Functional genomics using CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated endonuclease)-based approaches has revolutionized biomedical sciences. Gene editing is also widespread in parasitology generally and its use is increasing in studies on helminths including flatworm and roundworm parasites. Here, we survey the progress, specifically with experimental CRISPR-facilitated functional genomics to investigate helminth biology and pathogenesis, and also with the burgeoning use of CRISPR-based methods to assist in diagnosis of helminth infections. We also provide an historical timeline of the introduction and uses of CRISPR in helminth species to date.

Allometry explores the relationship between an organism's body size and its various components, offering insights into ecology, physiology, metabolism, and disease. The cell is the basic unit of biological systems, and yet the study of cell-type allometry remains relatively unexplored. Single-cell RNA sequencing (scRNA-seq) provides a promising tool for investigating cell-type allometry. Planarians, capable of growing and degrowing following allometric scaling rules, serve as an excellent model for these studies. We used scRNA-seq to examine cell-type allometry in asexual planarians of different sizes, revealing that they consist of the same basic cell types but in varying proportions. Notably, the gut basal cells are the most responsive to changes in size, suggesting a role in energy storage. We capture the regulated gene modules of distinct cell types in response to body size. This research sheds light on the molecular and cellular aspects of cell-type allometry in planarians and underscores the utility of scRNA-seq in these investigations.

It is impossible to overlook the effects of microplastics (MPs) on aquatic organisms as they continuously accumulate in water environment. Freshwater planarians, which exist in the benthic zone of water bodies and come into contact with the deposited MPs particles, provide a highly representative model for studying the effects of MPs on aquatic organisms. Anthocyanins (ANTs) have gained significant popularity in recent years for their diverse health benefits. In the current study, the median lethal concentration (LC50) of polystyrene (PS) to planarian Dugesia japonica was determined for the first time. Based on this, multiple toxic effects of single PS and PS in combination with ANTs on planarians were explored. The results showed that PS exposure disrupted the redox homeostasis and induced oxidative damage in planarians. Also, PS stress affected the neuromorphology, aggravated cell apoptosis in planarians probably by altering neural gene expressions as well as promoting the expression of apoptosis-related genes while inhibiting stem cell marker genes. In addition, the results also suggested that co-exposure of ANTs could effectively alleviate the toxicity of PS on planarians. Particularly, long-term environmentally relevant concentration PS exposure exhibited a higher propensity for inducing toxicity on planarians than short-term high concentration acute exposure, indicating that the harm of environmental MPs to humans and wildlife exposed to them should not be underestimated. Therefore, considering the recently rising and rapid development of ecotoxicomics, more in-depth research on the toxicity mechanism of environmentally relevant concentration PS-MPs to freshwater planarians from multi-omics levels will be our future work.

Programmed cell death plays a crucial role during tissue turnover in all animal species, and it is also essential during regeneration, serving as a key signalling mechanism to promote tissue repair and regrowth. In freshwater planarians, remarkable regenerative abilities are supported by neoblasts, a population of adult stem cells, which enable high somatic cell turnover. Cell death in planarians occurs continuously during regeneration and adult homeostasis, underscoring its critical role in tissue remodeling and repair. However, the exact mechanisms regulating cell death in these organisms remain elusive. In contrast, Drosophila melanogaster serves as a powerful model for studying programmed cell death in development, metamorphosis, and adult tissue maintenance, leveraging advanced genetic tools and visualization techniques. In Drosophila, cell death sculpts tissues, eliminates larval structures during metamorphosis, and supports homeostasis in adulthood. Despite limited regenerative capacity compared to planarians, Drosophila provides unique insights into cell death's regulatory mechanisms. Comparative analysis of these two systems highlights both conserved and divergent roles of programmed cell death in tissue renewal and regeneration. This review synthesizes the latest knowledge of programmed cell death in planarians and Drosophila, aiming to illuminate shared principles and system-specific adaptations, with relevance to tissue repair across biological systems.

Whole-body regeneration requires adult stem cells with high plasticity to differentiate into missing cell types. Planarians possess a unique configuration of organs embedded in a vast pool of pluripotent stem cells. How stem cells integrate positional information with discrete fates remains unknown. Here, we use the planarian pharynx to define the cell fates that depend on the pioneer transcription factor FoxA. We find that Roundabout receptor RoboA suppresses aberrant pharynx cell fates by altering foxA expression, independent of the canonical ligand Slit. An RNAi screen for extracellular proteins identifies Anosmin-1 as a potential partner of RoboA. Perturbing global patterning demonstrates that roboA / anosmin-1 functions locally in the brain. By contrast, altering pharynx fate with foxA knockdown induces head-specific neurons in the pharynx, indicating a latent plasticity of stem cells. Our data links critical extracellular cues with cell fate decisions of highly plastic stem cells, ensuring the fidelity of organ regeneration.

Regenerative abilities vary across species and developmental stages of animal life cycles. Determining mechanisms that promote or limit regeneration in certain life cycle stages may pinpoint the most critical factors for successful regeneration and suggest strategies for reverse-engineering regenerative responses in therapeutic settings. In contrast to many mammalian systems, which typically show a loss of regenerative abilities with age, planarian flatworms remain highly regenerative throughout adulthood. The robust reproductive and regenerative capabilities of the planarian Schmidtea polychroa (S. polychroa) make them an ideal model to determine when and how regeneration competence is established during development. We report that S. polychroa gradually acquires whole-body regenerative abilities during late embryonic and early juvenile stages. Anterior fragments are capable of regenerating missing trunk and tail tissues from stage 6.5 onward. By contrast, the ability of posterior fragments to make new head tissue depends on the developmental stage, tissue composition of the amputated fragment, and axial position of the cut plane. Irradiation-sensitive cells are required, but not sufficient, for the onset of head regeneration ability. We propose that regulation of the main body axis reset, specifically the ability to remake an anterior organizing center, determines when whole-body regeneration competence arises during development. Supporting this hypothesis, knockdown of the canonical Wnt pathway effector Spol-β-catenin-1, a posterior determinant, induces precocious head regeneration under conditions that are normally head regeneration-incompetent. Our results suggest that regeneration competence emerges through interactions between irradiation-sensitive cells, the cellular source of new tissue, and developing adult tissue(s) harboring axial patterning information.

