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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.

Planarians have a remarkable ability to undergo whole-body regeneration. Successful regeneration outcome is determined by processes like polarity establishment at the wound site, which is followed by pole (organizer) specification. Interestingly, these determinants are almost exclusively expressed by muscles in these animals. However, the molecular toolkit that enables the functional versatility of planarian muscles remains poorly understood. Here we report that SMED_DDX24, a D-E-A-D Box RNA helicase, is necessary for planarian survival and regeneration. We found that DDX24 is enriched in muscles and its knockdown disrupts muscle fiber organization. This leads to defective pole specification, which in turn results in misregulation of many positional control genes specifically during regeneration. ddx24 RNAi also upregulates wound-induced Wnt signalling. Suppressing this ectopic Wnt activity rescues the knockdown phenotype by enabling better anterior pole regeneration. To summarize, our work highlights the role of an RNA helicase in muscle fiber organization, and modulating amputation-induced wnt levels, both of which seem critical for pole re-organization, thereby regulating whole-body regeneration.

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.

Planarians are an accessible model system to study animal regeneration and stem cells. Over the last two decades, new molecular techniques have provided us with powerful tools to understand whole-body regeneration and pluripotent adult stem cells specifically. We describe a method for performing Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) on planarian cells that relies on FACS to isolate different cell populations followed by immunoprecipitation and library preparation for next-generation sequencing. Whole-genome profiling of histone modifications enables a greater understanding of epigenetic mechanisms in development, pluripotency, and differentiation. This protocol adds to the growing list of functional genomic approaches to study whole-body regeneration in animals.

Aeolosoma viride, a globally distributed freshwater annelid, has a semitransparent appearance with 10 to 12 segments, about 2 to 3 mm in length. It is easy to raise and handle in laboratory conditions. Due to its robust regenerative capacity and applicability of various molecular tools including EdU labeling, whole-mount in situ hybridization (WISH), and RNA interference (RNAi), it rises as a promising model for studying whole-body regeneration.

Alien land planarians have been recorded in most of Europe and, in Italy, in 17 out of 20 regions. In this work, after collecting new data through a citizen-science approach, we report for the first time the presence of at least one alien planarian species in Molise, Basilicata, and Aosta Valley and we update the distribution range provided in our previous work. Amongst new sites, alien planarians were also found on a small island in Southern Italy, Pantelleria, which is included in a National Park rich in endemic species, thus deserving particular attention. Moreover, we report the first record of Dolichoplana striata in Italy. The increase of distribution range of alien planarians in Italy needs to be tightly monitored, as these introduced worms are responsible for impacts on native soil biodiversity. Accordingly, an e-mail address has been created ad-hoc (planarieitaliane@gmail.com) to collect all the records and to keep the distribution of alien species updated.

Regeneration experiments can produce complex phenotypes including morphological outcomes and gene expression patterns that are crucial for the understanding of the mechanisms of regeneration. However, due to their inherent complexity, variability between individuals, and heterogeneous data spreading across the literature, extracting mechanistic knowledge from them is a current challenge. Toward this goal, here we present protocols to unambiguously formalize the phenotypes of regeneration and their experimental procedures using precise mathematical morphological descriptions and standardized gene expression patterns. We illustrate the application of the methodology with step-by-step protocols for planaria and limb regeneration phenotypes. The curated datasets with these methods are not only helpful for human scientists, but they represent a key formalized resource that can be easily integrated into downstream reverse engineering methodologies for the automatic extraction of mechanistic knowledge. This approach can pave the way for discovering comprehensive systems-level models of regeneration.

Over the past several decades, planarians have emerged as a powerful model system with which to study the cellular and molecular basis of whole-body regeneration. The best studied planarians belong to freshwater flatworm species that maintain their remarkable regenerative capacity partly through the deployment of a population of adult pluripotent stem cells. Assessment of gene function in planarian regeneration has primarily been achieved through RNA interference (RNAi), either through the feeding or injection of double-stranded RNA (dsRNA). RNAi treatment of planarians has several advantages, including ease of use, which allows for medium-throughput screens of hundreds of genes over the course of a single project. Here, I present methods for dsRNA synthesis and RNAi feeding, as well as strategies for follow-up assessment of both structural and functional regeneration of organ systems of planarians, with a special emphasis on neural regeneration.

