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We have identified two genes, smedwi-1 and smedwi-2, expressed in the dividing adult stem cells (neoblasts) of the planarian Schmidtea mediterranea. Both genes encode proteins that belong to the Argonaute/PIWI protein family and that share highest homology with those proteins defined by Drosophila PIWI. RNA interference (RNAi) of smedwi-2 blocks regeneration, even though neoblasts are present, irradiation-sensitive, and capable of proliferating in response to wounding; smedwi-2(RNAi) neoblast progeny migrate to sites of cell turnover but, unlike normal cells, fail at replacing aged tissue. We suggest that SMEDWI-2 functions within dividing neoblasts to support the generation of cells that promote regeneration and homeostasis.

Key Points Regeneration is widely distributed among the various phyla that compose the animal kingdom, including vertebrates. Limitations in the ability to interrogate this attribute at the molecular level have hampered efforts to delineate the mechanistic underpinnings of regeneration. Loss-of-function screens (using RNAi) and gain-of-function assays (transgenesis) have recently been introduced to study molecular pathways in traditional model systems of regeneration, overcoming past limitations to probe their biology at the molecular level. Studies in simple animals such as hydra and planarians are beginning to contribute to our understanding of tissue remodelling and adult somatic stem-cell regulation in animals. Studies in mammals and other vertebrates have highlighted central roles for the activation of specific signalling pathways in the processes of organ and limb regeneration. Comparative studies of regenerative processes among the various animals that are currently under investigation could provide important insights into the permissive and non-permissive mechanisms that underlie regenerative competence. Such information is likely to yield fundamental insights for our understanding of metazoan biology, and to expand the repertoire of therapeutic possibilities in the fields of regenerative medicine. Studies of regenerative biology have been hampered by a lack of tools for testing gene function in traditional model species. Recent advances have overcome this problem, leading to new insights into both basic regenerative biology and its biomedical potential. Significant progress has recently been made in our understanding of animal regenerative biology, spurred on by the use of a wider range of model organisms and an increasing ability to use genetic tools in traditional models of regeneration. This progress has begun to delineate differences and similarities in the regenerative capabilities and mechanisms among diverse animal species, and to address some of the key questions about the molecular and cell biology of regeneration. Our expanding knowledge in these areas not only provides insights into animal biology in general, but also has important implications for regenerative medicine and stem-cell biology.

After amputation, freshwater planarians properly regenerate a head or tail from the resulting anterior or posterior wound. The mechanisms that differentiate anterior from posterior and direct the replacement of the appropriate missing body parts are unknown. We found that in the planarian Schmidtea mediterranea, RNA interference (RNAi) of β-catenin or dishevelled causes the inappropriate regeneration of a head instead of a tail at posterior amputations. Conversely, RNAi of the β-catenin antagonist adenomatous polyposis coli results in the regeneration of a tail at anterior wounds. In addition, the silencing of β-catenin is sufficient to transform the tail of uncut adult animals into a head. We suggest that β-catenin functions as a molecular switch to specify and maintain anteroposterior identity during regeneration and homeostasis in planarians.

The Hedgehog (Hh) signaling pathway plays multiple essential roles during metazoan development, homeostasis, and disease. Although core protein components are highly conserved, the variations in Hh signal transduction mechanisms exhibited by existing model systems (Drosophila, fish, and mammals) are difficult to understand. We characterized the Hh pathway in planarians. Hh signaling is essential for establishing the anterior/posterior axis during regeneration by modulating wnt expression. Moreover, RNA interference methods to reduce signal transduction proteins Cos2/Kif27/Kif7, Fused, or Iguana do not result in detectable Hh signaling defects; however, these proteins are essential for planarian ciliogenesis. Our study expands the understanding of Hh signaling in the animal kingdom and suggests an ancestral mechanistic link between Hh signaling and the function of cilia.

The centrosome, a cytoplasmic organelle formed by cylinder-shaped centrioles surrounded by a microtubule-organizing matrix, is a hallmark of animal cells. The centrosome is conserved and essential for the development of all animal species described so far. Here, we show that planarians, and possibly other flatworms, lack centrosomes. In planarians, centrioles are only assembled in terminally differentiating ciliated cells through the acentriolar pathway to trigger the assembly of cilia. We identified a large set of conserved proteins required for centriole assembly in animals and note centrosome protein families that are missing from the planarian genome. Our study uncovers the molecular architecture and evolution of the animal centrosome and emphasizes the plasticity of animal cell biology and development.