Cytotoxic killing is an essential immune function, yet its cellular mechanisms have been characterized in only a few model species. Here, we show that planarian flatworms harness a unique cytotoxic strategy. In planarians, activin, a hormone regulating regeneration and reproduction, also acts as an inflammatory cytokine. Overactivation of activin signaling - through protein injection, genetic chimerism, or bacterial infection - triggers 'ruptoblasts', an undocumented immune cell type, to undergo 'ruptosis', a unique mode of cell bursting that eliminates nearby cells and bacteria in mere minutes, representing one of the fastest cytotoxic mechanisms observed. Ablating ruptoblasts suppresses inflammation but compromises bacterial clearance, highlighting ruptoblasts' broad-spectrum immune functions. We further identified ruptoblast-like cells in diverse basal bilaterians, unveiling an alternative strategy that couples hormonal regulation with immune defense and expanding the landscape of evolutionary immune innovations.

Phosphorylation plays a crucial role in the cellular response to radiation and cancer therapies, yet phosphoproteomics studies in planarians remain underexplored despite the organism's remarkable regenerative capacities. This study utilized advanced ion mobility mass spectrometry for 4D-label-free quantitative proteomics to identify phosphorylation sites associated with irradiation in planarians. A total of 33,284 phosphorylation sites from 15,505 phosphorylated peptides and 4710 unique phosphoproteins were identified. In the sub-lethal dose irradiation group, 1695 phosphoproteins with 3483 phosphorylation sites exhibited significant changes, while exposure to lethal doses of radiation led to significant changes in 2308 phosphoproteins with 6112 phosphorylation sites, including many kinases, transcription factors, and cytoskeletal proteins. Functional enrichment analysis revealed that the altered phosphoproteins were primarily involved in transcription, RNA biosynthesis, mRNA processing regulation, and spliceosomal complex assembly. Functional validation of five differentially phosphorylated proteins revealed that their depletion impaired stem cell regeneration after irradiation by disrupting DNA repair, suggesting that these proteins are critical to planarian biology and their radiation response. By identifying the phosphorylation state and specific sites of planarian proteins, our study lays the foundation for further research on protein phosphorylation in the radiation-induced DNA damage response. In addition, our findings provide preliminary insights into the role of calnexin, a protein involved in interacting with newly synthesized N-linked glycoproteins, in planarians.

The cell nuclei of Ophisthokonts, the eukaryotic supergroup defined by fungi and metazoans, is remarkable in the constancy of their double-membraned structure in both somatic and germ cells. Such remarkable structural conservation underscores common and ancient evolutionary origins. Yet, the dynamics of disassembly and reassembly displayed by Ophisthokont nuclei vary extensively. Besides closed mitosis in fungi and open mitosis in some animals, little is known about the evolution of nuclear envelope remodeling dynamics during oogenesis. Here, we uncovered a novel form of nuclear envelope remodeling as oocytes are formed in the flatworm Schmidtea mediterranea. From zygotene to metaphase II, both nuclear envelope (NE) and peripheral endoplasmic reticulum (ER) expand notably in size, likely involving de novo membrane synthesis. 3-D electron microscopy reconstructions demonstrated that the NE transforms itself into numerous double-membraned vesicles similar in membrane architecture to NE doublets in mammalian oocytes after germinal vesicle breakdown. The vesicles are devoid of nuclear pore complexes and DNA, yet are loaded with nuclear proteins, including a planarian homologue of PIWI, a protein essential for the maintenance of stem cells in this and other organisms. Our data contribute a new model to the canonical view of NE dynamics and suggest important roles of NE remodeling in planarian oogenesis.

The ability to regenerate after the loss of a part is a hallmark of living systems and occurs at both the tissue and organ scales, but also within individual cells. Regeneration entails many processes that are physical and mechanical in nature, including the closure of wounds, the repositioning of material from one place to another, and the restoration of symmetry following perturbations. However, we currently know far more about the genetics and molecular signaling pathways involved in regeneration, and there is a need to investigate the role of physical forces in the process. Here, we will provide an overview of how physical forces may play a role in wound healing and regeneration, in which we compare and contrast regenerative processes at the tissue and cell scales.

In holothurians, the regenerative process following evisceration involves the development of a 'rudiment' or 'anlage' at the injured end of the mesentery. This regenerating anlage plays a pivotal role in the formation of a new intestine. Despite its significance, our understanding of the molecular characteristics inherent to the constituent cells of this structure has remained limited. To address this gap, we employed state-of-the-art scRNA-seq and hybridization chain reaction fluorescent in situ hybridization analyses to discern the distinct cellular populations associated with the regeneration anlage. Through this approach, we successfully identified 13 distinct cell clusters. Among these, two clusters exhibit characteristics consistent with putative mesenchymal cells, while another four show features akin to coelomocyte cell populations. The remaining seven cell clusters collectively form a large group encompassing the coelomic epithelium of the regenerating anlage and mesentery. Within this large group of clusters, we recognized previously documented cell populations such as muscle precursors, neuroepithelial cells, and actively proliferating cells. Strikingly, our analysis provides data for identifying at least four other cellular populations that we define as the precursor cells of the growing anlage. Consequently, our findings strengthen the hypothesis that the coelomic epithelium of the anlage is a pluripotent tissue that gives rise to diverse cell types of the regenerating intestinal organ. Moreover, our results provide the initial view into the transcriptomic analysis of cell populations responsible for the amazing regenerative capabilities of echinoderms.

