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Regenerative capacities and strategies vary dramatically across animals, as well as between cell types, organs, and with age. In recent years, high-throughput single-cell transcriptomics and other single-cell profiling technologies have been applied to many animal models to gain an understanding of the cellular and molecular mechanisms underlying regeneration. Here, we review recent single-cell studies of regeneration in diverse contexts and summarize key concepts that have emerged. The immense regenerative capacity of some invertebrates, exemplified by planarians, is driven mainly by the differentiation of abundant adult pluripotent stem cells, whereas in many other cases, regeneration involves the reactivation of embryonic or developmental gene-regulatory networks in differentiated cell types. However, regeneration also differs from development in many ways, including the use of regeneration-specific cell types and gene regulatory networks., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The coincidence of cell cycle exit and differentiation has been described in a wide variety of stem cells and organisms for decades, but the causal relationship is still unclear due to the complicated regulation of the cell cycle. Here, we used the planarian Dugesia japonica since they may possess a simple cell cycle regulation in which Cdh1 is one of the factors responsible for exiting the cell cycle. When cdh1 was functionally inhibited, the planarians could not maintain their tissue homeostasis and could not regenerate their missing body parts. While the knockdown of cdh1 caused pronounced accumulation of the stem cells, the progenitor and differentiated cells were decreased. Further analyses indicated that the stem cells with cdh1 knockdown did not undergo differentiation even though they received ERK signaling activation as an induction signal. These results suggested that stem cells could not acquire differentiation competence without cell cycle exit. Thus, we propose that cell cycle regulation determines the differentiation competence and that cell cycle exit to G0 enables stem cells to undergo differentiation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Hox genes are highly conserved transcription factors renowned for their roles in the segmental patterning of the embryonic anterior-posterior (A/P) axis. We report functions for Hox genes in A/P tissue segmentation and transverse fission behavior underlying asexual reproduction in adult planarian flatworms, Schmidtea mediterranea. Silencing of each of the Hox family members identifies 5 Hox genes required for asexual reproduction. Among these, silencing of hox3 genes results in supernumerary fission segments, while silencing of post2b eliminates segmentation altogether. The opposing roles of hox3 and post2b in segmentation are paralleled in their respective regulation of fission behavior. Silencing of hox3 increases the frequency of fission behavior initiation while silencing of post2b eliminates fission behavior entirely. Furthermore, we identify a network of downstream effector genes mediating Hox gene functions, providing insight into their respective mechanisms of action. In particular, we resolve roles for post2b and effector genes in the functions of the marginal adhesive organ in fission behavior regulation. Collectively, our study establishes adult stage roles for Hox genes in the regulation of tissue segmentation and behavior associated with asexual reproduction., (© 2021. The Author(s).)

All animals, other than Platyhelminthes, produce eggs containing yolk, referred to as "entolecithal" eggs. However, only Neoophora, in the phylum Platyhelminthes, produce "ectolecithal" eggs (egg capsules), in which yolk is stored in the vitelline cells surrounding oocytes. Vitelline cells are derived from vitellaria (yolk glands). Vitellaria are important reproductive organs that may be studied to elucidate unique mechanisms that have been evolutionarily conserved within Platyhelminthes. Currently, only limited molecular level information is available on vitellaria. The current study identified major vitellaria-specific proteins in a freshwater planarian, Dugesia ryukyuensis, using peptide mass fingerprinting (PMF) and expression analyses. Amino acid sequence analysis and orthology analysis via OrthoFinder ver.2.3.8 indicated that the identified major vitellaria-specific novel yolk ferritins were conserved in planarians (Tricladida). Because ferritins play an important role in Fe (iron) storage, we examined the metal elements contained in vitellaria and ectolecithal eggs, using non-heme iron histochemistry, elemental analysis based on inductively coupled plasma mass spectrometry and transmission electron microscopy- energy-dispersive X-ray spectroscopy analysis. Interestingly, vitellaria and egg capsules contained large amounts of aluminum (Al), but not Fe. The knockdown of the yolk ferritin genes caused a decrease in the volume of egg capsules, abnormality in juveniles, and increase in Al content in vitellaria. Yolk ferritins of D. ryukyuensis may regulate Al concentration in vitellaria via their pooling function of Al and protect the egg capsule production and normal embryogenesis from Al toxicity., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Complex anatomical form is regulated in part by endogenous physiological communication between cells; however, the dynamics by which gap junctional (GJ) states across tissues regulate morphology are still poorly understood. We employed a biophysical modeling approach combining different signaling molecules (morphogens) to qualitatively describe the anteroposterior and lateral morphology changes in model multicellular systems due to intercellular GJ blockade. The model is based on two assumptions for blocking-induced patterning: (i) the local concentrations of two small antagonistic morphogens diffusing through the GJs along the axial direction, together with that of an independent, uncoupled morphogen concentration along an orthogonal direction, constitute the instructive patterns that modulate the morphological outcomes, and (ii) the addition of an external agent partially blocks the intercellular GJs between neighboring cells and modifies thus the establishment of these patterns. As an illustrative example, we study how the different connectivity and morphogen patterns obtained in presence of a GJ blocker can give rise to novel head morphologies in regenerating planaria. We note that the ability of GJs to regulate the permeability of morphogens post-translationally suggests a mechanism by which different anatomies can be produced from the same genome without the modification of gene-regulatory networks. Conceptually, our model biosystem constitutes a reaction-diffusion information processing mechanism that allows reprogramming of biological morphologies through the external manipulation of the intercellular GJs and the resulting changes in instructive biochemical signals., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

The ability to respond to light has profoundly shaped life. Animals with eyes overwhelmingly rely on their visual circuits for mediating light-induced coordinated movements. Building on previously reported behaviors, we report the discovery of an organized, eye-independent (extraocular), body-wide photosensory framework that allows even a head-removed animal to move like an intact animal. Despite possessing sensitive cerebral eyes and a centralized brain that controls most behaviors, head-removed planarians show acute, coordinated ultraviolet-A (UV-A) aversive phototaxis. We find this eye-brain-independent phototaxis is mediated by two noncanonical rhabdomeric opsins, the first known function for this newly classified opsin-clade. We uncover a unique array of dual-opsin-expressing photoreceptor cells that line the periphery of animal body, are proximal to a body-wide nerve net, and mediate UV-A phototaxis by engaging multiple modes of locomotion. Unlike embryonically developing cerebral eyes that are functional when animals hatch, the body-wide photosensory array matures postembryonically in "adult-like animals." Notably, apart from head-removed phototaxis, the body-wide, extraocular sensory organization also impacts physiology of intact animals. Low-dose UV-A, but not visible light (ocular-stimulus), is able to arouse intact worms that have naturally cycled to an inactive/rest-like state. This wavelength selective, low-light arousal of resting animals is noncanonical-opsin dependent but eye independent. Our discovery of an autonomous, multifunctional, late-maturing, organized body-wide photosensory system establishes a paradigm in sensory biology and evolution of light sensing., Competing Interests: The authors declare no competing interest.

