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.

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.

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.

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.

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.

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.

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.

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.

Mechanistic regulation of planarian shape during growth and degrowth.

Adult planarians can grow when fed and degrow (shrink) when starved while maintaining their whole-body shape. It is unknown how the morphogens patterning the planarian axes are coordinated during feeding and starvation or how they modulate the necessary differential tissue growth or degrowth. Here, we investigate the dynamics of planarian shape together with a theoretical study of the mechanisms regulating whole-body proportions and shape. We found that the planarian body proportions scale isometrically following similar linear rates during growth and degrowth, but that fed worms are significantly wider than starved worms. By combining a descriptive model of planarian shape and size with a mechanistic model of anterior-posterior and medio-lateral signaling calibrated with a novel parameter optimization methodology, we theoretically demonstrate that the feedback loop between these positional information signals and the shape they control can regulate the planarian whole-body shape during growth. Furthermore, the computational model produced the correct shape and size dynamics during degrowth as a result of a predicted increase in apoptosis rate and pole signal during starvation. These results offer mechanistic insights into the dynamic regulation of whole-body morphologies.

How neurobehavior and brain development in alternative whole-organism models can contribute to prediction of developmental neurotoxicity.

Developmental neurotoxicity (DNT) is not routinely evaluated in chemical risk assessment because current test paradigms for DNT require the use of mammalian models which are ethically controversial, expensive, and resource demanding. Consequently, efforts have focused on revolutionizing DNT testing through affordable novel alternative methods for risk assessment. The goal is to develop a DNT in vitro test battery amenable to high-throughput screening (HTS). Currently, the DNT in vitro test battery consists primarily of human cell-based assays because of their immediate relevance to human health. However, such cell-based assays alone are unable to capture the complexity of a developing nervous system. Whole organismal systems that qualify as 3 R (Replace, Reduce and Refine) models are urgently needed to complement cell-based DNT testing. These models can provide the necessary organismal context and be used to explore the impact of chemicals on brain function by linking molecular and/or cellular changes to behavioural readouts. The nematode Caenorhabditis elegans, the planarian Dugesia japonica, and embryos of the zebrafish Danio rerio are all suited to low-cost HTS and each has unique strengths for DNT testing. Here, we review the strengths and the complementarity of these organisms in a novel, integrative context and highlight how they can augment current cell-based assays for more comprehensive and robust DNT screening of chemicals. Considering the limitations of all in vitro test systems, we discuss how a smart combinatory use of these systems will contribute to a better human relevant risk assessment of chemicals that considers the complexity of the developing brain.

Integrative description of a new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from Shennongjia, Central China.

A new species of the genus Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from Xiangxi River, Shennongjia Forestry District, Hubei Province, China, is described on the basis of an integrative approach, involving morphology, and molecular systematics. The new species Dugesia saccaria A-T. Wang & Sluys, sp. nov. is characterized by the following features: a dumb-bell-shaped, muscularized hump located just anterior to the knee-shaped bend in the bursal canal; a ventrally displaced ejaculatory duct, which, however, opens terminally through the dorsal portion of the blunt tip of the penis papilla; a ventrally located seminal vesicle, giving rise to a vertically running duct that eventually curves downwards to communicate with the ejaculatory duct via a small diaphragm; oviducts opening asymmetrically into the dorsal portion of the common atrium and at the knee-shaped part of the bursal canal. The phylogenetic position of the new species was determined using four molecular markers (18S rDNA; ITS-1; 28S rDNA; COI), which suggested that it groups with other species of Dugesia from the Australasian and Oriental biogeographical regions.

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.

Assessing the in vivo toxicity of titanium dioxide nanoparticles in Schmidtea mediterranea: uptake pathways and (neuro)developmental outcomes.

Titanium dioxide nanoparticles (TiO2-NPs) in aquatic environments, originating from urban run-off, product use and post-consumer degradation, interact with aquatic organisms through water and sediments. Thorough toxicity assessment requires comprehensive data across all ecosystem compartments especially the benthic zone, which is currently lacking. Moreover, a proper physicochemical characterization of the particles is needed before and during toxicity assessment. In the present work, we used the planarian Schmidtea mediterranea to investigate the effects of TiO2-NPs (5 mg/L and 50 mg/L). Planarians are benthic organisms that play an important role in the food chain as predators. Our study integrated particle characterization with toxicokinetic and toxicodynamic parameters and showed that the uptake of TiO2-NPs of 21 nm occurred through the epidermis and intestine. Epidermal irritation and mucus production occurred immediately after exposure, and TiO2-NPs induced stronger effects in regenerating organisms. More specifically, TiO2-NPs interfered with neuroregeneration, inducing behavioral effects. A delay in the formation of the anterior commissure between the two brain lobes after seven and nine days of exposure to 50 mg/L was observed, probably as a result of a decrease in stem cell proliferation. Our findings underscore the need to incorporate multiple exposure routes in toxicity screenings. Additionally, we highlight the vulnerability of developing organisms and recommend their inclusion in future risk assessment strategies.

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.

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.

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.

Flatworm Transcriptomes Reveal Widespread Parasitism by Histophagous Ciliates.

Unicellular ciliates like Tetrahymena are best known as free-living bacteriovores, but many species are facultative or obligate parasites. These "histophages" feed on the tissues of hosts ranging from planarian flatworms to commercially important fish and the larvae of imperiled freshwater mussels. Here, we developed a novel bioinformatics pipeline incorporating the nonstandard ciliate genetic code and used it to search for Ciliophora sequences in 34 publicly available Platyhelminthes EST libraries. From 2,615,036 screened ESTs, we identified nearly 6,000 high-confidence ciliate transcripts, supporting parasitism of seven additional flatworm species. We also cultured and identified Tetrahymena from nine terrestrial and freshwater planarians, including invasive earthworm predators from the genus Bipalium and the widely studied regeneration models Dugesia japonica and Schmidtea mediterranea. A co-phylogenetic reconstruction provides strong evidence for the coevolution of histophagous Ciliophora with their Platyhelminthes hosts. We further report the antiprotozoal aminoglycoside paromomycin expels Tetrahymena from S. mediterranea, providing new opportunities to investigate the effects of this relationship on planarian biology. Together, our findings raise the possibility that invasive flatworms constitute a novel dispersal mechanism for Tetrahymena parasites and position the Platyhelminthes as an ideal model phylum for studying the ecology and evolution of histophagous ciliates.

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.

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.