Competence to regenerate lost tissues varies widely across species. The rat tapeworm, Hymenolepis diminuta, undergoes continual cycles of shedding and regenerating thousands of reproductive segments to propagate the species. Despite its prowess, H. diminuta can only regenerate posteriorly from a singular tissue: the neck or germinative region (GR). What cells and signaling pathways restrict regeneration competence to the GR? In this study, we show that the head regulates regeneration-competence by promoting maintenance of the GR and inhibiting proglottid formation in a distance-dependent manner. Anterior-posterior (A-P) patterning within the GR provide local signals that mediate these head-dependent responses. βcat1 is necessary for stem cell maintenance, proliferation and proglottidization. On the other hand, sfrp is necessary for maintaining the GR at its proper length. Our study demonstrates that the head organizes a balance of pro- and anti-regeneration signals that must be integrated together and therefore control competence to regenerate.

Planarian regeneration and tissue turnover involve fate specification in pluripotent stem cells called neoblasts. Neoblasts select fates through the expression of fate-specific transcription factors (FSTFs), generating specialized neoblasts. Specialized neoblasts are spatially intermingled and can be dispersed broadly, frequently being far from their target tissue. The post-mitotic progeny of neoblasts, serving as progenitors, migrate and differentiate into mature cell types. Pattern formation is thus strongly influenced by the migratory assortment and differentiation of fate-specified progenitors in precise locations, which we refer to as progenitor targeting. This central step of pattern maintenance and formation, however, is poorly understood. Here, we describe a requirement for the conserved map3k1 gene in targeting, restricting post-mitotic progenitor differentiation to precise locations. RNAi of map3k1 causes ectopic differentiation of eye progenitors along their migratory path, resulting in dispersed ectopic eyes and eye cells. Other neural tissues similarly display ectopic posterior differentiation and pharynx cells emerge dispersed laterally and anteriorly in map3k1 RNAi animals. Ectopic differentiated cells are also found within the incorrect organs after map3k1 RNAi, and ultimately teratomas form. These findings implicate map3k1 signaling in controlling the positional regulation of progenitor behavior - restricting progenitor differentiation to targeted locations in response to external cues in the local tissue environment.

Sox genes encode a family of transcription factors that regulate multiple biological processes during metazoan development, including embryogenesis, tissue homeostasis, nervous system specification, and stem cell maintenance. The planarian Dugesia japonica contains a reservoir of stem cells that grow and divide continuously to support cellular turnover. However, whether SOX proteins retain these conserved functions in planarians remains to be determined. In this study, three sox gene homologs, DjsoxP-1, DjsoxP-5, and Djsox5, were identified in the planarian transcriptome, and their roles were investigated. The results showed that the amino acids deduced from the three sox genes all contained high-mobility group (HMG) domain sequences, which are highly conserved in sox family members. Whole-mount in situ hybridization (WISH) and real-time quantitative PCR (RT-qPCR) results indicated that the three sox genes were mainly expressed in parenchymal tissues and regenerative blastema. Additionally, X-ray irradiation assay and dFISH suggested that the three Djsox genes were expressed in neoblasts and other cell types. Head regression in intact planarian and smaller blastemas in both head or tail fragments of regenerating planarians were exhibited with DjsoxP-1 and Djsox5 RNA interference (RNAi) compared to the control animals, suggesting that DjsoxP-1 and Djsox5 have essential roles during cellular turnover and regeneration in planarians; conversely, there was no obvious phenotypic abnormalities or regeneration defect in DjsoxP-5 RNAi animals. Knockdown of DjsoxP-1 or Djsox5 decreased neoblast proliferation and promoted cell apoptosis. In conclusion, our findings demonstrate that DjsoxP-1 and Djsox5 are involved in cellular turnover and regeneration in planarians by modulating coordination between cell proliferation and apoptosis.

Proper stem cell targeting and differentiation is necessary for regeneration to succeed. In organisms capable of whole body regeneration, considerable progress has been made identifying wound signals initiating this process, but the mechanisms that control the differentiation of progenitors into mature organs are not fully understood. Using the planarian as a model system, we identify a novel function for map3k1, a MAP3K family member possessing both kinase and ubiquitin ligase domains, to negatively regulate terminal differentiation of stem cells during eye regeneration. Inhibition of map3k1 caused the formation of multiple ectopic eyes within the head, but without controlling overall head, brain, or body patterning. By contrast, other known regulators of planarian eye patterning like wnt11-6/wntA and notum also regulate head regionalization, suggesting map3k1 acts distinctly. Consistent with these results, eye resection and regeneration experiments suggest that unlike Wnt signaling perturbation, map3k1 inhibition did not shift the target destination of eye formation in the animal. map3k1(RNAi) ectopic eyes emerged in the regions normally occupied by migratory eye progenitors, and these animals produced a net excess of differentiated eye cells. Furthermore, the formation of ectopic eyes after map3k1 inhibition coincided with an increase to numbers of differentiated eye cells, a decrease in numbers of ovo+ eye progenitors, and also was preceded by eye progenitors prematurely expressing opsin/tyosinase markers of eye cell terminal differentiation. Therefore, map3k1 negatively regulates the process of terminal differentiation within the eye lineage. Similar ectopic eye phenotypes were also observed after inhibition of map2k4, map2k7, jnk, and p38, identifying a putative pathway through which map3k1 prevents differentiation. Together, these results suggest that map3k1 regulates a novel control point in the eye regeneration pathway which suppresses the terminal differentiation of progenitors during their migration to target destinations.