Regeneration requires the coordination of stem cells, their progeny and distant differentiated tissues. Here, we present a comprehensive atlas of whole-body regeneration in Schmidtea mediterranea and identify wound-induced cell states. An analysis of 299,998 single-cell transcriptomes captured from regeneration-competent and regeneration-incompetent fragments identified transient regeneration-activated cell states (TRACS) in the muscle, epidermis and intestine. TRACS were independent of stem cell division with distinct spatiotemporal distributions, and RNAi depletion of TRACS-enriched genes produced regeneration defects. Muscle expression of notum, follistatin, evi/wls, glypican-1 and junctophilin-1 was required for tissue polarity. Epidermal expression of agat-1/2/3, cyp3142a1, zfhx3 and atp1a1 was important for stem cell proliferation. Finally, expression of spectrinβ and atp12a in intestinal basal cells, and lrrk2, cathepsinB, myosin1e, polybromo-1 and talin-1 in intestinal enterocytes regulated stem cell proliferation and tissue remodelling, respectively. Our results identify cell types and molecules that are important for regeneration, indicating that regenerative ability can emerge from coordinated transcriptional plasticity across all three germ layers.Benham-Pyle et al. present a single-cell analysis of planarian regeneration and identify rare amputation-specific cell states in the muscle, epidermis and intestine that are required for patterning and stem cell proliferation.

Planarian flatworms regenerate every organ after amputation. Adult pluripotent stem cells drive this ability, but how injury activates and directs stem cells into the appropriate lineages is unclear. Here we describe a single-organ regeneration assay in which ejection of the planarian pharynx is selectively induced by brief exposure of animals to sodium azide. To identify genes required for pharynx regeneration, we performed an RNAi screen of 356 genes upregulated after amputation, using successful feeding as a proxy for regeneration. We found that knockdown of 20 genes caused a wide range of regeneration phenotypes and that RNAi of the forkhead transcription factor FoxA, which is expressed in a subpopulation of stem cells, specifically inhibited regrowth of the pharynx. Selective amputation of the pharynx therefore permits the identification of genes required for organ-specific regeneration and suggests an ancient function for FoxA-dependent transcriptional programs in driving regeneration. Some animals can regrow whole limbs or organs after amputation. Flatworms called planaria, for example, can regenerate their whole body from small pieces. This remarkable ability depends on neoblasts—a type of stem cell found in planaria that can detect damaged or lost organs, migrate to the site of damage, produce the required cells, and integrate into the remaining tissues. Researchers hope that studying these animals will reveal ways to use stem cells to regenerate injured limbs or organs in humans. Planaria have been used in many studies of regeneration. However, manually amputating organs from the flatworms is time-consuming and the resulting wounds vary, which makes it hard to compare regeneration between animals treated in different ways. Now, Adler et al. have developed a new technique for studying regeneration in planaria. Placing the flatworms briefly into a solution of sodium azide causes the pharynx—an organ that is used for both eating and excretion–to drop off. Using chemicals in this way means the loss of the pharynx leaves a uniform wound, with no damage to the adjacent digestive system, and that large numbers of planaria with identical wounds can be produced rapidly. To ensure that treatment with sodium azide did not alter normal regeneration processes in planaria, Adler et al. carried out manual amputation of tissue in sodium azide-treated flatworms; regeneration in these flatworms was identical to that seen in untreated planaria. Planaria in which the pharynx had been removed by sodium azide exposure showed rapid recruitment of neoblasts to the wound site, where they formed epidermal, muscle and nerve cells, and organized into a functioning pharynx within a few days. Adler et al. then identified 20 genes that were required for various stages of regeneration. These experiments revealed that a transcription factor (a protein that controls gene expression) called FoxA was specifically required for the regrowth of the pharynx. This is a previously unknown function for FoxA.

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. A planarian fission protocol shows that the number of progeny and the frequency of fission initiation correlate with parent size, and TGFβ and Wnt signalling components are identified as regulators of fission behaviour.