Silver nanoparticles (AgNPs) are widely incorporated in household, consumer and medical products. Their unintentional release via wastewaters raises concerns on their environmental impact, particularly for aquatic organisms and their associated bacterial communities. It is known that the microbiome plays an important role in its host's health and physiology, e.g. by producing essential nutrients and providing protection against pathogens. A thorough understanding of the effects of AgNPs on bacterial communities and on their interactions with the host is crucial to fully assess AgNP toxicity on aquatic organisms. Our results indicate that the microbiome of the invertebrate Schmidtea mediterranea, a freshwater planarian, is affected by AgNP exposure at the tested 10 μg/ml concentration. Using targeted amplification of the bacterial 16S rRNA gene V3-V4 region, two independent experiments on the microbiomes of adult worms revealed a consistent decrease in Betaproteobacteriales after AgNP exposure, mainly attributed to a decrease in Curvibacter and Undibacterium. Although developing tissues and organisms are known to be more sensitive to toxic compounds, three independent experiments in regenerating worms showed a less pronounced effect of AgNP exposure on the microbiome, possibly because underlying bacterial community changes during development mask the AgNP induced effect. The presence of a polyvinyl-pyrrolidone (PVP) coating did not significantly alter the outcome of the experiments compared to those with uncoated particles. The observed variation between the different experiments underlines the highly variable nature of microbiomes and emphasises the need to repeat microbiome experiments, within and between physiological states of the animal., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Freshwater planarians are a powerful model organism for the study of animal regeneration, stem cell maintenance and differentiation, and the development and functions of several highly conserved complex tissues. At the same time, planarians are easy to maintain, inexpensive to propagate, and reasonably macroscopic (1 mm to 1 cm in length), making them excellent organisms to use in both complex academic research and hands-on teaching laboratories. Here, we provide a detailed description of how to maintain and propagate these incredibly versatile animals in any basic laboratory setting. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Salt solution preparation Basic Protocol 2: Cleaning planarian housing Basic Protocol 3: Food preparation Basic Protocol 4: Feeding planarians Basic Protocol 5: Expansion and amplification of colony., (© 2020 Wiley Periodicals LLC.)

The planarian flatworm is an ideal model to study the regeneration due to its robust regenerative ability. A variety of cellular response activities have been reported to be involved in the regeneration process, including the mitogen-activated protein kinase (MAPK) signaling. However, the mechanism of MAPK pathway in regenerative responses is still unclear. In this study, we employed the planarian, Dugesia japonica, as the model to investigate the function of MAP-extracellular signal-regulated kinase (MEK), an important component of MAPK signaling pathway, in the regeneration process. We found that MEK was required for the missing tissue response after several amputation and subsequent regeneration. MEK not only affected the size of blastema in the early stage of regeneration by regulating stem cell proliferation, but also determined the planarian's regeneration through balancing cell proliferation and apoptosis. In addition, the activation of Wnt pathway partially rescued regenerative defects induced by inhibition of MEK. Taken together, our results highlight a crucial role of MEK signaling in the planarian regeneration., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Some animals, such as planaria, can regenerate complex anatomical structures in a process regulated by genetic and biophysical factors, but additional external inputs into regeneration remain to be uncovered. Microbial communities inhabiting metazoan organisms are important for metabolic, immune, and disease processes, but their instructive influence over host structures remains largely unexplored. Here, we show that Aquitalea sp. FJL05, an endogenous commensal bacterium of Dugesia japonica planarians, and one of the small molecules it produces, indole, can influence axial and head patterning during regeneration, leading to regeneration of permanently two-headed animals. Testing the impact of indole on planaria tissues via RNA sequencing, we find that indole alters the regenerative outcomes in planarians through changes in expression to patterning genes, including a downregulation of Wnt pathway genes. These data provide a unique example of the product of a commensal bacterium modulating transcription of patterning genes to affect the host's anatomical structure during regeneration., Competing Interests: Declaration of competing interest We declare that none of the authors have competing financial or non-financial interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Regeneration involves regulating tissue proportionality across considerable size ranges through unknown mechanisms. In planarians, which scale reversibly over 40× through regeneration, we identify the Striatin-interacting phosphatase and kinase (STRIPAK) complex as a potent negative regulator of axis length. Inhibition of two proteins in the STRIPAK complex, mob4 and striatin, dramatically increased posterior length, through expansion of a posterior wnt1+ signaling center within midline muscle cells. wnt1 was required for tail expansion after mob4 inhibition and dynamically reestablishes proportionality after amputation in normal animals, indicating STRIPAK represses Wnt signaling for scaling. Regulation of wnt1 expansion was stem cell dependent, demonstrating that control of signaling-center production through stem cell differentiation underlies proportional growth in adult regenerative tissue., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The freshwater planarian mostly lives in the upper reaches of springs and rivers. Generally, it is realized as a suitable warning indicator of environmental toxicants. The freshwater planarian Dugesia japonica has a powerful regenerative capability and can regenerate a new individual including a complete central nervous system in one week. Rapamycin is an inhibitor of mammalian TORC1 (target of rapamycin complex-1) and used in the treatment of some diseases like cancer, cardiovascular and neurological diseases. However, the roles of rapamycin in the regulation of planarian regeneration remain to be elucidated. In present study, freshwater planarians D. japonica were firstly treated with 1 μM rapamycin for 18 h exposures and the expression patterns of Djtor was analyzed by the whole-mount in situ hybridization (WISH). Our results indicated rapamycin could strongly inhibit Djtor expression in planarian D. japonica and cause asymmetric blastemas and neuronal defects in planarians. Furthermore, knockdown of Djtor gene in planarians using RNA interference resulted in the suppression of downstream autophagy genes. These findings suggested that rapamycin might regulate freshwater planarian regeneration via Djtor signaling pathway., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Wnt signaling regulates primary body axis formation across the Metazoa, with high Wnt signaling specifying posterior identity. Whether a common Wnt-driven transcriptional program accomplishes this broad role is poorly understood. We identified genes acutely affected after Wnt signaling inhibition in the posterior of two regenerative species, the planarian Schmidtea mediterranea and the acoel Hofstenia miamia, which are separated by >550 million years of evolution. Wnt signaling was found to maintain positional information in muscle and regional gene expression in multiple differentiated cell types. sp5, Hox genes, and Wnt pathway components are down-regulated rapidly after β-catenin RNAi in both species. Brachyury, a vertebrate Wnt target, also displays Wnt-dependent expression in Hofstenia. sp5 inhibits trunk gene expression in the tail of planarians and acoels, promoting separate tail-trunk body domains. A planarian posterior Hox gene, Post-2d, promotes normal tail regeneration. We propose that common regulation of a small gene set-Hox, sp5, and Brachyury-might underlie the widespread utilization of Wnt signaling in primary axis patterning across the Bilateria., Competing Interests: The authors have declared that no competing interests exist.