Stem cells are of great importance in the maintenance and regeneration of tissues, with Forkhead box O (FoxO) proteins emerging as pivotal regulators of their functions. However, the precise impact of FoxO proteins on stem cell behavior within regenerative environments remains ambiguous. Planarians, renowned for their abundance of adult stem cells (neoblasts), serve as an excellent model for investigating the dynamics of stem cells during regeneration. In this study, we identified DjfoxO, a conserved foxO gene in the planarian Dugesia japonica, and demonstrated its expression in neoblasts, with elevated levels detected in the regenerative blastema during the regeneration process. Using a FoxO inhibitor (AS1842856) together with RNA interference techniques, we demonstrated that inhibition of FoxO signaling in planarians hinders the regeneration of missing tissues, including the central nervous system, eyespots, anterior intestinal branches, and pharynx. It is noteworthy that the knockdown of DjfoxO does not significantly affect the mitotic activity of neoblasts. Conversely, it impedes the production of lineage-specific progenitors, potentially via modulation of the Erk pathway. These findings elucidate the instructive function of FoxO signaling in regulating stem cell differentiation and provide valuable insights into its potential for improving stem cell-based regenerative therapies.

Animals vary in their ability to replace body parts lost to injury, a phenomenon known as restorative regeneration. Uncovering conserved signaling steps required for regeneration may aid regenerative medicine. Reactive oxygen species (ROS) are necessary for proper regeneration in species across a wide range of taxa, but it is unknown whether ROS are essential for annelid regeneration. As annelids are a widely used and excellent model for regeneration, we sought to determine whether ROS play a role in the regeneration of the highly regenerative annelid, Lumbriculus variegatus. Using a ROS-sensitive fluorescent probe we observed ROS accumulation at the wound site within 15 min after amputation; this ROS burst lessened by 6 h post-amputation. Chemical inhibition of this ROS burst delayed regeneration, an impairment that was partially rescued with exogenous ROS. Our results suggest that similar to other animals, annelid regeneration depends upon ROS signaling, implying a phylogenetically ancient requirement for ROS in regeneration.

Regeneration plays a key role in energy recycling and homeostasis maintenance. Planarians, as ideal model animals for studying regeneration, stem cell proliferation, and apoptosis, have the strong regenerative abilities. Considerable evidence suggests that ubiquitin plays an important role in maintaining homeostasis and regulating regeneration, but the function of Ubiquitin specific proteases 7 (Usp7) on regeneration in planarians remains elusive.

We identified an evolutionarily conserved gene, Usp7, and utilized RNA interference (RNAi), Quantitative real-time PCR (qRT-PCR), Whole-mount immunofluorescence, Tunnel, Whole-mount in situ hybridization (WISH), and western blotting to detect the function of Usp7 during the planarian regeneration.

In this study, we found that the regenerative trunk fragments in the Usp7 RNAi worms could not regenerate missing tails; meanwhile, the level of cell proliferation was decreased, while cell apoptosis was increased. Furthermore, the expression of Islet was inhibited in the Usp7 RNAi worms during planarian regeneration. The hybridization signal of wnt1/P-1 exhibited the dot-like pattern at the posterior of the regenerating planarians after Usp7 RNAi at regenerative 1 day (R 1 d). However, the concentrated expression pattern wnt1/P-1 dramatically declined at regenerative 3 days (R 3 d) and disappeared at regenerative 7 days (R 7 d). In addition, activating the Wnt pathway partially rescued regenerative defects induced by inhibition of Usp7.

Collectively, Usp7 is necessary for tissue regeneration and tail blastema formation partially by regulating the cell proliferation and apoptosis during planarian regeneration. It could also promote the posterior polarity reconstruction of the regenerative planarians via the Islet/Wnt1 axis.

Regeneration is the ability to accurately regrow missing body parts. The unparalleled regenerative capacity and incredible tissue plasticity of planarians, both resulting from the presence of abundant adult stem cells referred to as neoblasts, offer a unique opportunity to investigate the cellular and molecular principles underlying regeneration. Neoblasts are capable of self-renewal and differentiation into the desired cell types for correct replacement of lost parts after tissue damage. Positional information in muscle cells governs the polarity and patterning of the body plan during homeostasis and regeneration. For planarians, removal of neoblasts disables the regenerative feats and disruption of positional information results in the regeneration of inappropriate missing body regions, only the combination of neoblasts and positional information enables regeneration. Here, I summarize the current state of the field in neoblast lineage potential, subclasses and specification, and in the roles of positional information for proper tissue turnover and regeneration in planarians.

Eye loss occurs convergently in numerous animal phyla as an adaptation to dark environments. We investigate the cave planarian Girardia multidiverticulata (Gm), a representative species of the Spiralian clade, to study mechanisms of eye loss. We found that Gm, which was previously described as an eyeless species, retains rudimentary and functional eyes. Eyes are maintained in homeostasis and regenerated in adult planarians by stem cells, called neoblasts, through their fate specification to eye progenitors. The reduced number of eye cells in cave planarians is associated with a decreased rate of stem cell fate specification to eye progenitors during homeostasis and regeneration. Conversely, the homeostatic formation of new cells from stem cell-derived progenitors for other tissues, including for neurons, pharynx, and epidermis, is comparable between cave and surface species. These findings reveal a mode of evolutionary trait loss, with change in rate of fate specification in adult stem cells leading to tissue size reduction.

