily expressed in the pharynx and its expression could be significantly challenged upon stimulation with lipopolysaccharide, peptidoglycan, gram-positive and gram-negative bacteria. RNA interference results indicated that both DjTatD and DjDN2s play a role in pharyngeal regeneration and may serve as functional complements to each other. Additionally, we found that rDjTatD and recombinant T7DjTatD effectively reduce biofilm formation regardless of their bacterial origin. Together, our results demonstrated that DjTatD may be involved in the planarian immune response and pharyngeal regeneration. Furthermore, after further optimization in the future, rDjTatD and T7DjTatD can be considered highly effective antibiofilm agents.

Mutational profile of the regenerative process and de novo genome assembly of the planarian Schmidtea polychroa.

Planarians are organisms with a unique capacity to regenerate any part of their body. New tissues are generated in a process that requires many swift cell divisions. How costly is this process to an animal in terms of mutational load remains unknown. Using whole genome sequencing, we defined the mutational profile of the process of regeneration in the planarian species Schmidtea polychroa. We assembled de novo the genome of S. polychroa and analyzed mutations in animals that have undergone regeneration. We observed a threefold increase in the number of mutations and an altered mutational spectrum. High allele frequencies of subclonal mutations in regenerated animals suggested that most of the cells in the regenerated animal were descendants of a small number of stem cells with high expansion potential. We provide, for the first time, the draft genome assembly of S. polychroa, an estimation of the germline mutation rate for a planarian species and the mutational spectrum of the regeneration process of a living organism.

Ultrafast distant wound response is essential for whole-body regeneration.

Injury induces systemic responses, but their functions remain elusive. Mechanisms that can rapidly synchronize wound responses through long distances are also mostly unknown. Using planarian flatworms capable of whole-body regeneration, we report that injury induces extracellular signal-regulated kinase (Erk) activity waves to travel at a speed 10-100 times faster than those in other multicellular tissues. This ultrafast propagation requires longitudinal body-wall muscles, elongated cells forming dense parallel tracks running the length of the organism. The morphological properties of muscles allow them to act as superhighways for propagating and disseminating wound signals. Inhibiting Erk propagation prevents tissues distant to the wound from responding and blocks regeneration, which can be rescued by a second injury to distal tissues shortly after the first injury. Our findings provide a mechanism for long-range signal propagation in large, complex tissues to coordinate responses across cell types and highlight the function of feedback between spatially separated tissues during whole-body regeneration.

ACME Dissociation-Fixation, Flow Cytometry, and Cell Sorting of Freshwater Planarian Cells.

Planarian cell dissociation methods using enzymatic approaches are well established and have been widely used in the field. However, their use in transcriptomics and especially single-cell transcriptomics raises concerns as cells are dissociated alive, and this induces cellular stress responses. Here we describe a protocol for planarian cell dissociation using ACME, a dissociation-fixation approach based on acetic acid and methanol. ACME-dissociated cells are fixed, can be cryopreserved, and are amenable to modern methods of single-cell transcriptomics.

Live Imaging in Planarians: Immobilization and Real-Time Visualization of Reactive Oxygen Species.

Imaging of living animals allows the study of metabolic processes in relation to cellular structures or larger functional entities. To enable in vivo imaging during long-term time-lapses in planarians, we combined and optimized existing protocols, resulting in an easily reproducible and inexpensive procedure. Immobilization with low-melting-point agarose eliminates the use of anesthetics, avoids interfering with the animal during imaging-functionally or physically-and allows recovering the organisms after the imaging procedure. As an example, we used the immobilization workflow to image the highly dynamic and fast-changing reactive oxygen species (ROS) in living animals. These reactive signaling molecules can only be studied in vivo and mapping their location and dynamics during different physiological conditions is crucial to understand their role in developmental processes and regeneration. In the current protocol, we describe both the immobilization and ROS detection procedure. We used the intensity of the signals together with pharmacological inhibitors to validate the signal specificity and to distinguish it from the autofluorescent nature of the planarian.

TUNEL Staining in Sections of Paraffin-Enabled Planarians.

Planarians are a model animal for the study of regeneration and homeostasis. Understanding how planarians control their cellular balance is key to the knowledge of their plasticity. Both apoptotic and mitotic rates can be quantified in "whole mount" planarians. Apoptosis is usually analyzed through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), a technique that detects cell death by identifying DNA breaks. In this chapter we detail a protocol to analyze apoptotic cells in paraffin sections of planarians, which enables a more accurate cellular visualization and quantification than in "whole mount."