Differential coordination of growth and patterning across metazoans gives rise to a diversity of sizes and shapes at tissue, organ and organismal levels. Although tissue size and tissue function can be interdependent 1-5 , mechanisms that coordinate size and function remain poorly understood. Planarians are regenerative flatworms that bidirectionally scale their adult body size 6,7 and reproduce asexually, via transverse fission, in a size-dependent manner 8-10 . This model offers a robust context to address the gap in knowledge that underlies the link between size and function. Here, by generating an optimized planarian fission protocol in Schmidtea mediterranea, we show that progeny number and the frequency of fission initiation are correlated with parent size. Fission progeny size is fixed by previously unidentified mechanically vulnerable planes spaced at an absolute distance along the anterior-posterior axis. An RNA interference screen of genes for anterior-posterior patterning uncovered components of the TGFβ and Wnt signalling pathways as regulators of the frequency of fission initiation rather than the position of fission planes. Finally, inhibition of Wnt and TGFβ signalling during growth altered the patterning of mechanosensory neurons-a neural subpopulation that is distributed in accordance with worm size and modulates fission behaviour. Our study identifies a role for TGFβ and Wnt in regulating size-dependent behaviour, and uncovers an interdependence between patterning, growth and neurological function.

Silver nanoparticles (AgNPs) belong to the most commercialized nanomaterials, used in both consumer products and medical applications. Despite its omnipresence, in-depth knowledge on the potential toxicity of nanosilver is still lacking, especially for developing organisms. Research on vertebrates is limited due to ethical concerns, and planarians are an ideal invertebrate model to study the effects of AgNPs on stem cells and developing tissues in vivo , as regeneration mimics development by triggering massive stem cell proliferation. Our results revealed a strong interference of AgNPs with tissue- and neuroregeneration which was related to an altered stem cell cycle. The presence of a PVP-coating significantly influenced toxicity outcomes, leading to elevated DNA-damage and decreased stem cell proliferation. Non-coated AgNPs had an inhibiting effect on stem cell and early progeny numbers. Overall, regenerating tissues were more sensitive to AgNP toxicity, and careful handling and appropriate decision making is needed in AgNP applications for healing and developing tissues. We emphasize on the importance of AgNP characterization, as we showed that changes in physicochemical properties influence toxicity.

Freshwater planarians are well known for their remarkable plasticity and regenerative capabilities. Most studies of planarian regeneration have specifically examined regeneration after transverse or longitudinal sectioning or during homeostasis in intact adults. However, tissue transplantation, first performed over a century ago, constitutes another important tool in the study of regeneration in planarians, and can be easily performed given this species' extraordinary healing capacity and its lack of a circulatory system. Studies conducted to date have demonstrated the viability of transplantations involving a variety of tissue types of different positional identities, affecting any of the 3 main body axes. Moreover, these grafting experiments have shown that tissues possess axial positional identities, which are retained following transplantation. The confrontation between different positional identities that occurs after any type of tissue transplantation is resolved by the formation of a blastema, consisting of undifferentiated tissue produced by adult pluripotent stem cells (neoblasts). This blastema intercalates the positional identities of the graft and host tissues. The recent discovery of pathways involved in planarian growth, patterning, and organogenesis, as well as corresponding molecular markers, makes tissue transplantation a vital new tool with which to explore pattern formation. Here, we discuss the different grafting approaches used in planarians, and the corresponding intercalary regenerative response, placing particular emphasis on the respective contributions of donor and host tissue. Moreover, we discuss the temporal induction of blastema formation, and present new molecular data on the generation of an ectopic anterior/posterior axis in response to dorsal/ventral confrontations between host and donor tissue., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Planarians are flatworms capable of regenerating any missing body part in a process requiring stem cells and positional information. Muscle is a major source of planarian positional information and consists of several types of fibers with distinct regulatory roles in regeneration. The transcriptional regulatory programs used to specify different muscle fibers are poorly characterized. Using single-cell RNA sequencing, we define the transcriptomes of planarian dorsal-ventral muscle (DVM), intestinal muscle (IM), and pharynx muscle. This analysis identifies foxF-1, which encodes a broadly conserved Fox-family transcription factor, as a master transcriptional regulator of all non-body wall muscle. The transcription factors encoded by nk4 and gata4/5/6-2 specify two different subsets of DVM, lateral and medial, respectively, whereas gata4/5/6-3 specifies IM. These muscle types all express planarian patterning genes. Both lateral and medial DVM are required for medial-lateral patterning in regeneration, whereas medial DVM and IM have a role in maintaining and regenerating intestine morphology. In addition to the role in muscle, foxF-1 is required for the specification of multiple cell types with transcriptome similarities, including high expression levels of cathepsin genes. These cells include pigment cells, glia, and several other cells with unknown function. cathepsin + cells phagocytose E. coli, suggesting these are phagocytic cells. In conclusion, we describe a regulatory program for planarian muscle cell subsets and phagocytic cells, both driven by foxF-1. FoxF proteins specify different mesoderm-derived tissues in other organisms, suggesting that FoxF regulates formation of an ancient and broadly conserved subset of mesoderm derivatives in the Bilateria., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The planarian epidermis provides an excellent model to explore adult stem cell (ASC) lineage development due to well-characterized and distinct spatiotemporal phases during lineage progression. Using flow cytometry-isolated cells enriched in epidermal progenitors, we performed transcriptional profiling and RNAi screening to uncover regulators of epidermal differentiation. We identified a MYB-type transcription factor (Smed-myb-1) required for the specification of the first temporal phase of post-mitotic maturation. Knockdown of myb-1 abolished the early progenitor phase of differentiation without ceasing production of subsequent epidermal progenitor states or homeostatic turnover and regeneration of the epidermis. Further examination revealed accelerated maturation of ASC descendants, with premature entry into subsequent progeny phases and, ultimately, the epidermis. These results demonstrate that a spatiotemporal shift in lineage progression occurs in the absence of the early progenitor state after myb-1 RNAi, and identify myb-1 as a critical regulator of the early temporal window in stepwise specification during planarian epidermal differentiation., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Most animals hold the ability to regenerate damaged cells, tissues, and even any lost part of their bodies. To date, there is little known about the precise regulatory mechanism of regeneration and many fundamental questions remain unanswered. To further understand the precise regulatory mechanism of regeneration, we used planarian Dugesia japonica as a model and sequenced the transcriptomes of their regenerated tissues at different regeneration stages. Through de novo assembly and expression profiling, we found that Heat shock protein and MAPK pathway were involved into early response of regeneration in D. japonica. In addition, immune response, cell proliferation, and migration were activated during regeneration. Of notes, our results revealed a specific functional role of programmed cell death (PCD) in regeneration of D. japonica. PCD may not only remove the damaged and superfluous tissues for further patterning with regenerated tissues, but also provide signals to trigger neoblasts proliferation and differentiation directly. Together, our results revealed Heat shock protein and MAPK pathway mediated early response of regeneration and found a dual role of PCD in regeneration D. japonica. Meanwhile, we constructed regulatory networks of apoptosis, autophagy, and related signaling pathways and proposed a schematic model, which provided a global landscape of regeneration., (© 2018 Wiley Periodicals, Inc.)

