Planarians possess naturally occurring pluripotent adult somatic stem cells (neo-blasts) required for homeostasis and whole-body regeneration. However, no reliable neoblast culture methods are currently available, hindering mechanistic studies of pluripotency and the development of transgenic tools. We report robust methods for neoblast culture and delivery of exogenous mRNAs. We identify optimal culture media for the short-term maintenance of neoblasts in vitro and show via transplantation that cultured stem cells retain pluripotency for two days. We developed a procedure that significantly improves neoblast yield and purity by modifying standard flow cytometry methods. These methods enable the introduction and expression of exogenous mRNAs in neoblasts, overcoming a key hurdle impeding the application of transgenics in planarians. The advances in cell culture reported here create new opportunities for mechanistic studies of planarian adult stem cell pluripotency, and provide a systematic framework to develop cell culture techniques in other emerging research organisms.

Planarian flatworms are best known for their impressive regenerative capacity, yet this trait varies across species. In addition, planarians have other features that share morphology and function with the tissues of many other animals, including an outer mucociliary epithelium that drives planarian locomotion and is very similar to the epithelial linings of the human lung and oviduct. Planarians occupy a broad range of ecological habitats and are known to be sensitive to changes in their environment. Yet, despite their potential to provide valuable insight to many different fields, very few planarian species have been developed as laboratory models for mechanism-based research. Here we describe a previously undocumented planarian isolate, Girardia sp. (Guanajuato). After collecting this isolate from a freshwater habitat in central Mexico, we characterized it at the morphological, cellular, and mo-lecular level. We show that Girardia sp. (Guanajuato) not only shares features with animals in the Girardia genus but also possesses traits that appear unique to this isolate. By thoroughly characterizing this new planarian isolate, our work facilitates future comparisons to other flatworms and further molecular dissection of the unique and physiologically-relevant traits observed in this Girardia sp. (Guanajuato) isolate.

Hox genes are highly conserved and well-known for their role in segmental patterning during early development. Here, the authors identify an unreported role for Hox genes in the adult tissue patterning and fission behavior required for asexual reproduction in planarian flatworms.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.

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 spectrin beta 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.

As the planarian research community expands, the need for an interoperable data organization framework for tool building has become increasingly apparent. Such software would streamline data annotation and enhance cross-platform and cross-species searchability. We created the Planarian Anatomy Ontology (PLANA), an extendable relational framework of defined Schmidtea mediterranea (Smed) anatomical terms used in the field. At publication, PLANA contains over 850 terms describing Smed anatomy from subcellular to system levels across all life cycle stages, in intact animals and regenerating body fragments. Terms from other anatomy ontologies were imported into PLANA to promote interoperability and comparative anatomy studies. To demonstrate the utility of PLANA as a tool for data curation, we created resources for planarian embryogenesis, including a staging series and molecular fate-mapping atlas, and the Planarian Anatomy Gene Expression database, which allows retrieval of a variety of published transcript/gene expression data associated with PLANA terms. As an open-source tool built using FAIR (findable, accessible, interoperable, reproducible) principles, our strategy for continued curation and versioning of PLANA also provides a platform for community-led growth and evolution of this resource.

PIWI proteins utilize small RNAs called piRNAs to silence transposable elements, thereby protecting germline integrity. In planarian flatworms, PIWI proteins are essential for regeneration, which requires adult stem cells termed neoblasts. Here, we characterize planarian piRNAs and examine the roles of PIWI proteins in neoblast biology. We find that the planarian PIWI proteins SMEDWI-2 and SMEDWI-3 cooperate to degrade active transposons via the ping-pong cycle. Unexpectedly, we discover that SMEDWI-3 plays an additional role in planarian mRNA surveillance. While SMEDWI-3 degrades numerous neoblast mRNAs in a homotypic ping-pong cycle, it is also guided to another subset of neoblast mRNAs by antisense piRNAs and binds these without degrading them. Mechanistically, the distinct activities of SMEDWI-3 are primarily dictated by the degree of complementarity between target mRNAs and antisense piRNAs. Thus, PIWI proteins enable planarians to repurpose piRNAs for potentially critical roles in neoblast mRNA turnover.

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 TGF beta and Wnt signalling pathways as regulators of the frequency of fission initiation rather than the position of fission planes. Finally, inhibition of Wnt and TGF beta 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 TGF beta and Wnt in regulating size-dependent behaviour, and uncovers an interdependence between patterning, growth and neurological function.

Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals.

The epidermis is essential for animal survival, providing both a protective barrier and cellular sensor to external environments. The generally conserved embryonic origin of the epidermis, but the broad morphological and functional diversity of this organ across animals is puzzling. We define the transcriptional regulators underlying epidermal lineage differentiation in the planarian Schmidtea mediterranea, an invertebrate organism that, unlike fruitflies and nematodes, continuously replaces its epidermal cells. We find that Smed-p53, Sox and Pax transcription factors are essential regulators of epidermal homeostasis, and act cooperatively to regulate genes associated with early epidermal precursor cell differentiation, including a tandemly arrayed novel gene family (prog) of secreted proteins. Additionally, we report on the discovery of distinct and previously undescribed secreted organelles whose production is dependent on the transcriptional activity of soxP-3, and which we term Hyman vesicles.