The sexual strain of the planarian Schmidtea mediterranea , indigenous to Tunisia and several Mediterranean islands, is a hermaphrodite 1 , 2 . Here we isolate individual chromosomes and use sequencing, Hi-C 3 , 4 and linkage mapping to assemble a chromosome-scale genome reference. The linkage map reveals an extremely low rate of recombination on chromosome 1. We confirm suppression of recombination on chromosome 1 by genotyping individual sperm cells and oocytes. We show that previously identified genomic regions that maintain heterozygosity even after prolonged inbreeding make up essentially all of chromosome 1. Genome sequencing of individuals isolated in the wild indicates that this phenomenon has evolved specifically in populations from Sardinia and Corsica. We find that most known master regulators 5 – 13 of the reproductive system are located on chromosome 1. We used RNA interference 14 , 15 to knock down a gene with haplotype-biased expression, which led to the formation of a more pronounced female mating organ. On the basis of these observations, we propose that chromosome 1 is a sex-primed autosome primed for evolution into a sex chromosome. Assembly and analysis of the Schmidtea mediterranea genome indicate that this planarian’s chromosome 1 may be evolving into a sex chromosome.

Cystic kidney diseases (CKDs) affect millions of people worldwide. The defining pathological features are fluid-filled cysts developing from nephric tubules due to defective flow sensing, cell proliferation and differentiation. The underlying molecular mechanisms, however, remain poorly understood, and the derived excretory systems of established invertebrate models (Caenorhabditis elegans and Drosophila melanogaster) are unsuitable to model CKDs. Systematic structure/function comparisons revealed that the combination of ultrafiltration and flow-associated filtrate modification that is central to CKD etiology is remarkably conserved between the planarian excretory system and the vertebrate nephron. Consistently, both RNA-mediated genetic interference (RNAi) of planarian orthologues of human CKD genes and inhibition of tubule flow led to tubular cystogenesis that share many features with vertebrate CKDs, suggesting deep mechanistic conservation. Our results demonstrate a common evolutionary origin of animal excretory systems and establish planarians as a novel and experimentally accessible invertebrate model for the study of human kidney pathologies. Millions of people around the world are affected by cystic kidney diseases, which are amongst the most common inherited genetic disorders. Throughout their life, people with these diseases develop fluid-filled cysts in their kidneys, which stop these organs from working properly and can eventually lead to organ failure. Healthy kidneys perform many vital roles in the body, including removing waste products and keeping the concentration of salts in the blood in balance. These activities depend on the kidneys filtering the blood, and then reabsorbing useful chemicals from the filtered fluid as it passes through small tubes called tubules. Cysts disrupt both of these processes. Mutations in many different genes can cause cystic kidney diseases. Many of these genes encode proteins that are involved in the formation of cilia: hair-like structures that project from some cell membranes. Cilia on the cells that line tubules are thought to bend in response to the flow of fluid and then generate signals that dampen cell proliferation. This would explain how the loss of cilia could cause too many cells to develop, which would lead to the formation of cysts. But many of the molecular details are missing from this explanation. Previous studies in mammals and simple model organisms (such as fruit flies and roundworms) have sought to fill in the gaps, but each model has its own limitations. Now, Thi-Kim Vu et al. propose that a flat worm called a planarian could provide a new and experimentally accessible animal model to study cystic kidney diseases. These flat worms get rid of their waste products via an excretory system that consists of branched tubules that spread throughout the body. Thi-Kim Vu et al. found that, like the tubules in the kidney, these tubules filter and then reabsorb chemicals from body fluids. Moreover, these processes are performed in different parts of the tubules, exactly as they are in the tubules in kidneys. The genome of a flat worm called Schmidtea mediterranea contains many genes that cause cysts to form when their equivalents are mutated in humans. Reducing the expression of these genes (and others that are involved in cilia formation) also caused cysts to form in the flat worms. These findings indicate that it is likely that the excretory systems of different animals have a shared evolutionary history. If so, the findings support the idea that cilia in kidney tubules send signals in response to fluid flow that affect kidney-specific stem cells. They also suggest that problems with these signals could be at the core of some human cystic kidney diseases. One of the next challenges will be to identify these cilia-associated signals. Finally, given that studies involving thousands of flat worms can be carried out with minimal cost, the ultimate goal is to develop flat worms into a new model to discover and investigate genes linked to human kidney diseases.