Most animals undergo homeostatic tissue maintenance, yet those capable of robust regeneration in adulthood use mechanisms significantly overlapping with homeostasis. Here we show in planarians that modulations to body-wide patterning systems shift the target site for eye regeneration while still enabling homeostasis of eyes outside this region. The uncoupling of homeostasis and regeneration, which can occur during normal positional rescaling after axis truncation, is not due to altered injury signaling or stem cell activity, nor specific to eye tissue. Rather, pre-existing tissues, which are misaligned with patterning factor expression domains, compete with properly located organs for incorporation of migratory progenitors. These observations suggest that patterning factors determine sites of organ regeneration but do not solely determine the location of tissue homeostasis. These properties provide candidate explanations for how regeneration integrates pre-existing tissues and how regenerative abilities could be lost in evolution or development without eliminating long-term tissue maintenance and repair., Competing Interests: EH, CP No competing interests declared, (© 2018, Hill et al.)

Planarians have become widely recognized as one of the major animal models for regeneration studies in invertebrates. To induce RNA interference (RNAi) by feeding in planarians, the widely accepted protocol is one in which animals undergo two or three feedings of food containing double-stranded RNA (dsRNA) plus visible food coloring (e.g., blood) for confirmation of feeding by individual animals. However, one possible problem is that incorporated food coloring is often retained within the gut for several days, which makes it difficult to confirm the success of each round of dsRNA feeding based on the difference of the color density within the gut before and after feeding. As a consequence, the difference of appetite levels among individuals undergoing dsRNA feeding leads to phenotypic variability among them due to insufficient knockdown. In our attempts to overcome this problem, we have developed a novel method for achieving robust confirmation of the success of dsRNA feeding in individuals fed multiple times by means of including a combination of three different colored chalks (pink, yellow and blue) as food coloring. Notably, we found that this method is superior to the conventional method for positively marking individuals that actively consumed the dsRNA-containing food during four times of once-daily feeding. Using these selected animals, we obtained stable and sufficiently strong RNAi-induced phenotypes. We termed this improved multi-colored chalk-spiked method of feeding RNAi "Candi" and propose its benefits for gene function analysis in planarians., (© 2017 Japanese Society of Developmental Biologists.)

Planaria, particularly Schmidtea mediterranea and Dugesia japonica, are now established as research organisms in many laboratories across the life sciences community. Planarians are cheap and easy to keep in the lab. This chapter provides techniques and guidelines for establishing and maintaining a planarian colony. We provide sections on food preparation, housing, feeding, cleaning, culture expansion by amputation, and recognizing and responding to culture problems.

Many pigment cells acquire unique structural properties and gene expression profiles during animal development. The underlying differentiation pathways have been well characterized in cells formed during embryogenesis, such as the neural crest-derived melanocyte. However, much less is known about the developmental origins of pigment cells produced in adult organisms during tissue homeostasis and repair. Here we report a lineage analysis of ommochrome- and porphyrin-producing cells in the brown, freshwater planarian Schmidtea mediterranea Using an RNA-sequencing approach, we identified two classes of markers expressed in sequential fashion when new pigment cells are generated during regeneration or in response to pigment cell ablation. We also report roles for FOXF-1 and ETS-1 transcription factors, as well as for an FGFR-like molecule, in the specification and maintenance of this cell type. Together, our results provide insights into mechanisms of adult pigment cell development in the strikingly colorful Platyhelminthes phylum., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Asexual freshwater planarians reproduce by tearing themselves into two pieces by a process called binary fission. The resulting head and tail pieces regenerate within about a week, forming two new worms. Understanding this process of ripping oneself into two parts poses a challenging biomechanical problem. Because planarians stop "doing it" at the slightest disturbance, this remained a centuries-old puzzle. We focus on Dugesia japonica fission and show that it proceeds in three stages: a local constriction ("waist formation"), pulsation-which increases waist longitudinal stresses-and transverse rupture. We developed a linear mechanical model with a planarian represented by a thin shell. The model fully captures the pulsation dynamics leading to rupture and reproduces empirical time scales and stresses. It asserts that fission execution is a mechanical process. Furthermore, we show that the location of waist formation, and thus fission, is determined by physical constraints. Together, our results demonstrate that where and how a planarian rips itself apart during asexual reproduction can be fully explained through biomechanics., Competing Interests: The authors declare no conflict of interest.

β-Catenin, the core element of the Wnt/β-catenin pathway, is a multifunctional and evolutionarily conserved protein which performs essential roles in a variety of developmental and homeostatic processes. Despite its crucial roles, the mechanisms that control its context-specific functions in time and space remain largely unknown. The Wnt/β-catenin pathway has been extensively studied in planarians, flatworms with the ability to regenerate and remodel the whole body, providing a 'whole animal' developmental framework to approach this question. Here we identify a C-terminally truncated β-catenin (β-catenin4), generated by gene duplication, that is required for planarian photoreceptor cell specification. Our results indicate that the role of β-catenin4 is to modulate the activity of β-catenin1, the planarian β-catenin involved in Wnt signal transduction in the nucleus, mediated by the transcription factor TCF-2. This inhibitory form of β-catenin, expressed in specific cell types, would provide a novel mechanism to modulate nuclear β-catenin signaling levels. Genomic searches and in vitro analysis suggest that the existence of a C-terminally truncated form of β-catenin could be an evolutionarily conserved mechanism to achieve a fine-tuned regulation of Wnt/β-catenin signaling in specific cellular contexts.

Understanding how some animals are immortal and avoid the ageing process is important. We currently know very little about how they achieve this. Research with genetic model systems has revealed the existence of conserved genetic pathways and molecular processes that affect longevity. Most of these established model organisms have relatively short lifespans. Here we consider the use of planarians, with an immortal life-history that is able to entirely avoid the ageing process. These animals are capable of profound feats of regeneration fueled by a population of adult stem cells called neoblasts. These cells are capable of indefinite self-renewal that has underpinned the evolution of animals that reproduce only by fission, having disposed of the germline, and must therefore be somatically immortal and avoid the ageing process. How they do this is only now starting to be understood. Here we suggest that the evidence so far supports the hypothesis that the lack of ageing is an emergent property of both being highly regenerative and the evolution of highly effective mechanisms for ensuring genome stability in the neoblast stem cell population. The details of these mechanisms could prove to be very informative in understanding how the causes of ageing can be avoided, slowed or even reversed., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Regeneration requires the precise integration of cues that initiate proliferation, direct differentiation, and ultimately re-pattern tissues to the proper size and scale. Yet how these processes are integrated with wounding responses remains relatively unknown. The freshwater planarian, Schmidtea mediterranea, is an ideal model to study the stereotyped proliferative and transcriptional responses to injury due to its high capacity for regeneration. Here, we characterize the effector of the Hippo signalling cascade, yorkie, during planarian regeneration and its role in restricting early injury responses. In yki(RNAi) regenerating animals, wound responses are hyper-activated such that both stem cell proliferation and the transcriptional wound response program are heighted and prolonged. Using this observation, we also uncovered novel wound-induced genes by RNAseq that were de-repressed in yki(RNAi) animals compared with controls. Additionally, we show that yki(RNAi) animals have expanded epidermal and muscle cell populations, which we hypothesize are the increased sources of wound-induced genes. Finally, we show that in yki(RNAi) animals, the sensing of the size of an injury by eyes or the pharynx is not appropriate, and the brain, gut, and midline cannot remodel or scale correctly to the size of the regenerating fragment. Taken together, our results suggest that yki functions as a key molecule that can integrate multiple aspects of the injury response including proliferation, apoptosis, injury-induced transcription, and patterning.