In holothurians, the regenerative process following evisceration involves the development of a "rudiment" or "anlage" at the injured end of the mesentery. This regenerating anlage plays a pivotal role in the formation of a new intestine. Despite its significance, our understanding of the molecular characteristics inherent to the constituent cells of this structure has remained limited. To address this gap, we employed state-of-the-art scRNA-seq and HCR-FISH analyses to discern the distinct cellular populations associated with the regeneration anlage. Through this approach, we successfully identified thirteen distinct cell clusters. Among these, two clusters exhibit characteristics consistent with putative mesenchymal cells, while another four show features akin to coelomocyte cell populations. The remaining seven cell clusters collectively form a large group encompassing the coelomic epithelium of the regenerating anlage and mesentery. Within this large group of clusters, we recognized previously documented cell populations such as muscle precursors, neuroepithelial cells and actively proliferating cells. Strikingly, our analysis provides data for identifying at least four other cellular populations that we define as the precursor cells of the growing anlage. Consequently, our findings strengthen the hypothesis that the coelomic epithelium of the anlage is a pluripotent tissue that gives rise to diverse cell types of the regenerating intestinal organ. Moreover, our results provide the initial view into the transcriptomic analysis of cell populations responsible for the amazing regenerative capabilities of echinoderms.

The present, brief review paper summarizes previous studies on a new interpretation of the presence and absence of regeneration in invertebrates and vertebrates. Broad regeneration is considered exclusive of aquatic or amphibious animals with larval stages and metamorphosis, where also a patterning process is activated for whole-body regeneration or for epimorphosis. In contrast, terrestrial invertebrates and vertebrates can only repair injury or the loss of body parts through a variable "recovery healing" of tissues, regengrow or scarring. This loss of regeneration likely derives from the change in genomes during land adaptation, which included the elimination of larval stages and intense metamorphosis. The terrestrial conditions are incompatible with the formation of embryonic organs that are necessary for broad regeneration. In fact, no embryonic organ can survive desiccation, intense UV or ROS exposition on land, and rapid reparative processes without embryonic patterning, such as recovery healing and scarring, have replaced broad regeneration in terrestrial species. The loss of regeneration in land animals likely depends on the alteration of developmental gene pathways sustaining regeneration that occurred in progenitor marine animals. Terrestrial larval stages, like those present in insects among arthropods, only metamorphose using small body regions indicated as imaginal disks, a terrestrial adaptation, not from a large restructuring process like in aquatic-related animals. These invertebrates can reform body appendages only during molting, a process indicated as regengrow, not regeneration. Most amniotes only repair injuries through scarring or a variable recovery healing, occasionally through regengrow, the contemporaneous healing in conjunction with somatic growth, forming sometimes new heteromorphic organs.

Tissue regeneration is a complex process involving large changes in cell proliferation, fate determination, and differentiation. Mitochondrial dynamics and metabolism play a crucial role in development and wound repair, but their function in large-scale regeneration remains poorly understood. Planarians offer an excellent model to investigate this process due to their remarkable regenerative abilities. In this study, we examine mitochondrial dynamics during planarian regeneration. We find that knockdown of the mitochondrial fusion gene, opa1, impairs both tissue regeneration and stem cell pluripotency. Interestingly, the regeneration defects caused by opa1 knockdown are rescued by simultaneous knockdown of the mitochondrial fission gene, drp1, which partially restores mitochondrial dynamics. Furthermore, we discover that Mitolow stem cells exhibit an enrichment of pluripotency due to their fate choices at earlier stages. Transcriptomic analysis reveals the delicate mitonuclear balance in metabolism and mitochondrial proteins in regeneration, controlled by mitochondrial dynamics. These findings highlight the importance of maintaining mitochondrial dynamics in large-scale tissue regeneration and suggest the potential for manipulating these dynamics to enhance stem cell functionality and regenerative processes.

Taurine, a normal dietary component that is found in many tissues, is considered important for a number of physiological processes. It is thought to play a particular role in eye development and in the maturation of both the muscular and nervous systems, leading to its suggested use as a therapeutic for Alzheimer's and Parkinson's diseases. Taurine increases metabolism and has also been touted as a weight loss aid. Due to its possible benefits to health and development, taurine is added as a supplement to a wide array of products, including infant formula and energy drinks. Despite its pervasive use as a nutritional additive and implied physiological actions, there is little consensus on how taurine functions. This is likely because, mechanistically, taurine has been demonstrated to affect multiple metabolic pathways. Simple models and straightforward assay systems are required to make headway in understanding this complexity. We chose to begin this work using the planarian because these animals have basic, well-understood muscular and nervous systems and are the subjects of many well-tested assays examining how their physiology is influenced by exposure to various environmental, nutritional, and therapeutic agents. We used a simple behavioral assay, the planarian locomotor velocity test (pLmV), to gain insight into the stimulant properties of taurine. Using this assay, we observed that taurine is a mild stimulant that is not affected by sugars or subject to withdrawal. We also provide evidence that taurine makes use of the dopamine D1 receptor to mediate this stimulant effect. Given the pervasiveness of taurine in many commercial products, our findings using the planarian system provide needed insight into the stimulant properties of taurine that should be considered when adding it to the diet.