Comet assay provides the opportunity to detect and characterize DNA strand breaks. Cellular lysing followed by embedding in agarose slide is used to visualize under an electrical current migration patterns corresponding to DNA fragments of different sizes. Here we describe the process of detecting and characterizing DNA damage by Comet assay on planarians, which is a model organism commonly used to understand the process of whole-body regeneration, stem cell regulation, and adult tissue maintenance.

Deoxyribonuclease II (DNase II) has been found to regulate inflammation, autoimmunity and apoptosis in vertebrates and invertebrates. The strong capacity of degrading DNA makes DNase II play an important role in the immune process. Planarian has become one of the model references due to its strong immune system, the environment they live makes planarians face the threat of microorganisms and injury, the strong immune system can protect planarians from the threat of bacterial and infection. In this study, we found that there was DNase in the lysis buffer of planarians, then we acquired the sequence of DjDN2s (Dugesia japonica DNase2s) and confirmed the DjDN2s were conserved DNase IIs. The predicted structure showed the active sites and binding patterns of DjDN2s. Whole-mount in situ hybridization results showed DjDN2s mainly expressed in immune organs. Quantitative real-time PCR revealed that the expression of DjDN2s upregulated in varying degrees when got hurt and challenged with bacteria, and the knockdown of DjDN2s led to the slower repair of wound. The recombinant phages which take DjDN2 also had the ability to degrade DNA and clear young biofilm of Gram-negative bacteria. Collectively, DNase II of planarian might play a role in the antimicrobial response and wound-induced response.

The migration of adult stem cells in vivo is an important issue, but the complex tissue structures involved, and limited accessibility of the cells hinder a detailed investigation. To overcome these problems, the freshwater planarian Dugesia japonica was used because it has a simple body plan and abundant adult pluripotent stem cells (neoblasts) distributed uniformly throughout its body. To investigate the migratory mechanisms of neoblasts, two planarian homologous genes of metastatic tumor antigen (MTA-A and MTA-B), a protein involved in cancer metastasis that functions through histone deacetylation, were identified, and their function was analyzed using RNA interference (RNAi). MTA-A or MTA-B knockdown disrupted homeostatic tissue turnover and regeneration in planarians. Whereas neoblasts in MTA-A (RNAi) and MTA-B (RNAi) animals were maintained, neoblast differentiation was inhibited. Furthermore, the normal uniform neoblast distribution pattern changed to a branch-like pattern in MTA-A (RNAi) and MTA-B (RNAi) animals. To examine the neoblast migratory ability, a partial X-ray irradiation assay was performed in D. japonica. Using this assay system, the MTA-A knockdown neoblasts migrated collectively in a branch-like pattern, and the MTA-B knockdown neoblasts were not able to migrate. These results indicated that MTA-A was required for the exit of neoblasts from the branch-like region, and that MTA-B was required for neoblast migration. Thus, the migration mediated by MTA-A and MTA-B enabled uniform neoblast distribution and was required for neoblast differentiation to achieve tissue homeostasis and regeneration.

International audience; The planarian species Schmidtea mediterranea is a flatworm living in freshwater that is used in the research laboratory as a model to study developmental and regeneration mechanisms, as well as antibacterial mechanisms. However, the cultivable microbial repertoire of the microbes comprising its microbiota remains unknown. Here, we characterized the bacterial constituents of a 10-year-old laboratory culture of planarian species S. mediterranea via culturomics analysis. We isolated 40 cultivable bacterial species, including 1 unidentifiable species. The predominant phylum is Proteobacteria , and the most common genus is Pseudomonas . We discovered that parts of the bacterial flora of the planarian S. mediterranea can be classified as fish pathogens and opportunistic human pathogens.

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.

Although adult pluripotent stem cells (aPSCs) are found in many animal lineages, mechanisms for their formation during embryogenesis are unknown. Here, we leveraged Hofstenia miamia, a regenerative worm that possesses collectively pluripotent aPSCs called neoblasts and produces manipulable embryos. Lineage tracing and functional experiments revealed that one pair of blastomeres gives rise to cells that resemble neoblasts in distribution, behavior, and gene expression. In Hofstenia, aPSCs include transcriptionally distinct subpopulations that express markers associated with differentiated tissues; our data suggest that despite their heterogeneity, aPSCs are derived from one lineage, not from multiple tissue-specific lineages during development. Next, we combined single-cell transcriptome profiling across development with neoblast cell-lineage tracing and identified a molecular trajectory for neoblast formation that includes transcription factors Hes, FoxO, and Tbx. This identification of a cellular mechanism and molecular trajectory for aPSC formation opens the door for in vivo studies of aPSC regulation and evolution.