Cytoplasmic polyadenylation is a mechanism of mRNA regulation prevalent in metazoan germ cells; it is largely dependent on Cytoplasmic Polyadenylation Element Binding proteins (CPEBs). Two CPEB homologs were identified in the planarian Schmidtea mediterranea. Smed-CPEB1 is expressed in ovaries and yolk glands of sexually mature planarians, and required for oocyte and yolk gland development. In contrast, Smed-CPEB2 is expressed in the testes and the central nervous system; its function is required for spermatogenesis as well as non-autonomously for development of ovaries and accessory reproductive organs. Transcriptome analysis of CPEB knockdown animals uncovered a comprehensive collection of molecular markers for reproductive structures in S. mediterranea, including ovaries, testes, yolk glands, and the copulatory apparatus. Analysis by RNA interference revealed contributions for a dozen of these genes during oogenesis, spermatogenesis, or capsule formation. We also present evidence suggesting that Smed-CPEB2 promotes translation of Neuropeptide Y-8, a prohormone required for planarian sexual maturation. These findings provide mechanistic insight into potentially conserved processes of germ cell development, as well as events involved in capsule deposition by flatworms., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Puromycin-sensitive aminopeptidase (PSA) belongs to the M1 zinc metallopeptidase family. PSA is the most abundant aminopeptidase in the brain and plays a role in the metabolism of neuropeptides including those involved in neurodegeneration. A cDNA DjPsa was identified from the planarian Dugesia japonica cDNA library. It contains a 639-bp open reading frame corresponding to a deduced protein of 212 amino acids. Whole mount in situ hybridization revealed that DjPsa is expressed in the brain and ventral nerve cords of intact and regenerating animals and demonstrates a tissue and stage-specific expression pattern of DjPsa in developing embryos and larvae. Knocking down DjPsa gene expression with RNA interference during planarian regeneration inhibits the brain reformation completely. The results suggest that DjPsa is required for planarian brain regeneration.

Placenta specific protein 8 (Plac8) has been well studied in vertebrates, yet little is known in invertebrates. In this study, a novel Plac8 from the planarian Dugesia japonica was identified and its functions in immune responses and development were characterized. Our results show that Djplac8 was expressed in the pharynx, epidermis and intestine of intact adult planarian. The expression of DjPlac8 increased significantly upon lipopolysaccharide (LPS) challenge, and inhibited the growth of the Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), suggesting the role of Plac8 in immune response. Spatial and temporal expression and distribution of DjPlac8 mRNA in regenerated planarians indicates that DjPlac8 was mainly expressed in the pharynx. In situ hybridization also revealed the elevated expression of the DjPlac8 gene in the embryonic pharynx, germ band and parenchyma cells, indicating an important role in embryonic development of D. japonica. When DjPlac8 was deactivated by RNA interference-mediated knockdown, the head of planarians underwent abnormal development. In summary, we identified DjPlac8 as a novel multifunctional protein that plays essential roles in immune response and development of planarians., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Planarians have a long history in the fields of developmental and regenerative biology. These animals have also sparked interest in neuroscience due to their neuroanatomy, spectrum of simple behaviors, and especially, their almost unparalleled ability to generate new neurons after any type of injury. Research in adult planarians has revealed that neuronal subtypes homologous to those found in vertebrates are generated from stem cells throughout their lives. This feat is recapitulated after head amputation, wherein animals are capable of regenerating whole brains and regaining complete neural function. In this review, we summarize early studies on the anatomy and function of the planarian nervous system and discuss our present knowledge of the molecular mechanisms governing neurogenesis in planarians. Modern studies demonstrate that the transcriptional programs underlying neuronal specification are conserved in these remarkable organisms. Thus, planarians are outstanding models to investigate questions about how stem cells can replace neurons in vivo. WIREs Dev Biol 2017, 6:e266. doi: 10.1002/wdev.266 For further resources related to this article, please visit the WIREs website., (© 2017 Wiley Periodicals, Inc.)

Many metazoans start germ cell development during embryogenesis, while some metazoans possessing pluripotent stem cells undergo postembryonic germ cell development. The latter reproduce asexually but develop germ cells from pluripotent stem cells or dormant primordial germ cells when they reproduce sexually. Sexual induction of the planarian Dugesia ryukyuensis is an important model for postembryonic germ cell development. In this experimental system, hermaphroditic reproductive organs are differentiated in presumptive gonadal regions by the administration of a crude extract from sexual planarians to asexual ones. However, the substances involved in the first event during postembryonic germ cell development, i.e., ovarian development, remain unknown. Here, we aimed to identify a bioactive compound associated with postembryonic ovarian development. Bioassay-guided fractionation identified ʟ-tryptophan (Trp) on the basis of electrospray ionization-mass spectrometry, circular dichroism, and nuclear magnetic resonance spectroscopy. Originally masked by a large amount of ʟ-Trp, ᴅ-Trp was detected by reverse-phase high-performance liquid chromatography. The ovary-inducing activity of ᴅ-Trp was 500 times more potent than that of ʟ-Trp. This is the first report describing a role for an intrinsic ᴅ-amino acid in postembryonic germ cell development. Our findings provide a novel insight into the mechanisms of germ cell development regulated by low-molecular weight bioactive compounds.