Most of annelids grow all over their asexual life through the continuous addition of segments from a special zone called "segment addition zone" (SAZ) adjacent to the posterior extremity called pygidium. Amputation of posterior segments leads to regeneration (posterior regeneration-PR) of the pygidium and a new SAZ, as well as new segments issued from this new SAZ. Amputation of anterior segments leads some species to regeneration (anterior regeneration-AR) of the prostomium and a SAZ which produces new segments postero-anteriorly as during PR. During the 1960s and 1970s decades, experimental methods on different species (Syllidae, Nereidae, Aricidae) showed that the function of SAZ depends on the presence and number of mesodermal regeneration cells. Selective destruction of mesodermal regeneration cells in AR had no effect on the regeneration of the prostomium, but as for PR, it inhibited segment regeneration. Thus, worms deprived of mesodermal regeneration cells are always able to regenerate the pygidium or the prostomium, but they are unable to regenerate segments, a result which indicates that the SAZ functions only if these regeneration cells are present during PR or AR. Additionally, during AR, nerve fibres regenerate from the cut nerve cord toward the newformed brain, a situation which deprives the SAZ of local regenerating nerve fibres and their secreted growth factors. In contrast, during PR, nerve fibres regenerate both during the entire regeneration phase and then in normal growth. This review summarizes the experimental evidence for mesoderm cell involvement in segment regeneration, and the differential impact of the digestive tube and the regenerated nerve cord during PR vs AR.

Tissue homeostasis and regeneration involve complex cellular changes. The role of rRNA modification-dependent translational regulation in these processes remains largely unknown. Planarians, renowned for their ability to undergo remarkable tissue regeneration, provide an ideal model for the analysis of differential rRNA regulation in diverse cell types during tissue homeostasis and regeneration. We investigated the role of RNA 2'-O-methyltransferase, Fibrillarin (FBL), in the planarian Schmidtea mediterranea and identified two FBL homologs: Smed-fbl-1 (fbl-1) and Smed-fbl-2 (fbl-2). Both are essential for planarian regeneration, but play distinct roles: fbl-1 is crucial for progenitor cell differentiation, while fbl-2 is important for late-stage epidermal lineage specification. Different 2'-O-methylation patterns were observed upon fbl-1 and fbl-2 knockdown, suggesting their roles in translation of specific mRNA pools during regeneration. Ribo-seq analysis further revealed differing impacts of fbl-1 and fbl-2 knockdown on gene translation. These findings indicate divergent roles of the duplicate fbl genes in specific cell lineage development in planarians and suggest a role of rRNA modifications in translational regulation during tissue maintenance and 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.

Live imaging of regenerative processes can reveal how animals restore their bodies after injury through a cascade of dynamic cellular events. Here, we present a comprehensive toolkit for live imaging of tissue regeneration in the flatworm Macrostomum lignano, including a high-throughput cloning pipeline, targeted cellular ablation, and advanced microscopy solutions. Using tissue-specific reporter expression, we examine how various structures regenerate. Enabled by a custom luminescence/fluorescence microscope, we overcome intense stress-induced autofluorescence to demonstrate genetic cellular ablation and reveal the limited regenerative capacity of neurons and their essential role during wound healing, contrasting muscle cells' rapid regeneration after ablation. Finally, we build an open-source tracking microscope to continuously image freely moving animals throughout the week-long process of regeneration, quantifying kinetics of wound healing, nerve cord repair, body regeneration, growth, and behavioral recovery. Our findings suggest that nerve cord reconnection is highly robust and proceeds independently of regeneration.

Regeneration of missing body parts can be observed in diverse animal phyla, but it remains unclear to which extent these capacities rely on shared or divergent principles. Research into this question requires detailed knowledge about the involved molecular and cellular principles in suitable reference models. By combining single-cell RNA sequencing and mosaic transgenesis in the marine annelid Platynereis dumerilii, we map cellular profiles and lineage restrictions during posterior regeneration. Our data reveal cell-type specific injury responses, re-expression of positional identity factors, and the re-emergence of stem cell signatures in multiple cell populations. Epidermis and mesodermal coelomic tissue produce distinct putative posterior stem cells (PSCs) in the emerging blastema. A novel mosaic transgenesis strategy reveals both developmental compartments and lineage restrictions during regenerative growth. Our work supports the notion that posterior regeneration involves dedifferentiation, and reveals molecular and mechanistic parallels between annelid and vertebrate regeneration.

Despite significant improvements in the comprehension of neuro-regeneration, restoring nerve injury in humans continues to pose a substantial therapeutic difficulty. In the peripheral nervous system (PNS), the nerve regeneration process after injury relies on Schwann cells. These cells play a crucial role in regulating and releasing different extracellular matrix proteins, including laminin and fibronectin, which are essential for facilitating nerve regeneration. However, during regeneration, the nerve is required to regenerate for a long distance and, subsequently, loses its capacity to facilitate regeneration during this progression. Meanwhile, it has been noted that nerve regeneration has limited capabilities in the central nervous system (CNS) compared to in the PNS. The CNS contains factors that impede the regeneration of axons following injury to the axons. The presence of glial scar formation results from this unfavourable condition, where glial cells accumulate at the injury site, generating a physical and chemical barrier that hinders the regeneration of neurons. In contrast to humans, several species, such as axolotls, polychaetes, and planarians, possess the ability to regenerate their neural systems following amputation. This ability is based on the vast amount of pluripotent stem cells that have the remarkable capacity to differentiate and develop into any cell within their body. Although humans also possess these cells, their numbers are extremely limited. Examining the molecular pathways exhibited by these organisms has the potential to offer a foundational understanding of the human regeneration process. This review provides a concise overview of the molecular pathways involved in axolotl, polychaete, and planarian neuro-regeneration. It has the potential to offer a new perspective on therapeutic approaches for neuro-regeneration in humans.