Understanding the molecular mechanisms underlying thermal tolerance of aquatic invertebrates can inform predictions about the effects of thermal regime changes on these species. While gene expression and protein abundance changes underlie compensatory responses, little is known about the role of post-translational modifications as thermal tolerance mechanisms. To test the hypothesis that protein phosphorylation changes in response to thermal acclimation, we studied the phosphoproteome of the freshwater planarian Crenobia alpina. This species has a supposedly limited thermal tolerance and is found in cold-stable habitats. We systematically investigated phosphopeptide abundances following 168 h acclimation to 11, 14, 17, and 20 °C, using label-free quantitative phosphoproteomics. We provide a comparative analysis of 2115 phosphosites from 1049 phosphoproteins, whereby little to no differences could be observed between 11, 14, and 17 °C. However, more than 130 phosphopeptides were significantly more abundant and 40 were less abundant following acclimation to 20 °C. These phosphoproteins were functionally associated with the regulation of neuronal processes, cilia, DNA damage repair, aquaporins, and mitochondrial fission and fusion. These data support the hypothesis that phosphorylation plays a role in thermal acclimation responses, suggesting that PTMs are of significance in invertebrate thermal acclimation. PTMs may therefore offer an alternative route of transient protein adaptation to temperature increase in invertebrates, and should not be neglected when trying to understand how molecular system dynamics in response to elevated temperatures.

Regeneration, the restoration of body parts after injury, is quite widespread in the animal kingdom. Species from virtually all Phyla possess regenerative abilities. Human beings, however, are poor regenerators. Yet, the progress of knowledge and technology in the fields of bioengineering, stem cells, and regenerative biology have fostered major advancements in regenerative medical treatments, which aim to regenerate tissues and organs and restore function. Human induced pluripotent stem cells can differentiate into any cell type of the body; however, the structural and cellular complexity of the human tissues, together with the inability of our adult body to control pluripotency, require a better mechanistic understanding. Planarians, with their capacity to regenerate lost body parts thanks to the presence of adult pluripotent stem cells could help providing such an understanding. In this paper, we used a top-down approach to shortlist blastema transcription factors (TFs) active during anterior regeneration. We found 44 TFs—31 of which are novel in planarian—that are expressed in the regenerating blastema. We analyzed the function of half of them and found that they play a role in the regeneration of anterior structures, like the anterior organizer, the positional instruction muscle cells, the brain, the photoreceptor, the intestine. Our findings revealed a glimpse of the complexity of the transcriptional network governing anterior regeneration in planarians, confirming that this animal model is the perfect playground to study in vivo how pluripotency copes with adulthood.

We investigated the effect of weak combined magnetic field (CMF) on stem cell proliferation and regeneration of the planarian Schmidtea mediterranea. CMF parameters were set in accordance with Valery Lednev's theory of magnetic parametric resonance. It was shown that CMF with an amplitude of 74 μT and a frequency of 30 Hz accelerated the growth of the planarian head blastema by 25%. Alterations of the frequency in range from 27 to 33 Hz led to a complete disappearance of the effect. A further decrease in the CMF frequency inhibited regeneration. The maximum inhibition (24%) was observed at a frequency of 16 Hz. A further decrease in the CMF frequency (down to 13 Hz) led to disappearance of the described effect. Regeneration rate changes under the CMF are influenced by alterations in stem cell mitotic activity, which in turn depends on the wound-induced gene expression level. Thus, the CMF, preset in accordance to the Lednev's theory, can specifically influence the expression of regeneration-related genes and regeneration itself, what can find biomedical applications.

A new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from northern China is described on the basis of an integrative approach, involving morphology, karyology, histology, molecular distance, molecular phylogeny, and mitochondrial gene order. Here, we present the complete mitogenome of the new species Dugesia constrictiva ChenDong, sp. nov. This new species is mainly characterized by the presence of the following features: asymmetrical openings of the oviducts; large, cuboidal copulatory bursa; vasa deferentia opening through the ventro-lateral wall of the seminal vesicle; laterally compressed seminal vesicle; ventrally displaced ejaculatory duct, opening at the blunt tip of the penis papilla; long duct intercalated between seminal vesicle and diaphragm; chromosome complement diploid, with 16 metacentric chromosomes; mitochondrial gene order as follows: cox1-E-nad6-nad5-S2-D-R-cox3-I-Q-K-atp6-V-nad1-W-cox2-P-nad3-A-nad2-M-H-F-rrnS-L1-Y-G-S1-rrnL-L2-T-atp8-C-N-cob-nad4l-nad4. In triclads, mitochondrial gene order is considerably conserved between freshwater planarians and land flatworms, whereas it is variable between marine planarians and both freshwater and land flatworms. The secondary structures of tRNAs are all equipped with 4 arms, excepting tRNA S1 and tRNA S2, which lack the D arm and have excessively enlarged loops. Numerous transpositions of tRNA are present between D. constrictiva and its congeners. Mitochondrial gene arrangements may form a new, additional tool for taxonomic studies. The phylogenetic tree based on analysis of the mitochondrial genome basically corroborates current classification of the higher taxa of planarian flatworms.