Planarians provide the ideal model for studying eye development, with their simple eye structure and exceptionally rapid regeneration. Here, we observed the eye morphogenesis, photophobic behavior, spectral sensitivity and expression pattern of Djopsin in the freshwater planarian Dugesia japonica. The results showed that: (i) Djopsin encoding the putative protein belonged to the rhabdomeric opsins group and displayed high conservation during animal evolution; (ii) planarians displayed diverse photophobic response to different visible wavelengths and were more sensitive to light blue (495 nm) and yellow (635 nm); (iii) the morphogenesis and functional recovery of eyes were related to the expression pattern of Djopsin during head regeneration; and (iv) Djopsin gene plays a major role in functional recovery during eye regeneration and visual system maintenance in adult planarians., (© 2016 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)

The β-catenin-dependent Wnt pathway exerts multiple context-dependent roles in embryonic and adult tissues. In planarians, β-catenin-1 is thought to specify posterior identities through the generation of an anteroposterior gradient. However, the existence of such a gradient has not been directly demonstrated. Here, we use a specific polyclonal antibody to demonstrate that nuclear β-CATENIN-1 exists as an anteroposterior gradient from the pre-pharyngeal region to the tail of the planarian Schmidtea polychroa High levels in the posterior region steadily decrease towards the pre-pharyngeal region but then increase again in the head region. During regeneration, β-CATENIN-1 is nuclearized in both anterior and posterior blastemas, but the canonical WNT1 ligand only influences posterior nuclearization. Additionally, β-catenin-1 is required for proper anterior morphogenesis, consistent with the high levels of nuclear β-CATENIN-1 observed in this region. We further demonstrate that β-CATENIN-1 is abundant in developing and differentiated organs, and is particularly required for the specification of the germline. Altogether, our findings provide the first direct evidence of an anteroposterior nuclear β-CATENIN-1 gradient in adult planarians and uncover novel, context-dependent roles for β-catenin-1 during anterior regeneration and organogenesis., (© 2016. Published by The Company of Biologists Ltd.)

The molecular mechanisms underlying whole-body regeneration are best understood in the planarian flatworm Schmidtea mediterranea, where a heterogeneous population of somatic stem cells called neoblasts provides new tissue for regeneration of essentially any missing body part. Studies on Schmidtea have provided a detailed description of neoblasts and their role in regeneration, but comparatively little is known about the evolutionary history of these cells and their underlying developmental programs. Acoels, an understudied group of aquatic worms that are also capable of extensive whole-body regeneration, have arisen as an attractive group to study the evolution of regenerative processes due to their phylogenetically distant position relative to flatworms. Here, we review the phylogenetic distribution of neoblast cells and compare their anatomical locations, transcriptional profiles, and roles during regeneration in flatworms and acoels to understand the evolution of whole-body regeneration. While the general role of neoblasts appears conserved in species separated by 550 million years of evolution, the extrinsic inputs they receive during regeneration can vary, making the distinction between homology and convergence of mechanism unclear. A more detailed understanding of the precise mechanisms behind whole-body regeneration in diverse phyla is necessary to understand the evolutionary history of this powerful process., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Collectively, planarian stem cells (neoblasts) are totipotent and are required for tissue homeostasis and regeneration. Recent work has begun to test the long-standing question of whether all neoblasts have the same potential, or whether they actually represent molecularly distinct subpopulations with distinct tissue restriction. Here, we summarize the current state of the field in neoblast lineage organization. It is clear that at least some neoblasts are totipotent, whereas other neoblasts represent functionally distinct molecular subclasses with restricted potential. In addition to neoblast subclasses, tissue-specific progenitors have also been identified, though their ability to proliferate is largely unknown. Together, neoblast lineage development, subclasses, and cell hierarchies are becoming elucidated, showing the complex regulation required for proper tissue homeostasis and regeneration in planarians., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Planarians display remarkable plasticity in maintenance of their germline, with the ability to develop or dismantle reproductive tissues in response to systemic and environmental cues. Here, we investigated the role of G protein-coupled receptors (GPCRs) in this dynamic germline regulation. By genome-enabled receptor mining, we identified 566 putative planarian GPCRs and classified them into conserved and phylum-specific subfamilies. We performed a functional screen to identify NPYR-1 as the cognate receptor for NPY-8, a neuropeptide required for sexual maturation and germ cell differentiation. Similar to NPY-8, knockdown of this receptor results in loss of differentiated germ cells and sexual maturity. NPYR-1 is expressed in neuroendocrine cells of the central nervous system and can be activated specifically by NPY-8 in cell-based assays. Additionally, we screened the complement of GPCRs with expression enriched in sexually reproducing planarians, and identified an orphan chemoreceptor family member, ophis, that controls differentiation of germline stem cells (GSCs). ophis is expressed in somatic cells of male and female gonads, as well as in accessory reproductive tissues. We have previously shown that somatic gonadal cells are required for male GSC specification and maintenance in planarians. However, ophis is not essential for GSC specification or maintenance and, therefore, defines a secondary role for planarian gonadal niche cells in promoting GSC differentiation. Our studies uncover the complement of planarian GPCRs and reveal previously unappreciated roles for these receptors in systemic and local (i.e., niche) regulation of germ cell development.

Few animals are known to lay eggs in the absence of ovulation or copulation, as it is presumably energetically wasteful and subjected to negative selection. Characterization of Smed-boule, a member of the DAZ family of germline RNA-binding proteins, revealed that egg capsule (or capsule) production and deposition occurs independently of the presence of gametes in the planarian flatworm Schmidtea mediterranea. Reduction of Smed-boule expression by RNA-interference (RNAi) causes ablation of spermatogonial stem cells and the inability of ovarian germline stem cells to undergo oogenesis. Although animals subjected to Smed-boule RNAi lose their gametes and become sterile, they continue to lay egg capsules. Production of sterile capsules is even observed in virgin Smed-boule(RNAi) and control planarians maintained in complete isolation, demonstrating that egg production in S. mediterranea occurs independently of ovulation, fertilization, or mating. Evidence suggests that this is a conserved feature amongst Platyhelminthes, and therefore relevant to the pathology and dissemination of parasitic flatworms. These findings demonstrate that Smed-boule functions at different stages during male and female germline stem cell development, and also demonstrate that egg capsule production by planarian flatworms occurs independently of signals produced by mating or ova.

How positional information instructs adult tissue maintenance is poorly understood. Planarians undergo whole-body regeneration and tissue turnover, providing a model for adult positional information studies. Genes encoding secreted and transmembrane components of multiple developmental pathways are predominantly expressed in planarian muscle cells. Several of these genes regulate regional identity, consistent with muscle harboring positional information. Here, single-cell RNA-sequencing of 115 muscle cells from distinct anterior-posterior regions identified 44 regionally expressed genes, including multiple Wnt and ndk/FGF receptor-like (ndl/FGFRL) genes. Two distinct FGFRL-Wnt circuits, involving juxtaposed anterior FGFRL and posterior Wnt expression domains, controlled planarian head and trunk patterning. ndl-3 and wntP-2 inhibition expanded the trunk, forming ectopic mouths and secondary pharynges, which independently extended and ingested food. fz5/8-4 inhibition, like that of ndk and wntA, caused posterior brain expansion and ectopic eye formation. Our results suggest that FGFRL-Wnt circuits operate within a body-wide coordinate system to control adult axial positioning.