In order to address a biological explanation for the different regenerative abilities present among animals, a new evolutionary speculation is presented. It is hypothesized that epigenetic mechanisms have lowered or erased regeneration during the evolution of terrestrial invertebrates and vertebrates. The hypothesis indicates that a broad regeneration can only occur in marine or freshwater conditions, and that life on land does not allow for high regeneration. This is due to the physical, chemical and microbial conditions present in the terrestrial environment with respect to those of the aquatic environment. The present speculation provides examples of hypothetic evolutionary animal lineages that colonized the land, such as parasitic annelids, terrestrial mollusks, arthropods and amniotes. These are the animals where regeneration is limited or absent and their injuries are only repaired through limited healing or scarring. It is submitted that this loss derived from changes in the developmental gene pathways sustaining regeneration in the aquatic environment but that cannot be expressed on land. Once regeneration was erased in terrestrial species, re-adaptation to freshwater niches could not reactivate the previously altered gene pathways that determined regeneration. Therefore a broad regeneration was no longer possible or became limited and heteromorphic in the derived, extant animals. Only in few cases extensive healing abilities or regengrow, a healing process where regeneration overlaps with somatic growth, have evolved among arthropods and amniotes. The present paper is an extension of previous speculations trying to explain in biological terms the different regenerative abilities present among metazoans.

Microcystin-leucine arginine (MC-LR), a representative cyanobacterial toxin, poses an increasing and serious threat to aquatic ecosystems. Despite investigating its toxic effects in various organisms and cells, the toxicity to tissue regeneration and stem cells in vivo still needs to be explored. Planarians are ideal regeneration and toxicology research models and have profound implications in ecotoxicology evaluation. This study conducted a systemic toxicity evaluation of MC-LR, including morphological changes, growth, regeneration, and the underlying cellular and molecular changes after MC-LR exposure, which were investigated in planarians. The results showed that exposure to MC-LR led to time- and dose-dependent lethal morphological changes, tissue damage, degrowth, and delayed regeneration in planarians. Furthermore, MC-LR exposure disturbed the activities of antioxidants, including total superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, and total antioxidant capacity, leading to oxidative stress and DNA damage, and then reduced the number of dividing neoblasts and promoted apoptosis. The results demonstrated that oxidative stress and DNA damage induced by MC-LR exposure caused apoptosis. Excessive apoptosis and suppressed neoblast activity led to severe homeostasis imbalance. This study explores the underlying mechanism of MC-LR toxicity in planarians and provides a basis for the toxicity assessment of MC-LR to aquatic organisms and ecological risk evaluation.

This review explores the regenerative capacity of Xenopus laevis, focusing on tail regeneration, as a model to uncover cellular, molecular, and developmental mechanisms underlying tissue repair. X. laevis tadpoles provide unique insights into regenerative biology due to their regeneration-competent and -incompetent stages and ability to regrow complex structures in the tail, including the spinal cord, muscle, and skin, after amputation. The review delves into the roles of key signaling pathways, such as those involving reactive oxygen species (ROS) and signaling molecules like BMPs and FGFs, in orchestrating cellular responses during regeneration. It also examines how mechanotransduction, epigenetic regulation, and metabolic shifts influence tissue restoration. Comparisons of regenerative capacity with other species shed light on the evolutionary loss of regenerative abilities and underscore X. laevis as an invaluable model for understanding the constraints of tissue repair in higher organisms. This comprehensive review synthesizes recent findings, suggesting future directions for exploring regeneration mechanisms, with potential implications for advancing regenerative medicine.

Proper stem cell targeting and differentiation is necessary for regeneration to succeed. In organisms capable of whole body regeneration, considerable progress has been made identifying wound signals initiating this process, but the mechanisms that control the differentiation of progenitors into mature organs are not fully understood. Using the planarian as a model system, we identify a novel function for map3k1, a MAP3K family member possessing both kinase and ubiquitin ligase domains, to negatively regulate terminal differentiation of stem cells during eye regeneration. Inhibition of map3k1 caused the formation of multiple ectopic eyes within the head, but without controlling overall head, brain, or body patterning. By contrast, other known regulators of planarian eye patterning like WntA and notum also regulate head regionalization, suggesting map3k1 acts distinctly. Eye resection and regeneration experiments suggest that unlike Wnt signaling perturbation, map3k1 inhibition did not shift the target destination of eye formation in the animal. Instead, map3k1(RNAi) ectopic eyes emerge in the regions normally occupied by migratory eye progenitors, and the onset of ectopic eyes after map3k1 inhibition coincides with a reduction to eye progenitor numbers. Furthermore, RNAi dosing experiments indicate that progenitors closer to their normal target are relatively more sensitive to the effects of map3k1, implicating this factors in controlling the site of terminal differentiation. Eye phenotypes were also observed after inhibition of map2k4, map2k7, jnk, and p38, identifying a putative pathway through which map3k1 prevents differentiation. Together, these results suggest that map3k1 regulates a novel control point in the eye regeneration pathway which suppresses the terminal differentiation of progenitors during their migration to target destinations.

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.