The growing number of commercially used chemicals that are under-evaluated for developmental neurotoxicity (DNT) combined with the difficulty in describing the etiology of exposure-related neurodevelopmental toxicity has created a reticent threat to human health. Current means of screening chemicals for DNT are limited to expensive, time-consuming, and labor-intensive traditional laboratory animal models. In this study, we hypothesize that exposed head-regenerating planarian flatworms can effectively and efficiently categorize DNT in known developmental neurotoxins (ethanol and bisphenol A [BPA]). Planarian flatworms are an established alternative animal model for neurodevelopmental studies and have remarkable regenerative abilities allowing neurodevelopment to be induced via head resection. Here, we observed changes in photophobic behavior and central nervous system (CNS) morphology to evaluate the impact of exposure to low concentrations of ethanol, BPA, and BPA industry alternatives bisphenol F, and bisguaiacol on neurodevelopment. Our studies show that exposure to 1% v/v ethanol during regeneration induces a recoverable 48-h delay in the development of proper CNS integrity, which aligns with behavioral assessments of cognitive ability. Exposure to BPA and its alternatives induced deviations to neurodevelopment in a range of severities, distinguished by suppressions, delays, or a combination of the 2. These results suggest that quick and inexpensive behavioral assessments are a viable surrogate for tedious and costly immunostaining studies, equipping more utility and resolution to the planarian model for neurodevelopmental toxicity in the future of mass chemical screening. These studies demonstrate that behavioral phenotypes observed following chemical exposure are classifiable and also temporally correlated to the anatomical development of the CNS in planaria. This will facilitate and accelerate toxicological screening assays with this alternative animal model.

Freshwater planarian Dugesia japonica is an excellent model organism for investigating stem cell behavior during regeneration. Despite studies showing that numerous genetic factors are involved in regeneration, much more research is required to fully understand the molecular mechanisms that orchestrate regeneration. In this study, we identified an evolutionarily conserved gene DjPtpn11(DjShp2). DjPtpn11 transcripts are expressed in neoblasts and some differentiated cells, with a high expression at the newly formed blastema. Its silencing by RNA interference (RNAi) affected anterior regeneration and inhibited the regeneration of posterior regions, including cholinergic and serotonergic neuron regeneration. In adult planarians, DjPtpn11 knockdown did not affect neoblast survival and proliferation but might prevent the stem cell migration and differentiation through ERK signaling. DjPtpn11 was demonstrated to be necessary for the anterior blastema cell differentiation partially via regulating ERK-DjMkpA activity. DjPtpn11 also influenced posterior specification via DjIslet, suggesting that DjPtpn11 may be involved in regulating the Wnt signaling pathway during the development of posterior blastema. Together, these data identified that DjPtpn11 is an essential modulator for the regeneration of planarians, and it may influence the appropriate differentiation of blastema cells.

Ionising radiation causes the death of the most actively dividing cells, thus leading to depletion of the stem cell pool. Planarians are invertebrate flatworms that are unique in that their stem cells, called neoblasts, constantly replace old, damaged, or dying cells. Amenability to efficient RNAi treatments, the rapid development of clear phenotypes, and sensitivity to ionising radiation, combined with new genomic technologies, make planarians an outstanding tool for the discovery of potential radioprotective agents. In this work, using the well-known antioxidant N-acetylcysteine, planarians are, for the first time, shown to be an excellent model system for the fast and effective screening of novel radioprotective and radio-sensitising substances. In addition, a panel of measurable parameters that can be used for the study of radioprotective effects on this model is suggested.

© 2021 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.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.