Introduction of alien species may significantly affect soil ecosystems, through predation or disruption of components of native ecosystems (Winsor et al. 2004; Álvarez-Presas et al. 2014; Justine et al. 2014). Land planarians have been reported as alien species in soils throughout the world and, among those, some species are considered to be successful invaders, e.g. Platydemus manokwari de Beauchamp, 1963, Arthurdendyus triangulatus (Dendy, 1894), Bipalium adventitium Hyman, 1943, Bipalium kewense Moseley, 1878 and Dolichoplana striata Moseley, 1877 (Winsor et al. 2004; Álvarez-Presas et al. 2014; Justine et al. 2014, 2015). Soil moisture status seems to be an important element for their successful invasion (Fraser & Boag 1998). In Europe at least 18 species of alien land planarians have been recorded since now and some of them are considered as invasive ones, e.g. P. manokwari (cf. Justine et al. 2014). Although the alien land planarian B. kewense has been reported to occur in many greenhouses in Italy (Bello et al. 1995), no data are available on its establishment and/or impact on natural environments. On 28 th September 2014, 20 specimens (~1 individual/m 2 ) of the land planarian Diversibipalium multilineatum (Makino & Shirasawa, 1983) (Fig. 1), native to Japan, were collected under pots, branches and plastic materials in a private garden located in the center of Bologna (Emilia Romagna, Central Italy), near the urban park Giardini Margherita (44°29' N, 11°21' E; WGS84). Thirty plant species (both indigenous and alien), mainly cultivated as bonsai (e.g. Lagerstroemia indica L., Juniperus procumbens (Siebold ex Endl.) Miquel), were present in this shady, wet garden (25 m 2 ). Between March 2014 and June 2015, 70 more specimens of D. multilineatum were collected at the same site, mainly at dusk and dawn after rain. Reproduction by fission and regeneration processes were observed in several of those specimens, which were kept for some time in captivity. A specimen of D. multilineatum was also collected in a garden in Léguevin (Haute-Garonne, France), which will be described in a forthcoming paper by Justine et al. (in prep.) (see also Kawakatsu et al. 2014). Specimens without a genital pore were initially ascribed to D. multilineatum on the basis of their external appearance: the dorsal surface was brownish yellow and presented five longitudinal stripes at the head plate and the neck, showing the typical appearance of the species. The middorsal stripe was widened at its anterior end, on the head plate, and at the pharynx level. The ventral pattern of the animals at the pharyngeal region was also characteristic, with the middorsal stripe widened at this level. The Italian Diversibipalium specimens used for the molecular analysis were fixed and preserved in absolute ethanol. Fragments of the mitochondrial gene COI and 28S ribosomal RNA nuclear gene (GenBank Acc. Numbers KU245358 and KU245357, respectively) were obtained using the procedure and COI primers described in Álvarez-Presas et al. (2008) and Solà et al. (2013). The French specimen's COI (Specimen MNHN JL177, GenBank Acc. Number KT922162) was obtained as described in Justine et al. (2015). 28S sequences of 14 Bipaliinae specimens and four Microplana species (outgroup) retrieved from GenBank were included in the phylogenetic analyses (Fig. 2). Sequence alignment was obtained by using the online software MAFFT version 7 (Katoh & Standley 2013), while ambiguously aligned positions were removed using the program Gblocks (Talavera & Castresana 2007) with default settings, excepting the minimum number of sequences for a flank position at the minimum value (set at 10) and with half of the allowed gap positions. The final alignment had a length of 1589 bp. We used two phylogenetic inference approaches: maximum likelihood (ML), using the RaxML 8.2.3 software (Stamatakis 2014), and Bayesian inferences (BI), using MrBayes 3.2.4 (Ronquist et al. 2012). The evolutionary model used, GTR+I+G, was estimated to be the best with the software jModeltest 2.1.7 (Darriba et al. 2012; Guindon & Gascuel 2003), using the Akaike Information Criterion (AIC). MrBayes analyses were performed for 10-milion generation with sampling parameters every 10 3 and a 25% default burn-in value for the final trees. Convergence of the two runs (average standard deviation of split frequencies << 0.01) and likelihood stationarity were checked. The maximum likelihood analyses were performed under 1000 bootstrap pseudoreplicates. The phylogenetic results show a close and highly supported relationship of the Italian Diversibipalium specimens with those from Japan and South Korea that have been identified as D. multilineatum (Fig. 2). Diversibipalium multilineatum is the sister-group of B. nobile Kawakatsu & Makino, 1982, but with low support. The COI sequences of the French (MNHN JL177) and the Italian Diversibipalium specimens were compared in Geneious v. 8.0.5 (http://www.geneious.com, Kearse et al. 2012) and were found to be identical. These results indicate that the species introduced in both countries is the same, and most probably concerns the species D. multilineatum. The pathways of introduction of D. multilineatum are currently unknown, although a relationship between the horticultural trade and the introduction of alien land planarians is well known (Álvarez-Presas et al. 2014 and references therein). Here we report the first occurrence of individuals of D. multilineatum outside Asia. The GenBank sequence of D. multilineatum from South Korea is not yet supported by a published description of the specimen, while it is debatable whether South Korea should be considered part of the natural range of D. multilineatum, which only seems to include Japan. In the present paper, we consider the South Korean animal to be an introduced specimen. Soil moisture status, temperature, and food availability are considered to be the main factors determining the presence of terrestrial planarians (Boag et al. 1998); the microclimatic conditions of the Italian garden were similar to plant nurseries and greenhouses, while an abundance of food was available, such as isopods [Porcellionides pruinosus (Brandt, 1833)], oligochaetes [Dendrobaena attemsi (Michaelsen, 1902) and several juveniles of Lumbricus spp.] and gastropods [Cernuella cisalpina (Rossmassler, 1837), Cornu aspersum (O.F. Müller 1774), Deroceras reticulatum (O.F. Müller, 1774), Discus rotundatus (O.F. Müller, 1774), Limacus flavus (Linnaeus, 1758), Milax nigricans (Philippi, 1836), Papillifera papillaris (Linnaeus, 1758), Pomatias elegans (O.F. Müller, 1774)]. Moreover, winter 2014 reached the highest temperatures and rainfall of the last two decades (source: CNR-ISAC, Bologna), thus favouring establishment and spread of D. multilineatum. The potential environmental impacts of some invasive flatworms are well documented (Álvarez-Presas et al. 2014; Justine et al. 2014) and, even if these effects have not yet been assessed for D. multilineatum, the adoption of precautionary measures and of early intervention is here strongly recommended (Genovesi & Shine 2004). Finally, knowledge of the introduction pathway(s), together with the analysis of prey preference and possible impact on the invertebrate fauna, will be essential to halt or at least to limit the spread of this introduced land flatworm.

The growth and patterning of anatomical structures from specific cellular fields in developing organisms relies on organizing centers that instruct surrounding cells to modify their behavior, namely migration, proliferation, and differentiation. We discuss here how organizers can form in adult organisms, a process of utmost interest for regenerative medicine. Animals like Hydra and planarians, which maintain their shape and fitness thanks to a highly dynamic homeostasis, offer a useful paradigm to study adult organizers in steady-state conditions. Beside the homeostatic context, these model systems also offer the possibility to study how organizers form de novo from somatic adult tissues. Both extracellular matrix remodeling and caspase activation play a key role in this transition, acting as promoters of organizer formation in the vicinity of the wound. Their respective roles and the crosstalk between them just start to be deciphered., (© 2016 Elsevier Inc. All rights reserved.)