Understanding how neural circuits are regenerated following injury is a fundamental question in neuroscience. Hydra is a powerful model for studying this process because it has a simple neural circuit structure, significant and reproducible regenerative abilities, and established methods for creating transgenics with cell-type-specific expression. While Hydra is a long-standing model for regeneration and development, little is known about how neural activity and behavior is restored following significant injury. In this study, we ask if regenerating neurons terminally differentiate prior to reforming functional neural circuits, or if neural circuits regenerate first and then guide the constituent naive cells toward their terminal fate. To address this question, we developed a dual-expression transgenic Hydra line that expresses a cell-type-specific red fluorescent protein (tdTomato) in ec5 peduncle neurons, and a calcium indicator (GCaMP7s) in all neurons. With this transgenic line, we can simultaneously record neural activity and track the reappearance of the terminally-differentiated ec5 neurons. Using SCAPE (Swept Confocally Aligned Planar Excitation) microscopy, we monitored both calcium activity and expression of tdTomato-positive neurons in 3D with single-cell resolution during regeneration of Hydra's aboral end. The synchronized neural activity associated with a regenerated neural circuit was observed approximately 4 to 8 hours after expression of tdTomato in ec5 neurons. These data suggest that regenerating ec5 neurons undergo terminal differentiation prior to re-establishing their functional role in the nervous system. The combination of dynamic imaging of neural activity and gene expression during regeneration make Hydra a powerful model system for understanding the key molecular and functional processes involved in neural regeneration following injury.

Due to their tissue structure similar to mammalian skin and their close evolutionary relationship with chordates, holothurians (Echinodermata: Holothuroidea) are particularly interesting for studies on wound healing. However, previous studies dealing with holothuroid wound healing have had limited approaches, being restricted to tissue repair or perivisceral immune response. In this study, we combined tissue, cellular and humoral parameters to study the wound healing process of Holothuria grisea. The immune responses of the perivisceral coelom were assessed by analyzing the number, proportion and viability of coelomocytes and the volume and protein concentration of the coelomic fluid. Additionally, the morphology of the healing tissue and number of coelomocytes in the connective tissue of different body wall layers were examined over 30 days. Our results showed that perivisceral reactions started 3 h after injury and decreased to baseline levels within 24 h. In contrast, tissue responses were delayed, beginning after 12 h and returning to baseline levels only after day 10. The number of coelomocytes in the connective tissue suggests a potential cooperation between these cells during wound healing: phagocytes and acidophilic spherulocytes act together in tissue clearance/homeostasis, whereas fibroblast-like and morula cells cooperate in tissue remodeling. Finally, our results indicate that the major phases observed in mammalian wound healing are also observed in H. grisea, despite occurring at a different timing, which might provide insights for future studies. Based on these data, we propose a model that explains the entire healing process in H. grisea.

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.

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.

Planarians are an excellent model for investigating molecular mechanisms necessary for regenerating a functional nervous system. Numerous studies have led to the generation of extensive genomic resources, especially whole-animal single-cell RNA-seq resources. These have facilitated in silico predictions of neuronal subtypes, many of which have been anatomically mapped by in situ hybridization. However, our knowledge of the function of dozens of neuronal subtypes remains poorly understood. Previous investigations identified that polycystic kidney disease (pkd)-like genes in planarians are strongly expressed in sensory neurons and have roles in mechanosensation. Here, we examine the expression and function of all the pkd genes found in the Schmidtea mediterranea genome and map their expression in the asexual and hermaphroditic strains. Using custom behavioral assays, we test the function of pkd genes in response to mechanical stimulation and in food detection. Our work provides insight into the physiological function of sensory neuron populations and protocols for creating inexpensive automated setups for acquiring and analyzing mechanosensory stimulation in planarians.

Organ injuries, such as acute kidney injury, ischemic stroke, and spinal cord injury, often result in complications that can be life-threatening or even fatal. Recently, many nanomaterials have emerged as promising agents for repairing various organ injuries. In this review, we present the important developments in the field of nanomaterial-based repair medicine, herein referred to as 'nanorepair medicine'. We first introduce the disease characteristics associated with different types of organ injuries and highlight key examples of relevant nanorepair medicine. We then provide a summary of existing strategies in nanorepair medicine, including organ-targeting methodologies and potential countermeasures against exogenous and endogenous pathologic risk factors. Finally, we offer our perspectives on current challenges and future expectations for the advancement of nanomedicine designed for organ injury repair.

Regeneration in many animals involves the formation of a blastema, which differentiates and organizes into the appropriate missing body parts. Although the mechanisms underlying blastema formation are often fundamental to regeneration biology, information on the cellular and molecular basis of blastema formation remains limited. Here, we focus on a fragmenting potworm (Enchytraeus japonensis), which can regenerate its whole body from small fragments. We find soxC and mmpReg as upregulated genes in the blastema. RNAi of soxC and mmpReg reduce the number of blastema cells, indicating that soxC and mmpReg promote blastema formation. Expression analyses show that soxC-expressing cells appear to gradually accumulate in blastema and constitute a large part of the blastema. Additionally, similar expression dynamics of SoxC orthologue genes in frog (Xenopus laevis) are found in the regeneration blastema of tadpole tail. Our findings provide insights into the cellular and molecular mechanisms underlying blastema formation across species.

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.

Planarians are an excellent model for investigating molecular mechanisms necessary for regenerating a functional nervous system. Numerous studies have led to the generation of extensive genomic resources, especially whole-animal single-cell RNA-seq resources. These have facilitated in silico predictions of neuronal subtypes, many of which have been anatomically mapped by in situ hybridization. However, our knowledge of the function of dozens of neuronal subtypes remains poorly understood. Previous investigations identified that polycystic kidney disease (pkd)-like genes in planarians are strongly expressed in sensory neurons and have roles in mechanosensation. Here, we examine the expression and function of all the pkd genes found in the Schmidtea mediterranea genome and map their expression in the asexual and hermaphroditic strains. Using custom behavioral assays, we test the function of pkd genes in response to mechanical stimulation and in food detection. Our work provides insight into the physiological function of sensory neuron populations and protocols for creating inexpensive automated setups for acquiring and analyzing mechanosensory stimulation in planarians.