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.

Pluripotent cells in the embryo can generate all cell types, but lineage-restricted cells are generally thought to replenish adult tissues. Planarians are flatworms and regenerate from tiny body fragments, a process requiring a population of proliferating cells (neoblasts). Whether regeneration is accomplished by pluripotent cells or by the collective activity of multiple lineage-restricted cell types is unknown. Using ionizing radiation and single-cell transplantation, we identified neoblasts that can form large descendant-cell colonies in vivo. These clonogenic neoblasts (cNeoblasts) produce cells that differentiate into neuronal, intestinal, and other known post-mitotic cell types and are distributed throughout the body. Single transplanted cNeoblasts restored regeneration in lethally irradiated hosts. We conclude that broadly distributed, adult pluripotent stem cells underlie the remarkable regenerative abilities of planarians.

The transcriptome of a cell dictates its unique cell type biology. We used single-cell RNA sequencing to determine the transcriptomes for essentially every cell type of a complete animal: the regenerative planarian Planarians contain a diverse array of cell types, possess lineage progenitors for differentiated cells (including pluripotent stem cells), and constitutively express positional information, making them ideal for this undertaking. We generated data for 66,783 cells, defining transcriptomes for known and many previously unknown planarian cell types and for putative transition states between stem and differentiated cells. We also uncovered regionally expressed genes in muscle, which harbors positional information. Identifying the transcriptomes for potentially all cell types for many organisms should be readily attainable and represents a powerful approach to metazoan biology.

The principles underlying regeneration in planarians have been explored for over 100 years through surgical manipulations and cellular observations. Planarian regeneration involves the generation of new tissue at the wound site via cell proliferation (blastema formation), and the remodeling of pre-existing tissues to restore symmetry and proportion (morphallaxis). Because blastemas do not replace all tissues following most types of injuries, both blastema formation and morphallaxis are needed for complete regeneration. Here we discuss a proliferative cell population, the neoblasts, that is central to the regenerative capacities of planarians. Neoblasts may be a totipotent stem-cell population capable of generating essentially every cell type in the adult animal, including themselves. The population properties of the neoblasts and their descendants still await careful elucidation. We identify the types of structures produced by blastemas on a variety of wound surfaces, the principles guiding the reorganization of pre-existing tissues, and the manner in which scale and cell number proportions between body regions are restored during regeneration.

Planarians are flatworms capable of regenerating any missing body region. This capacity is mediated by neoblasts, a proliferative cell population that contains pluripotent stem cells. Although population-based studies have revealed many neoblast characteristics, whether functionally distinct classes exist within this population is unclear. Here, we used high-dimensional single-cell transcriptional profiling from over a thousand individual neoblasts to directly compare gene expression fingerprints during homeostasis and regeneration. We identified two prominent neoblast classes that we named ζ (zeta) and σ (sigma). Zeta-neoblasts encompass specified cells that give rise to an abundant postmitotic lineage including epidermal cells, and are not required for regeneration. By contrast, sigma-neoblasts proliferate in response to injury, possess broad lineage capacity, and can give rise to zeta-neoblasts. These findings present a new view of planarian neoblasts, in which the population is comprised of two major and functionally distinct cellular compartments.

Planarians have been a classic model system for the study of regeneration, tissue homeostasis, and stem cell biology for over a century, but they have not historically been accessible to extensive genetic manipulation. Here we utilize RNA-mediated genetic interference (RNAi) to introduce large-scale gene inhibition studies to the classic planarian system. 1065 genes were screened. Phenotypes associated with the RNAi of 240 genes identify many specific defects in the process of regeneration and define the major categories of defects planarians display following gene perturbations. We assessed the effects of inhibiting genes with RNAi on tissue homeostasis in intact animals and stem cell (neoblast) proliferation in amputated animals identifying candidate stem cell, regeneration, and homeostasis regulators. Our study demonstrates the great potential of RNAi for the systematic exploration of gene function in understudied organisms and establishes planarians as a powerful model for the molecular genetic study of stem cells, regeneration, and tissue homeostasis.

How animals establish and pattern the primary body axis is one of the most fundamental problems in biology. Data from diverse deuterostomes (frog, fish, mouse, and amphioxus) and from planarians (protostomes) suggest that Wnt signaling through beta-catenin controls posterior identity during body plan formation in most bilaterally symmetric animals. Wnt signaling also influences primary axis polarity of pre-bilaterian animals, indicating that an axial patterning role for Wnt signaling predates the evolution of bilaterally symmetric animals. The use of posterior Wnt signaling and anterior Wnt inhibition might be a unifying principle of body plan development in most animals.

Planarian flatworms can regenerate heads at anterior-facing wounds and tails at posterior-facing wounds throughout the body. How this regeneration polarity is specified has been a classic problem for more than a century. We identified a planarian gene, Smed-betacatenin-1, that controls regeneration polarity. Posterior-facing blastemas regenerate a head instead of a tail in Smed-betacatenin-1(RNAi) animals. Smed-betacatenin-1 is required after wounding and at any posterior-facing wound for polarity. Additionally, intact Smed-betacatenin-1(RNAi) animals display anteriorization during tissue turnover. Five Wnt genes and a secreted Frizzled-related Wnt antagonist-like gene are expressed in domains along the anteroposterior axis that reset to new positions during regeneration, which suggests that Wnts control polarity through Smed-betacatenin-1. Our data suggest that beta-catenin specifies the posterior character of the anteroposterior axis throughout the Bilateria and specifies regeneration polarity in planarians.