The wnt signaling pathway is an intercellular communication mechanism essential in cell-fate specification, tissue patterning and regional-identity specification. A βcatenin-dependent signal specifies the AP (Anteroposterior) axis of planarians, both during regeneration of new tissues and during normal homeostasis. Accordingly, four wnts (posterior wnts) are expressed in a nested manner in central and posterior regions of planarians. We have analyzed the specific role of each posterior wnt and the possible cooperation between them in specifying and patterning planarian central and posterior regions. We show that each posterior wnt exerts a distinct role during re-specification and maintenance of the central and posterior planarian regions, and that the integration of the different wnt signals (βcatenin dependent and independent) underlies the patterning of the AP axis from the central region to the tip of the tail. Based on these findings and data from the literature, we propose a model for patterning the planarian AP axis.

Traditional toxicology testing has relied on low-throughput, expensive mammalian studies; however, timely testing of the large number of environmental toxicants requires new in vitro and in vivo platforms for inexpensive medium- to high-throughput screening. Herein, we describe the suitability of the asexual freshwater planarian Dugesia japonica as a new animal model for the study of developmental neurotoxicology. As these asexual animals reproduce by binary fission, followed by regeneration of missing body structures within approximately 1 week, development and regeneration occur through similar processes allowing us to induce neurodevelopment "at will" through amputation. This short time scale and the comparable sizes of full and regenerating animals enable parallel experiments in adults and developing worms to determine development-specific aspects of toxicity. Because the planarian brain, despite its simplicity, is structurally and molecularly similar to the mammalian brain, we are able to ascertain neurodevelopmental toxicity that is relevant to humans. As a proof of concept, we developed a 5-step semiautomatic screening platform to characterize the toxicity of 9 known neurotoxicants (consisting of common solvents, pesticides, and detergents) and a neutral agent, glucose, and quantified effects on viability, stimulated and unstimulated behavior, regeneration, and brain structure. Comparisons of our findings with other alternative toxicology animal models, such as zebrafish larvae and nematodes, demonstrated that planarians are comparably sensitive to the tested chemicals. In addition, we found that certain compounds induced adverse effects specifically in developing animals. We thus conclude that planarians offer new complementary opportunities for developmental neurotoxicology animal models., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

We discuss the expediency of using invertebrates, such as flatworms and planarians, as experimental objects. Free-living planarian flatworms (phylum Platyhelminthes, class Turbellaria) are invertebrate animals in which a bilateral symmetry appears for the first time in evolution and organs and tissues form. As the highest ecological link of the food chain--predators--these animals are characterized by a set of behavioral reactions controlled by a differentiated central nervous system. Planarians have unsurpassed ability to regenerate lost or damaged body parts. Owing to the ease of their breeding and their convenience for manipulations, these animals are used to study the influence of chemical and physical factors on the processes of life, growth, and reproduction. Currently, planarians are recognized as a model for biological research in the field of regeneration, stem cell biology, study of their proliferation and differentiation, as well as the regulatory mechanisms of morphogenetic processes. The genome of the planarian Schmidtea mediterranea was fully sequenced, which opened up the opportunity to work with this object at the molecular biological level. Furthermore, planarians are used in neurobiological and toxicological studies, in studying the evolutionary aspects of centralization of the nervous system, mechanisms of muscle contraction, and in the development of new antiparasitic drugs. This review aims to demonstrate the relevance and diversity of research conducted on simple biological objects--planarians--to awider audience to show the historical continuity of these studies and their wide geographical distribution and to focus on the studies carried out in Russia, which, as a rule, are not included in the foreign reviews on planarian regeneration.

Members of the COE family of transcription factors are required for central nervous system (CNS) development. However, the function of COE in the post-embryonic CNS remains largely unknown. An excellent model for investigating gene function in the adult CNS is the freshwater planarian. This animal is capable of regenerating neurons from an adult pluripotent stem cell population and regaining normal function. We previously showed that planarian coe is expressed in differentiating and mature neurons and that its function is required for proper CNS regeneration. Here, we show that coe is essential to maintain nervous system architecture and patterning in intact (uninjured) planarians. We took advantage of the robust phenotype in intact animals to investigate the genetic programs coe regulates in the CNS. We compared the transcriptional profiles of control and coe RNAi planarians using RNA sequencing and identified approximately 900 differentially expressed genes in coe knockdown animals, including 397 downregulated genes that were enriched for nervous system functional annotations. Next, we validated a subset of the downregulated transcripts by analyzing their expression in coe-deficient planarians and testing if the mRNAs could be detected in coe+ cells. These experiments revealed novel candidate targets of coe in the CNS such as ion channel, neuropeptide, and neurotransmitter genes. Finally, to determine if loss of any of the validated transcripts underscores the coe knockdown phenotype, we knocked down their expression by RNAi and uncovered a set of coe-regulated genes implicated in CNS regeneration and patterning, including orthologs of sodium channel alpha-subunit and pou4. Our study broadens the knowledge of gene expression programs regulated by COE that are required for maintenance of neural subtypes and nervous system architecture in adult animals.

During adult homeostasis and regeneration, the freshwater planarian must accomplish a constant balance between cell proliferation and cell death, while also maintaining proper tissue and organ size and patterning. How these ordered processes are precisely modulated remains relatively unknown. Here we show that planarians use the downstream effector of the Hippo signaling cascade, yorkie (yki; YAP in vertebrates) to control a diverse set of pleiotropic processes in organ homeostasis, stem cell regulation, regeneration and axial patterning. We show that yki functions to maintain the homeostasis of the planarian excretory (protonephridial) system and to limit stem cell proliferation, but does not affect the differentiation process or cell death. Finally, we show that Yki acts synergistically with WNT/β-catenin signaling to repress head determination by limiting the expression domains of posterior WNT genes and that of the WNT-inhibitor notum. Together, our data show that yki is a key gene in planarians that integrates stem cell proliferation control, organ homeostasis, and the spatial patterning of tissues.

A fundamental assumption of today's molecular genetics paradigm is that complex morphology emerges from the combined activity of low-level processes involving proteins and nucleic acids. An inherent characteristic of such nonlinear encodings is the difficulty of creating the genetic and epigenetic information that will produce a given self-assembling complex morphology. This 'inverse problem' is vital not only for understanding the evolution, development and regeneration of bodyplans, but also for synthetic biology efforts that seek to engineer biological shapes. Importantly, the regenerative mechanisms in deer antlers, planarian worms and fiddler crabs can solve an inverse problem: their target morphology can be altered specifically and stably by injuries in particular locations. Here, we discuss the class of models that use pre-specified morphological goal states and propose the existence of a linear encoding of the target morphology, making the inverse problem easy for these organisms to solve. Indeed, many model organisms such as Drosophila, hydra and Xenopus also develop according to nonlinear encodings producing linear encodings of their final morphologies. We propose the development of testable models of regeneration regulation that combine emergence with a top-down specification of shape by linear encodings of target morphology, driving transformative applications in biomedicine and synthetic bioengineering.