Optimization and calibration of behavioural tests on different species of planaria for ecotoxicological studies.

Freshwater planarian are emerging as a valuable in vivo model for (eco) toxicological studies, but the lack of harmonization of procedures between laboratories remains a challenge. This study aimed to optimize, automate and select the best behavioural tests and analyse the potential of different planarian species as models for toxicological assessment. We implemented four tests: exploration, photomotor response, Tapping and Planarian Light Dark Test, on different planaria species using the DanioVision system. We conclude that the exploration assay performed in 24 well-plate at 10,000 lux is the one that is robust and reliable for toxicological studies with planaria. Dugesia japonica and Schmidtea mediterranea have proved to be sensitive models for toxicological screening of organophosphorus pesticides through behavioural analysis. Under necessary experimental conditions, the motility baseline in controls, for both species allowed the detection of behavioural changes, making both good models for behavioural testing in (eco) toxicological context.

Neurotoxicological mechanisms of carbon quantum dots in a new animal model Dugesia japonica.

As luminescent nanomaterials, the carbon quantum dots (CQDs) research focused on emerging applications since their discovery. However, their toxicological effects on the natural environment are still unclear. The freshwater planarian Dugesia japonica is distributed extensively in aquatic ecosystems and can regenerate a new brain in 5 days after amputation. Therefore it can be used as a new model organism in the field of neuroregeneration toxicology. In our study, D. japonica was cut and incubated in medium treated with CQDs. The results showed that the injured planarian lost the neuronal ability of brain regeneration after treatment with CQDs. Its Hh signalling system was interfered with at Day 5, and all cultured pieces died on or before Day 10 due to head lysis. Our work reveals that CQDs might affect the nerve regeneration of freshwater planarians via the Hh signalling pathway. The results of this study improve our understanding of CQD neuronal development toxicology and can aid in the development of warning systems for aquatic ecosystem damage.

Characterization of the planarian surface electroencephalogram.

BACKGROUND: Despite large morphological differences between the nervous systems of lower animals and humans, striking functional similarities have been reported. However, little is known about how these functional similarities translate to cognitive similarities. As a first step towards studying the cognitive abilities of simple nervous systems, we here characterize the ongoing electrophysiological activity of the planarian Schmidtea mediterranea. One previous report using invasive microelectrodes describes that the ongoing neural activity is characterized by a 1/fx power spectrum with the exponent 'x' of the power spectrum close to 1. To extend these findings, we aimed to establish a recording protocol to measure ongoing neural activity safely and securely from alive and healthy planarians under different lighting conditions using non-invasive surface electrodes. RESULTS: As a replication and extension of the previous results, we show that the ongoing neural activity is characterized by a 1/fx power spectrum, that the exponent 'x' in living planarians is close to 1, and that changes in lighting induce changes in neural activity likely due to the planarian photophobia. CONCLUSIONS: We confirm the existence of continuous EEG activity in planarians and show that it is possible to noninvasively record this activity with surface wire electrodes. This opens up broad possibilities for continuous recordings across longer intervals, and repeated recordings from the same animals to study cognitive processes.

Multiple toxicity evaluations of perfluorooctane sulfonate on intact planarian Dugesia japonica.

Perfluorooctane sulfonate (PFOS) is gaining widespread attention as a persistent organic pollutant with multiple mechanisms of toxicity. In this study, PFOS at different concentrations and different exposure times was used to evaluate the multiple toxicities on intact planarian Dugesia japonica. The proliferation of neoblasts, apoptosis, DNA damage and the expression levels of neuronal genes and the major genes of the Wnt pathway were effectively studied. The results demonstrated that the balance between proliferation and apoptosis of intact planarian cells was disrupted after PFOS exposure, which in turn affected tissue homeostasis and differentiation. PFOS exposure led to increased DNA damage and altered neuronal gene expression. In addition, PFOS exposure could down-regulate the expression of Wnt pathway genes, but the inhibition of the Wnt pathway by PFOS was time- and concentration-dependent. These findings suggest that PFOS has multiple toxic effects on planarians and may interfere with cell proliferation and neurodevelopment by affecting the key gene expression in the Wnt pathway, providing estimable information on the neurodevelopmental toxicity and ecotoxicity of PFOS toxicity in aquatic animals and environments.