Planarians require ced-12/elmo-1 to clear dead cells by excretion through the gut.

Cell corpse removal is a critical component of both development and homeostasis throughout the animal kingdom. Extensive research has revealed many of the mechanisms involved in corpse removal, typically involving engulfment and digestion by another cell; however, the dynamics of cell corpse clearance in adult tissues remain unclear. Here, we track cell death in the adult planarian Schmidtea mediterranea and find that, following light-induced cell death, pigment cell corpses transit to the gut and are excreted from the animal. Gut phagocytes, previously only known to phagocytose food, are required for pigment cells to enter the gut lumen. Finally, we show that the planarian ortholog of ced-12/engulfment and cell motility (ELMO) is required for corpse phagocytosis and removal through the gut. In total, we present a mechanism of cell clearance in an adult organism involving transit of dead cells to the gut, transport into the gut by phagocytes, and physical excretion of debris.

An RNA/DNA-Based Flow Cytometry Approach for Isolating Slow-Cycling Stem Cells.

Flow cytometry methods for sorting specific populations of cells based on fluorescence or physical properties have been a widely used technique for decades. Flow cytometry has been particularly vital to the study of planarians, which remain refractory to transgenic transformation, as it has provided a work-around solution for studying stem cell biology and lineage relationships in the context of regeneration. Many flow cytometry applications have been published in planarians, beginning with broad Hoechst-based strategies for isolating cycling stem cells and progressing to more function-based approaches involving vital dyes and surface antibodies. In this protocol, we look to build on the classic DNA-labeling Hoechst staining strategy by adding pyronin Y staining to label RNA. While Hoechst labeling alone allows for the isolation of stem cells in the S/G2/M phases of the cell cycle, heterogeneity within the population of stem cells with 2 C DNA content is not resolved. By considering RNA levels, this protocol can further divide this population of stem cells into two groups: G1 stem cells with relatively high RNA content and a slow-cycling population with low RNA content, which we call RNAlow stem cells. In addition, we provide instruction for combining this RNA/DNA flow cytometry protocol with EdU labeling experiments and describe an optional step for incorporating immunostaining prior to cell sorting (in this case with the pluripotency marker TSPAN-1). This protocol adds a new staining strategy and examples of combinatorial flow cytometry approaches to the repertoire of flow cytometry techniques for studying planarian stem cells.

Identification of TOR-responsive slow-cycling neoblasts in planarians.

Epimorphic regeneration commonly relies on the activation of reserved stem cells to drive new cell production. The planarian Schmidtea mediterranea is among the best regenerators in nature, thanks to its large population of adult stem cells, called neoblasts. While neoblasts have long been known to drive regeneration, whether a subset of neoblasts is reserved for this purpose is unknown. Here, we revisit the idea of reserved neoblasts by approaching neoblast heterogeneity from a regulatory perspective. By implementing a new fluorescence-activated cell sorting strategy in planarians, we identify a population of neoblasts defined by low transcriptional activity. These RNAlow neoblasts are relatively slow-cycling at homeostasis and undergo a morphological regeneration response characterized by cell growth at 48 h post-amputation. At this time, RNAlow neoblasts proliferate in a TOR-dependent manner. Additionally, knockdown of the tumour suppressor Lrig-1, which is enriched in RNAlow neoblasts, results in RNAlow neoblast growth and hyperproliferation at homeostasis, and ultimately delays regeneration. We propose that slow-cycling RNAlow neoblasts represent a regeneration-reserved neoblast population.

Planarian EGF repeat-containing genes megf6 and hemicentin are required to restrict the stem cell compartment.