Simultaneous longitudinal imaging across multiple conditions and replicates has been crucial for scientific studies aiming to understand biological processes and disease. Yet, imaging systems capable of accomplishing these tasks are economically unattainable for most academic and teaching laboratories around the world. Here, we propose the Picroscope, which is the first low-cost system for simultaneous longitudinal biological imaging made primarily using off-the-shelf and 3D-printed materials. The Picroscope is compatible with standard 24-well cell culture plates and captures 3D z-stack image data. The Picroscope can be controlled remotely, allowing for automatic imaging with minimal intervention from the investigator. Here, we use this system in a range of applications. We gathered longitudinal whole organism image data for frogs, zebrafish, and planaria worms. We also gathered image data inside an incubator to observe 2D monolayers and 3D mammalian tissue culture models. Using this tool, we can measure the behavior of entire organisms or individual cells over long-time periods., Ly et al. report a multi-well imaging system (the Picroscope) that can perform longitudinal bright-field z-stack imaging for 24 wells simultaneously. They demonstrate its capability by imaging zebrafish embryos, planaria worms, development of Xenopus tropicalis over a 28-h period and embryonic stem cell monolayer and organoids inside an incubator.

C-shapes are stereotyped movements in planarians that are elicited by diverse stimuli (e.g. acidity, excitatory neurotransmitters, psychostimulants, and pro-convulsants). Muscle contraction and seizure contribute to the expression of C-shape movements, but a causative role for pain is understudied and unclear. Here, using nicotine-induced C-shapes as the endpoint, we tested the efficacy of three classes of antinociceptive compounds - an opioid, NSAID (non-steroidal anti-inflammatory drug), and transient receptor potential ankyrin 1 (TRPA1) channel antagonist. For comparison we also tested effects of a neuromuscular blocker. Nicotine (0.1-10 mM) concentration-dependently increased C-shapes. DAMGO (1-10 µM), a selective µ-opioid agonist, inhibited nicotine (5 mM)-induced C-shapes. Naloxone (0.1-10 µM), an opioid receptor antagonist, prevented the DAMGO (1 µM)-induced reduction of nicotine (5 mM)-evoked C-shapes, suggesting an opioid receptor mechanism. C-shapes induced by nicotine (5 mM) were also reduced by meloxicam (10-100 µM), a NSAID; HC 030,031 (1-10 µM), a TRPA1 antagonist; and pancuronium (10-100 µM), a neuromuscular blocker. Evidence that nicotine-induced C-shapes are reduced by antinociceptive drugs from different classes, and require opioid receptor and TRPA1 channel activation, suggest C-shape etiology involves a pain component.

Research on individual behaviors can help to reveal the processes and mechanisms that mediate an animal's habits and interactions with the environment. Importantly, individual behaviors arise as outcomes of genetic programs, morphogenesis, physiological processes, and neural functions; thus, behavioral analyses can be used to detect disorders in these processes. Planarians belong to an early branching bilateral group of organisms that possess a simple central nervous system. Furthermore, planarians display various behavioral responses to the environment via their nervous system. Planarians also have remarkable regenerative abilities, including whole-brain regeneration. Therefore, the combination of planarians' phylogenetic position, behavioral properties, regenerative ability, and genetic accessibility provides a unique opportunity to understand the basic mechanisms underlying the anatomical properties of neural morphogenesis and the dynamic physiological processes and neural function. Here, we describe a step-by-step protocol for conducting simple behavioral analyses in planarians with the aim of helping to introduce researchers to the utility of performing behavioral analyses in planarians. Since the conditions of planarians impact experimental results and reproducibility, this protocol begins with a method for maintaining planarians. Next, we introduce the behavioral tests as well as the methods for quantifying them using minimal and cost-effective equipment and materials. Finally, we present a unique RNAi technique that enables conditional silencing of neural activity in the brain of planarians.

Spermatogenesis is one of the most dramatic cellular differentiation events observed in animals. In particular, spermiogenesis (the final stage of spermatogenesis) involves extensive shedding of cytoplasmic organelles, dramatic nuclear rearrangements, and assembly of long flagellar structures. In planarian flatworms, the spherical nucleus present in round spermatids elongates to produce the filamentous nucleus of mature sperm. Newly formed cortical microtubules participate in cytoskeletal rearrangements observed during spermiogenesis and remain present in sperm. In addition, a pair of flagella assemble at one end of each spermatid in a process that likely involves de novo formation of centrioles. This chapter includes a brief introduction to planarian spermatogenesis and current tools for the analysis of molecular players in this process. Step-by-step protocols for isolating and imaging spermatogenic cells are provided with enough detail to be carried out by newcomers to the field who would like to study this unique organism in the laboratory.