The extracellular matrix (ECM) is important for maintaining the boundaries between tissues. This role is particularly critical in the stem cell niche, as pre-neoplastic or cancerous stem cells must pass these boundaries in order to invade into the surrounding tissue. Here, we examine the role of the ECM as a regulator of the stem cell compartment in the planarian Schmidtea mediterranea, a highly regenerative, long-lived organism with a large population of adult stem cells. We identify two EGF repeat-containing genes, megf6 and hemicentin, with identical knockdown phenotypes. We find that megf6 and hemicentin are needed to maintain the structure of the basal lamina, and in the absence of either gene, pluripotent stem cells migrate ectopically outside of their compartment and hyper-proliferate, causing lesions in the body wall muscle. These muscle lesions and ectopic stem cells are also associated with ectopic gut branches, which protrude from the normal gut towards the dorsal side of the animal. Interestingly, both megf6 and hemicentin knockdown worms are capable of regenerating tissue free of both muscle lesions and ectopic cells, indicating that these genes are dispensable for regeneration. These results provide insight into the role of planarian ECM in restricting the stem cell compartment, and suggest that signals within the compartment may act to suppress stem cell hyperproliferation.

The true colours of the flatworm: Mechanisms of pigment biosynthesis and pigment cell lineage development in planarians.

Pigment cells serve a variety of important uses across the animal kingdom, and in many species can change and regenerate throughout the lifetime of the organism. The functions of these cells, as well as their origins in both embryonic development and adult regeneration, are not fully understood. Here, we review advances in the study of pigment cells in the freshwater planarian, a model system for stem cell biology and regeneration. Freshwater planarians produce at least three pigment types to generate brown eye and body colouration: melanin, porphyrin, and ommochrome. The body pigments of planarians are produced and contained by a specialized, highly dendritic cell type located in the subepidermal parenchymal space. This cell type is specifically ablated following intense light exposure, a characteristic which has been exploited to discover the gene expression and regeneration of planarian pigment cells. Regenerating pigment cells progress through an immature state marked by upregulation of pigment synthesis genes before differentiating into mature pigment cells; these two states are dynamically regulated in homeostasis to maintain constant body pigmentation. The transcription factors Albino, FoxF-1, and Ets-1, as well as an FGFR-like molecule, are required for proper maintenance of the pigment lineage in both regeneration and homeostasis. These discoveries set the stage for research into external signals that regulate the pigment lineage, as well as possible functions for pigment cells in planarians, including the extra-ocular light response. These insights will address outstanding questions about the evolutionarily-conserved biology of pigment cells.

Comparative transcriptomic analyses and single-cell RNA sequencing of the freshwater planarian Schmidtea mediterranea identify major cell types and pathway conservation.

BACKGROUND: In the Lophotrochozoa/Spiralia superphylum, few organisms have as high a capacity for rapid testing of gene function and single-cell transcriptomics as the freshwater planaria. The species Schmidtea mediterranea in particular has become a powerful model to use in studying adult stem cell biology and mechanisms of regeneration. Despite this, systematic attempts to define gene complements and their annotations are lacking, restricting comparative analyses that detail the conservation of biochemical pathways and identify lineage-specific innovations. RESULTS: In this study we compare several transcriptomes and define a robust set of 35,232 transcripts. From this, we perform systematic functional annotations and undertake a genome-scale metabolic reconstruction for S. mediterranea. Cross-species comparisons of gene content identify conserved, lineage-specific, and expanded gene families, which may contribute to the regenerative properties of planarians. In particular, we find that the TRAF gene family has been greatly expanded in planarians. We further provide a single-cell RNA sequencing analysis of 2000 cells, revealing both known and novel cell types defined by unique signatures of gene expression. Among these are a novel mesenchymal cell population as well as a cell type involved in eye regeneration. Integration of our metabolic reconstruction further reveals the extent to which given cell types have adapted energy and nucleotide biosynthetic pathways to support their specialized roles. CONCLUSIONS: In general, S. mediterranea displays a high level of gene and pathway conservation compared with other model systems, rendering it a viable model to study the roles of these pathways in stem cell biology and regeneration.

FOX and ETS family transcription factors regulate the pigment cell lineage in planarians.

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.