Planarians are flatworms capable of whole-body regeneration, able to regrow any missing body part after injury or amputation. The extraordinary regenerative capacity of planarians is based upon the presence in the adult of a large population of somatic pluripotent stem cells. These cells, called neoblasts, offer a unique system to study the process of stem cell specification and differentiation in vivo. In recent years, FACS-based isolation of neoblasts, RNAi functional analyses as well as high-throughput approaches such as single-cell sequencing have allowed a rapid progress in our understanding of many different aspects of neoblast biology. Here, we summarize our current knowledge on the molecular signatures that define planarian neoblasts heterogeneity, which includes a percentage of truly pluripotent stem cells, and guide the commitment of pluripotent neoblasts into lineage-specific progenitor cells, as well as their differentiation into specific planarian cell types.

Accessibility to germ cells allows the study of germ cell development, meiosis, and recombination. The sexual biotype of the freshwater planarian, Schmidtea mediterranea, is a powerful invertebrate model to study the epigenetic specification of germ cells. Unlike the large number of testis and male germ cells, planarian oocytes are relatively difficult to locate and examine, as there are only two ovaries, each with 5-20 oocytes. Deeper localization within the planarian body and lack of protective epithelial tissues also make it challenging to dissect planarian ovaries directly. This protocol uses a brief fixation step to facilitate the localization and dissection of planarian ovaries for downstream analysis to overcome these difficulties. The dissected ovary is compatible for ultrastructural examination by transmission electron microscopy (TEM) and antibody immunostaining. The dissection technique outlined in this protocol also allows for gene perturbation experiments, in which the ovaries are examined under different RNA interference (RNAi) conditions. Direct access to the intact germ cells in the ovary achieved by this protocol will greatly improve the imaging depth and quality and allow cellular and subcellular interrogation of oocyte biology.

Many biological organisms regenerate structure and function after damage. Despite the long history of research on molecular mechanisms, many questions remain about algorithms by which cells can cooperate towards the same invariant morphogenetic outcomes. Therefore, conceptual frameworks are needed not only for motivating hypotheses for advancing the understanding of regeneration processes in living organisms, but also for regenerative medicine and synthetic biology. Inspired by planarian regeneration, this study offers a novel generic conceptual framework that hypothesizes mechanisms and algorithms by which cell collectives may internally represent an anatomical target morphology towards which they build after damage. Further, the framework contributes a novel nature-inspired computing method for self-repair in engineering and robotics. Our framework, based on past in vivo and in silico studies on planaria, hypothesizes efficient novel mechanisms and algorithms to achieve complete and accurate regeneration of a simple in silico flatwormlike organism from any damage, much like the body-wide immortality of planaria, with minimal information and algorithmic complexity. This framework that extends our previous circular tissue repair model integrates two levels of organization: tissue and organism. In Level 1, three individual in silico tissues (head, body, and tail—each with a large number of tissue cells and a single stem cell at the centre) repair themselves through efficient local communications. Here, the contribution extends our circular tissue model to other shapes and invests them with tissue-wide immortality through an information field holding the minimum body plan. In Level 2, individual tissues combine to form a simple organism. Specifically, the three stem cells form a network that coordinates organism-wide regeneration with the help of Level 1. Here we contribute novel concepts for collective decision-making by stem cells for stem cell regeneration and large-scale recovery. Both levels (tissue cells and stem cells) represent networks that perform simple neural computations and form a feedback control system. With simple and limited cellular computations, our framework minimises computation and algorithmic complexity to achieve complete recovery. We report results from computer simulations of the framework to demonstrate its robustness in recovering the organism after any injury. This comprehensive hypothetical framework that significantly extends the existing biological regeneration models offers a new way to conceptualise the information-processing aspects of regeneration, which may also help design living and non-living self-repairing agents.

Energy drinks pose consumer and environmental risks. One of the few organisms suitable for investigating both risks are planarians, which display mammalian-like behavioral effects during drug exposure and reside in aquatic environments. We investigated effects of Monster Energy® (0.001 - 10%) on planarian behaviors using established assays. For acute exposure, only higher concentrations reduced motility (1%) and caused stereotypies (1%). Lower concentrations (0.1-1%) enhanced light avoidance, a measure of defensive responding. In place conditioning experiments conducted with low concentrations (0.0001%-0.1%), planarians avoided the energy drink-paired side. These results suggest that Monster Energy® causes aversive effects in aquatic life such as planarians.